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Marine Biological Laboratory Library 
Woods Hole, Mass. 


Presented by 


“r. Hideo Moriyama 
Shonan Hygiene Institute 
Kamakura, Japan 


April 23, 1956 


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THE NATURE OF VIRUSES 
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THE ORIGIN OF LIFE 


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THE NATURE OF VIRUSES 
AND 


THE ORIGIN OF LIFE 


BY 


HIDEO MORTYAMA 
Shonan Hygiene Institute, Kamakura, Japan 


TOYO 
is 5 


PUBLISHED BY 
Shonan Hygiene Institute, Kamakura, Japan 


SOLE AGENCY 


Igaku-Shoin Company, Limited 
20, Hongo-6, Bunkyo-ku, Tokyo, Japan 


Printed in Japan by Kato—Bunmei-Sha Printing Co., Tokyo 


PREFACE 


If one were possessed with the geocentric theory, innumerable 
astronomical facts ever known would appear to him to be extremely 
confused. At present an immense number of phenomena concerning 
living matter have been investigated and almost every detail of them 
has been fully explored. However, the author felt that they might 
mostly be not duly understood, so that biology might appear only an 
enormous accumulation of facts not to be entitled to be called a 
“‘science.’’ Where numbers of related facts are fully understood and 
arranged orderly on system there may emerge a “‘science.”’ 

Indeed, most of numberless biological phenomena known appear 
to be extremely complicated and incomprehensible. Such a chaos might 
have prevailed the brain of the geocentrist. The Copernican theory 
may be needed in order for biology to become a “‘science.”’ 

The author felt that he found a theory by which all the life 
phenomena might be explained most simply and by which every known 
fact without exception might be arranged on system orderly. The 
theory was founded on the numerous facts found by the author and 
his collaborator, Dr. Ohashi, and has been advanced, as he believes, 
by applying modern researches carried out by other workers especially 
in virology. 

The manner whereby the theory has developed as well as the way 
in which numerous biological phenomena are arranged orderly accord- 
ing to the theory are described in this book. The more fundamental 
parts of the theory are treated in detail in the sister volume of this 
book, ‘‘Immunity.’’ 

It is very regrettable that the author’s concept may not be fully 
expressed in these two books, because English is not his mother 
tongue and besides he was unfortunately never blessed with any 
aptitude for languages. 


Hideo Moriyama. 


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CONTENTS 


Ler gees ee NL Binet on aa a Pe Sam i Aid ue: SEU, mais. At Rew. f 


PART I 
INTRODUCTION TO VIRUSES 


SHAE TER: 1. VIRUS PAR TICEES: ioc) «ues 4) ise sa keys eects ; 


li Dheekey tovoolvine the Riddleson [iter ise uci acl essen) ie 
Sem ACLeEIAlD Vitis er 2.4 ceteris. blige Ne tkecs oo\ Gea Ge ala emoeioe Mietedoieees 


GHAPTER II, THE SIZE OF-VIRUS PARTICLES. 0 265 cw we ew fe eos 


1. Relation between the Kind of Host Cells and the Size of Virus Particles 
2. Difference in the Resistance of Virus due to Particle Size ...... 
3. Water Quantity Combining with Virus Particles ........... 


CRAPTER IE (‘THE ACTIVITY OF VIRUS PARTICLES. ......0 2 Se 
1. The Difference in Phage Activity due to the Cultivating Condition of 
HOStUBACtenniaiam here airs scorer Sis meee cane at eine ee Ne el co omue cite ees 

2. The Effect of Difference in Bacterial Strains on Phage Activity ... 
Swerne wAClivItys Ole VACCINIA VALU 55.4, Supe ee a) ie. ee 


CHAPTER IV. THE TRANSMISSION OF PROTEIN CHANGE. ..... 


i Muitinniecabls Denatiease oo 82 oxi) gecesi ey & pone so). Sink ae eee 
2 similarity, between henninvand \VaGiuSes.) ... ss: @ suc 16 «6 «6 
Serle nfectionsor Menaturationys eect nac ve) oucuie) een «ah ea sme 


PART II 


THE FUNDAMENTAL STRUCTURE OF 
PROTOPALSM AND OF VIRUSES 


CHAPTER I. THE STRUCTURE AND ACTION OF PROTOPLASM .. . 


ivam@ne StructuresoL Protoplasm = s- ih a ee ee SS 
Za bhewAction Of-Frotvoplasims =.~c8 ee ek tee eres ee Ase cos. ek Rew es 


CHAPTER II. POWERFUL FORCE, GENERATED BY PROTEIN 


Pe ORIVICRIZ ATION: 005 3.) kf Ren cue sicemaNen ate one eee ale ats 
1. Water Molecule Layer Surrounding Virus Particles. ......... 
2. ene; horcesGeneratedsby Polimerization® .. 9.72) Go. ss 2 3, Se 


Vii 


39 
39 
43 


45 
45 
48 


viii CONTENTS 


CHAPTER III. THE PROPAGATION OF TRANSNATURATION ahi: 
AND <THE MULE TIPLICATIONJOBSVIRGSE Siege a0 cule. eee es 52 
1. Propagation of Transnaturation in Protoplasm by Viruses... ..... 52 
2.) opread of .Denaturation an ked -BbloodiCellssame im. |S ee 54 

GHAPTER IV. THE CRYSTALDINEDY ORSVEIRUSES) fale.) Llosa 58 
I.) The Fusion of Pagtieles:! 412) 6. se) a eee tee Babes ike ere 58 
2. Lhe. Expulsionvorshipids: <5 6.5 3/12; (es ee ee ee ee tee 59 
3. he LengthvofiGrystalline Warnus Particles sae arn en ee 62 
4. Various Shapes of Plant Varases:. 4. cuccun x nied, cee ee 66 

CHATER V. FINER STRUCTURE OF VIRUS PARTICLES AND ITS 
SIGNIFICANCE: P91 4% Moa 2a ta a ee ete cane ae Te) eee 68 
1. Decomposition and Fusion of Elementary Bodies of Protoplasm. ... 68 
2>. Virus Particles: Containing Dipids (> oh. ets. BOA ti Bee ele eee 70 
3. The Size of Virus Particles as Coagulated Elementary Bodies .... 74 

CHARTER Wis” -GHE SHARE, OF Vilkklio@eAteit Gl Boy ee an is ene 77 
i caled and Hilamentous Virus (Particles: s* =) au. a eee 77 
2. Particles Elusive in Electron*Micropraphy -- 2 == ae ee 82 

CHAPTER VII. THE MODE OF VIRUS MULTIPLICATION. ...... 87 
1. Multiplication of Phage within Bacterial Cell Protoplasm. ...... 87 
2. The Difference in Nucleic Acid Content between Virus and Host Cell 

PLOtOp las rmcts he thn SBR eae ae i hie Bec Rana 2) te Cee 93 
3::\ The Mode’ of: Produttionof Virus Structure <-. - f° te ee 95 

CHAPTER Vill, (REPLICATION OB VIRUS TEASE RING {yess 99 
em otrictural Constitution Ob WiGuSeSi) 10) nein ener ne oes 99 
2. Change of Viruses Following Their Combination with Host Cells. . . 101 
3. Essential Factors for the Spread of the Structural Change Caused by 

SOVIETS Sole Tue ye: Gag aoe GA eles aay nels eerie rene 104 

CHAPTER LS = VIRUS AND NUCLEIC ZACIDS) 225 255 eee 108 
ive ucleic ANeids an: Virus Particles. ot ciel c 5 hoes se Me eee 108 
2 “PREC ACHON Ob INMCICIC NACIES 2M, ec y, Seay, ec se ee 111 

CHAPTER? X. -“FHE SUMMARY OF PART TE. 20h). f75 lay eee ete 116 

REFERENCES * [2¢5) S0CUtep eee US as Eee stort here | ee ar re 123 

PART III 
THE EVOLUTION OF VIRUSES AND THE GENERATION 
OF THE SECONDARY ORGANISMS 

CHAPTER. vIHE ‘ORIGIN OF VIRUSES. 7 Gaia wee etc os 129 
ie he (Getieration ‘of Phage <5 207.09 Sees Pe ee eee 129 
2. The Reason for the Phage Production from Healthy Bacteria .... 132 
8. Non-Pathogemic Viruses. 2. casisnc eee wie beetles) 136 
Acleeate abv IOMBeH rg Aico tay talisnat aac Sic ei aa eo ie. tae 139 


CONTENTS 


( 


GHAPTERSIL SDHE- GENERATION OR VIRUSES-.... oe eee ss ese 
-1. Changes in the Structure of Protoplasm Protein Leading to Virus 

GON ETATION GAR en eh occ 5 crs ghee ne tec fe Tauneh LOM Sete LN Ree Ch gel tm 

>. Environmental Chaneé and Virus Generation. ............ 

3. The Seasonal Change in the Virus Infection. ............ 


Geir Mier Orr RheGi VIRUSES cis 2 Agi 2's co She sa meal 
imesezemavoralierpiG Dermatitis’... ccc soe 6 al ole aces cue 
PA GCANICCLS Bre Bowe tol ne etal veils heise. Sd aolio, ol is Bae os Wovens: a 4a) oh tens es 
3. .The Maintenance of Assimilase Action in Protoplasm Fragments. . . 


CHAPTER LV. “LHe VARTABILERY“Of VIRUSES: = =) a5 aun ee oe 
len sbhe inheritance or Altered: Structures - of ses co ee 
2 che Variationsot Newly Proauced’ Viruses 7). 287 '.) 2.2 lsh 
3. Increase and Decrease in the Virulence of Viruses. ......... 


(CEU S95 82g iekilee op Ba BO 4 ES) oS a ee as 


el MMUNity ACaINStWVATISES vit eek Ue pis yes cu cudeeatelce) leh al -otubo ge A Pusvy umole 
DeemeheahexationnOl: VinUSES 2. JA apie a te eee ee ano Se 


CHAPTER VI. DEVELOPMENT FROM VIRUSES TO ORGANISMS ... 
RTC KELLSIA SUN Hah ee en Wal nt es ane ENS Well eps SaStSeLMN Sind gion be Mayet wiakee s 
Peeingispiutable- Organisms). +. . fih)6 6 ye oe oy OE cued eee a 


CHAPTER VII. CAUSES OF THE EVOLUTION OF VIRUSES. ..... 
1. Development of Instincts Indispensable for Organisms. ....... 
2. Difficult Situation of Newly Generated Viruses to Continue Their 

ERIS LENCE Mere iny Ot ee tel 4 ering eI nee eae: sh ae ae Be eee. sae 


CLARE Rev LileweViILR USE Oc AN DMINS Bb Gai Smge see oe is. a wey arcu 
i. The Multiplication. of Viruses.in Insects . 2... .0.-.0< te wotd sso 2 ee 
Zeb neeReVersipility.OL Pcotein olructure-. 2 <2. <2 +. + + 6 sles 
Deeeritame. Changed srruceure 4s Se br Pe ase et ok kote! Coe ke 
4. The Cause of the Fixation of Viruses 


empty hav Lcey wip, Be)! Le) ey asi ee, Ree) Me fe) fer ra 


CHAPTER IX. THE REJUVENATION OF VIRUSES 
1. .The Transmission of Viruses by Insects 
2. The Mechanims of Rejuvenation 
3. Various Means for Rejuvenation 


si we it err ey ef timy le he “ec 
Oe, tel AF, 8) Sh Se) Be eer Fey” ples. 6 Km 
ame nate) rele eG ef het ter "Sy er, e , we a) em wTEAe | je ene 


CHAPTER EX. THE, SECONDARY ORGANISMSs 3 0) a coulis 6 een 
(eeeParasirism: and Commensalismeisnyo Se se ite Meare eo ohio Maka gun a 
Pe important woignilicance Of Paraswismiai Wau, cue syeus! see 6 cS 
Je tne hmit of the,secondary,, Organisms ig: .i.cee ceiebletis (o vecta ue! a. Eos 
AM inclusion. bodies) and Metamorphosis 1) suet se cubes. eke naette 


CoAPTER XI. TRE SUMMARY "OF PART Ties ps, etre acie’ 
PoE E INGE okies a5). + eee Re REP Cate Pia d 6s Tete aT 


x CONTENTS 


PART sty 


THE GENERATION OF THE PRIMARY ORGANISMS AND 
THE FUNDAMENTAL PRINCIPLES 
OF LIFE PHENOMENA 


Page 

CHAPTER I. THE MATRIX FOR THE GENERATION OF THE PRIMARY 
ORGANISMS: sen esa: Se a Se ey ne eens ee 223 
1. The Sedimentation of Globulin and Lipids in the Primeval Oceans. , 223 
2. The Propertaes of Astificial/Cells. 2% 2 4.4.5 =) 6 Se ee 225 
3. The Evolution of the Prinitive/Organisms . 2 =... 4 2.s.ce en eee 228 
CHAPTER ) THE PATTERNS ORSEROROREASM. 2). eee 231 
1. Protoplasm as ‘a’ Maxed '‘Srystals jas tr oh aS ce le See 231 
2 Lhe» volution Of eroteins Molecules): ese hae ee) eee 233 

3. The Reason for the Presence of Optically Active Amino Acids in 
Protoplasm tt > te the te cue ah ee oa eee eae ee 234 
CHAPTER Wilt=e- DEB a WAR URE OB) (GENIES a see) eee en 237 
Jie sbhe (Generarion cor, Genes.) < fer 5s) 65 acces Ber oe ee ea oe A ee 237 
2. Phe Strueture OF Genes ye) 5) 6) “os sek Sons: ei Seem ae io, Le 238 
Say MHeCSIZe OF (GENES. pork ek ett se Se a rarity bed hee eet 241 
CHAPTER.) CHE SUPREMACY OR GENES mee. ee eee ee 244 
1. The Type of Nucleic Acids and Its Biological Significance. ..... 244 
Ze EdaSMageneSae len sear oe we accel) eS hos Ea at Me rhie Rhee Soe te a ck oe 248 
CHAPTER V2 “RELATIONSHIP-OR GENES OVE NZYMESS > cele 251 
i he Nature of Razymes (36.0 005° Set ee ee ee eee 251 
2. The Relationship between Genes and Enzymes. ........... 252 
CHAPTER VI. FACTORS INTERFERING WITH GENES. ........ 257 
fe wadormones a. 3 ee Rae a ee, 257 
Pp PaMOPO ATC: ARGS HY eee ys | cats “ex akan eo ct de uke Dae SER eo 259 
CHAPTER VII. SEXUAL REPRODUCTION AND REJUVENESCENCE . . 267 
le ebherOrigin Of sexttal Reproduction: sae een meen een ee 267 
2. Rejuvenescence of Microorganisms by Making Use of the Sexual 

Reproduction of the Hoest..<. 5... 025.4 shes Beles ee ee 271 

CHAPTER VIII. REJUVENESCENCE OF MICROORGANISMS WITHOUT 
SEXUAL REPRODUCTION <-.. 5-850 20 eee eee 275 
i) they Microorganisms Parasiticvon inSe Cts ssa) aan retro neanre 275 
2.) Che; Rejuvenescence of ;Cubercle Bacillus’ 2oaicw. nt a oe lets ciene 276 
3. The Significance of Filtrable Forms of Bacteria ........... 279 
CHAPTER IX: ; METABOLISM: 5) .3:ceacn 4 Po eth 8 a alee 284 
1. Energy Requirement by Extremely Primitive Organisms. ...... 284 


2: Nucleie Acid. as: Energy’ Donor. 2 2c ole a iocs dare eee 288 


CONTENTS xi 


Page 
3. Dimensions of the Mass of Assimilase with Special Reference to 
PROLEINGSVRUNESIS Pio siz) Scie hale acon te tat eae IRN tay aS ad eect 293 


CHAPTER X. THE CHANGE OF PROTOPLASM STRUCTURE AND THE 


REVELATION OF LIFE:-PHENOMENALH YS Oo. .< oe 298 

i; Zhe Oscillation of Protoplasm Structure*. 45) 5... 5. 0... 298 
Ae ReESOPpuOncanG MXCrelion:, 0s 0 as 4 heed eee eee ee 300 
SwaubhosVovementtor Orcanisms.. 1 |) ct) ey ees eee 304 
4454 ne: Mechanism of Blood Coagulation. .....-. « 2 «2 .s.. dm ae 309 
Brune Meckanism: OF -MibOgis, 509 is. Sey ee Bee ee TE ee 312 
SHAPLER XI. THE SUMMARY OF PART I¥>".. 2) Se ae eS 318 
Ls DLL BIS Di | OF 25 RR ODT Pek Sid oooh ie AR a pe WA pe SF I he ge tet Ae pee ol 328 

PART. Wi 
THE NATURE OF EVOLUTION 

CHAPTER I. THE THEORY OF MEMORY. .... Bie Is) ele phate any has 
1. The Faculty of Protein to Memorize Its Structure. ......... 335 
2. Training Effect and the Oblivion of Memory. ...... Be are ws WSL, 
Seteebee) CASS OL CASING 0) o., 5 Selina sulle Gey iptlan 5g site get ote 
Beet Nee Poe UE yc CPE ONG EINN oe feria), Sune bel edad RAS ods MR UME ieee tee See os 347 
i; Lhe Principle of Individual. Developments 2-2. ... 6 sa be See 347 
2.8 Vne- Mechanism of, Reseneration.. 595° 2% S95 Ys CP eRe 349 
BS melnewnormationyorOreansy .- ue keene Sen ee es ee ee 351 
An. Lhe Significance.of Rertilization. <0 <i x Gao we wthna. a Bea De ee 358 
CHAPTER Ill. THE DEVELOPMENT OF CANCERS: . . 2... -c-.-0« 361 
1. The Reduction of Protoplasm to Primitive Structure. ........ 361 
wee there redspesition to Cancer. o- 2 iol Wray Pe ees ee 364 


CHAPTER IV. THE MANNER OF THE GRADUAL CHANGE OF GENES 367 


1. The Explanation of the Biogenetic Law or the Theory of Recapitulation 367 
Peeiientiveraon Of Active Groups. < 4.4.0) fos phe oe Sele 368 
3. The Establishment of the Strong Reversibility in Gene Structure. . . 373 


CHAPTER V. GRADUAL ALTERATION OF GENE AND ITS 


REC ALIONSHIP RO ViUreADION]) py 4 cs. cr eee. ee cene 375 

eae COLUNOREHCSIS: a Hamid ik Se tamk ol tettn feo be Gs oes ee, pilin au om eines A ake ee 375 
anager Ad a RrCCuGene ay 1. Gv tue ae ec Donte Ring yt aro) 376 
3. The Production of Fitted Characters by Adaptation ......... 379 
ShArere Vi. SELECTION AND ISOGATION.. ooo cic cy oe be os 382 
Pence be itecep Of the Selection, 2 cet Geta. buses Gos oe ol 382 
2. The Effect of Isolation and the Origin of Species. .......... 385 
CHAPTER VII. THE MECHANISM OF ADAPTATION. ......... 387 
i) “the Adaptation.as a Reversible -Chanve, 7... Sw we 387 
Be Aver moaiicmiONeT.. , . . gaeen a esees ty Re atl kee 2alS 388 


xii CONTENTS 


Page 
CHAPTER VIII. THE INHERITANCE OF ACQUIRED CHARACTERS . . 394 
i) “Lhe: Forgetting of the Oricinal(Structures 5°. 2. 2 fee 394 
2. The Transmission of the Acquired Pattern to Germ Cells. ..... 397 
3, , Lhe Effect of Wsevand?Disuse.s.2- se eee cee fl eh co eee 400 
CHAPTER-IX.. EVOLUTION AND MUTATION. 3.0... 2 ee ee 402 
I. “The: Significancezof; Mutation. >). sence ee ee 402 
2. ~Genic Changes) dueto (Chemical: Agents Wa. see eee 403 
CHAPTER X. LHE MECHANISM OR EVOLUDIONS 25s. arenas 407 
J. The Evolution without sNaturaleSelection -) 2s. nebian eee Ge eee 407 
2. The Evolution of Evolution Mechanism: ~ . . 44. i .-) oeeeeemeee 408 
J.) The Survival. of the .Pittests 0's a0 see ete ta. ko 410 
4.” The Degeneration of ‘Orcans) and Atavism 272 22 asec) se su emmeme 4i1 
CHAPTER XI. THE CHANGE IN HUMAN CHARACTER BY 
ENVIRONMENT = 2 wet e 3e% Soda eh Betas awe lemons 414 
iL: “Climate and Manikcind serie wien ote. Gar ote Salta Wa emer ae Joy yas 
2. “The Inheritance of Habitude © 3207s ask Wb me eet eae ee 416 
CHAPTER XII. THE EVOLUTION OF MANKIND AND ITS FUTURE. . 419 
1. The Orthogenesis of Protein Molecules. .... Beg a SO 
2. The Orthogenesis' of Mankind nao eee terete vd Soe) ae heii make eee 
3. The Cause of the Orthogenesis of Man. ...... ab ite Tae eB ee eee 
4. Viruses, the Fatal Enemy of Man ....... race Saree eee cr. 
CHAPTER XI. ~ THE SUMMARY, OF PART AV x55 555 912 gree tee eaten 440 


REFERENCES ES. Sil ars, adel aha oo) ot Dae mr ene Pe Aiolaieees 451 


PARTY. 4 


INTRODUCTION TO VIRUSES 


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CHAPTER I 
VIRUS PARTICLES 


1. The Key to Solving the Riddle of Life 


There may be no riddle so fascinating and so difficult as that of 
life. Around us,—on the land, in the sea, under the ground, or in the 
air, life is wriggling, jostling, thronging in every possible shape. What 
is living? Even to think about this question is the very manifestation 
of life itself. A great number of inquisitive human beings have tried 
and even now are trying to pierce its mystery. Viruses are the key to 
solve this fascinating riddle. Without this key it may be impossible 
to open the mysterious chamber of life and, accordingly, to find out the 
nature of viruses is indispensable for the solution of the riddle. 

It has long been known that the majority of infectious diseases are 
caused by microorganisms such as bacteria and protozoa. There are, 
however, many infectious diseases not originated by microorganisms 
observable under the ordinary microscope, and such infectious diseases 
appear to be rather more numerous. Among these are common cold, 
measles, and mumps, the diseases by which almost every one of us is 
affected. Smallpox, influenza, trachoma, yellow fever, poliomyelitis, 
many types of encephalitis, rabies, efc., are also counted among them. 
Not only human beings but also many other creatures, for instance, 
domestic animals, insects, and vegetables, are also infected with them. 

The agents producing such diseases are now known to pass through 
bacteria-proof filters, and are called filtrable viruses or shortly viruses. 

The characteristic features of viruses are firstly their too small size 
as compared with other pathogenic microbes, and secondly their in- 
ability to grow in vitro with ordinary bacteriological method; for their 
multiplication are needed always living cells, in which only they can 
grow. For the multplication, however, usually a virus needs a certain 
kind of living cells; that is, certain cells of a certain creature are 
necessary for the multiplication. This fact is of great importance, 
since it reminds us of the relation between an enzyme and its substrate. 
Thus, the relationship between a virus and the cells in which it can 
grow appears similar to that between diastase and starch, or between 
pepsin and protein. These enzymes, however, cannot multiply by 
affecting the respective substrate in contrast to viruses which can mul- 


4 I. INTRODUCTION TO VIRUSES 


tiply in the cells. 

To the belief of the writer, viruses are a kind of enzymes, and the 
protoplasm protein of the cells is their substrate, and when the protein 
is affected by a virus, an active structure or structures are produced 
which can act as the virus; this change of the protein is to spread 
through the protoplasm as a chain reaction. The reason why the writer 
has reached such a conclusion will be mentioned step by step. 


2. Bacterial Viruses 


The virus which affects bacteria is called phage. Since there are 
numerous kinds of bacteria and, moreover, since a certain strain of 
bacteria is generally affected by a number of different viruses, the kind 
of phage should be very numerous. Phage has formerly been regarded 
by many workers as a special kind of viruses or virus-like agent, not 
as a true virus, but now it has become evident that it is a typical virus 
having no peculiar property dissimilar to the ordinary. As we could 
ascertain this fact long ago, we chose phage as our object of virus 
researches. Besides phage, our experiments were carried out with 
vaccinia virus and the experimental results obtained with this virus 
were well consistent, without exception, with those obtained with phage, 
and vice versa. 

Since viruses affect living cells only, it is a rather difficult task to 
investigate into their function. Phage, however, affects bacteria which 
can easily be cultivated and handled zz vitro, so that the employment 
of phage as an experimental object offers a vast advantage. 

When bacterial cells are infected with a phage they will generally 
be dissolved into minute particles and such particles carry the virus 
activity; the particles may, therfore, in turn affect other normal cells 
causing the latter again to dissolve into minute particles. In sucha 
way phage may multiply. 

The chief reason for which viruses were regarded as a microorgan- 
ism might be dependent on their multiplicability but, besides this, their 
particulate nature might have a share with it. As will be mentioned 
later, the fact that viruses exist in minute particles involves a striking 
significance. 

A high speed centrifuge is commonly used to isolate virus, but as 
we unfortunately had no such an apparatus we contrived a chemical 
method for the isolation. Thus, we were able to make virus particles 
sedimentable by an ordinary centrifuge by taking advantate of the fact 
that the virus particles have the property to agglutinate’ at a weakly 
acid pH (1). Although no fundamental differences seemed to exist in 
their property between particles obtained by a high speed centrifuge 


I. VIRUS PARTICLES 5 


and those isolated by our method, there is a good reason to believe 
that the former particles are apparently a portion of the latter having 
peculiar properties as will be fully discussed in a later chapter. Our 
application of such a method in virus investigation might enable us to 
succeed in finding facts other researchers might have failed to observe. 

Although the optimal agglutination pH varied to more or less 
extent with varying kind of viruses, it was found to be about 5.5 with 
vaccinia and also with many plant viruses, and 4.6 with phage. This 
pH was markedly influenced by both the kind and the concentration of 
inorganic salts present in the media. If due regard was not paid to 
the salt, occasionally virus particles were isolated in an entirely in- 
activated state, whereas application of distilled water containing no salt 
yielded usually good results. It should be noted in this connection that 
sometimes Ringer’s solution proved to be better than distilled water 
while physiological saline solution proved unfavourable (2) (3). Ringer’s 
solution contains, besides NaCl, minute quantities of other salts, espe- 
cially that of Ca, which may play a réle in this case as will fully be 
discussed later. 

When a desirable precipitate cannot be obtained by a mere adjust- 
ment of a virus solution to its optimal pH, the addition of alcohol at 
about thirty per cent is recommended, thereby viruses may become 
readily sedimentable, although alcohol tends to inactivate the virus. 

The substances isolated by our method are the aggregate of minute 
particles composed of a protein and lipids. In order to prove these 
particles to be the virus itself, we carried out a series of experiments 
from various points of view. A number of reports, however, have been 
made afterwards by some workers informing that considerable results 
were obtained by a similar chemical method (4) (5) (6)(7). They used 
mainly methanol instead of ethanol, and manipulations were always 
carried out at extremely low temperatures which must have been very 
effective to prevent the virus from inactivation. 

It is most important, however, that such particles, composed of a 
protein and lipids, are never peculiar to viruses, but that they can be 
obtained from normal cells having no concern with viruses. For example, 
bacterial culture lysed by a phage may give rise to a precipitate by a 
proper addition of acetic acid, and the precipitate thus produced is 
composed of the particles with phage activity, whereas similar particles 
with no virus activity can be isolated from normal bacterial cells ground 
up mechanically or lysed by proper chemicals. The particles never have 
virus activity when the cells are decomposed by agents other than 
viruses (8). 

Whether viruses are involved or not, the yield of the particles by 
our method is so high that almost the total protoplasm appears to have 


6 I. INTRODUCTION TO VIRUSES 


been changed into such particles and isolated as such. According to 
our experiment, for example, phage particles weighing about 11077 
in the dry state were obtained per cell of E. coli, whereas the dry 
weight of a single cell was estimated to be 1.11077 (9). Since the 
dry weight of a single coli-phage particle obtained by us was measured 
to be about 1X10~7, some 10° particles would be produced from a single 
bacterial cell, if its whole protoplasm was changed into phage particles. 
On the other hand, Delbriick estimated the number of phage particles 
produced from a single cell to be 100 to 300, and designated as ‘‘burst 
size’’ (10); this figure seems to be in accord with our estimation. 
Likewise in the case of vaccinia virus, from the infected skin tissue of 
a rabbit or of a calf are obtained amounts of particles so large that 
the whole protoplasm of the tissue seems to have been converted into 
the particles. In addition, it can actually be observed under the micro- 
scope by the dark field illumination that the cells are fully packed with 
virus-like particles (11). 

From these facts we have concluded that the protoplasm has the 
property to be readily changed into minute particles by physical or 
chemical effects and that when produced by a virus the particles are 
endowed with the virus activity. Our theory of the virus was based 
on the fact above stated that virus-like particles can be obtained from 
the normal cells, but most workers seemed to pay little attention to 
this important fact when it was first found by us. In recent years, 
however, this became generally accepted and at present particles are 
called ‘“‘normal components”’ or ‘‘healthy particles’’ (12), although no 
workers seem as yet to value this fact so high as we do. 

When the normal particles were first recoginzed, the writer expressed 
the opinion that the relationship between the particles and the virus 
might be comparable to that between serum globulins and the antibody. 
This belief has become at present still much firmer to the writer; the 
reason will be made clear by degrees with the progress of the 
description. 


3. Virus Particles as Denaturase 


Under the microscope by dark field illumination, normal particles, 
even when not liberated from the cell, can be observed in the cell 
itself. When cells, such as those of the rabbit brain or liver, are pressed 
between cover-glasses and observed, almost the total cell-body appears 
to be decomposed by the mechanical pressure into minute particles. 

Phage particles can be seen under the microscope, but in the 
growing young cells no virus-like particles may be observed. However, 
if bacteria are treated with such chemicals as a salt of heavy metal, 


I. VIRUS PARTICLES if 


alcohol, acetic acid, efc., or exposed to heat or ground up mechanically, 
particles will appear in the cell, and sometimes some of them will be 
discharged and found outside the cell-body (13). Under the microscope 
this phenomenon appears to be just a coagulation of the protoplasm into 
minute particles. When phage is added instead of such a chemical or 
physical aggression, the bacterial cell may undergo a similar change 
with the production of minute particles. Even without any particular 
aggression, an incubation of coli-bacteria for a prolonged period may 
lead to the production of particles inside the cell body. Since the bac- 
teria in which particles have so appeared are not motile, they may be 
in a dead state. At any rate, it should be borne in mind that the 
particles produced by any other causes than phage never possess the 
phage action and that only the particles which appear in the presence 
of phage are provided with the action. 

Lepeschkin (13) emphasized the fact that the protoplasm, in general, 
coagulates into minute bodies on the exposure to chemical or physical 
agents, and that when the coagulation is still reversible, the particle 
formation is not so distinct, but that when a stimulus sufficient to kill 
the cells is given, the particles are vigorously formed, the total proto- 
plasm being coagulated into minute bodies. Sometimes minute particle 
thus formed are set free from the cells and exist in the surrounding 
medium. According to him, such coagulation of the protoplasm of a 
spirogyra-cell caused by a pressure of the cover-glass will spread suc- 
cessively to another part of the protoplasm until the total is coagualted 
into minute particles; sometimes the coagulation will be transmitted to 
another cell, not limited to a single cell. 

In view of these facts, the writer began to form the opinion that 
viruses were a sort of denaturase of protoplasm protein, and that the 
denaturation or the change in the protein structure would spread 
successively in the protoplasm as a chain reaction, followed by the 
coagulation of the protoplasm, and that a group or a structure concern- 
ing the virus action would be produced on the structural change or the 
denaturation of the protoplasm protein. As pointed out already, the 
characteristic feature of viruses exists in the fact that they cannot 
multiply without living cells. This fact may lead to the view that for 
the generation of the structure capable of acting as a virus, the proto- 
plasm protein is needed to be in a “‘living state’’. 

As stated above, virus-like particles can be obtained from normal 
cells. This is, however, not always the case; for example, while virus 
particles may be readily isolated from the rabbit skin tissue affected by 
vaccinia, the isolation is impossible from the normal skin. It is also 
known that papilloma virus particles can be separated from the infected 
skin tissue regardless of the failure of the isolation of similar particles 


8 I. INTRODUCTION TO VIRUSES 


from the normal healthy skin. It is evident, therefore, that no virus- 
like particles are present in the normal skin tissue. The writer was 
able, however, to ascertain that, on the injection of toxic agencies such 
as staphylococcal toxin or sapotoxin, the akin tissue would give rise to 
particles similar to vaccinia virus which could readily be separated as 
in the case of virus infection. Thus virus-like particles can also be 
produced by a proper stimulus, without virus, in the cell in which no 
particles can be detected normally. In the case of the skin tissue, 
therefore, it may be said that protein capable of coagulating into minute 
bodies is produced by the effect of a virus which provides to the protein 
with the virus activity. 


CHAPTER II 
THE SIZE OF VIRUS PARTICLES 


1. Relationship between the Kind of Host Cells and 
the Size of Virus Particles 


A vast number of researches have been carried out concerning the 
size of viruses, presumably mainly because viruses were believed to be 
a type of microbes. If enzymes were similarly considered to be a kind 
of microbes, profound attentions might have likewise been paid to their 
particle, or molecular, sizes. However, of course, since no one ever 
regarded them as microbes, little attention was paid to their sizes; 
nontheless, it is known that enzymes such as_ succinooxidase and 
cytochrome exist in a particulate state like a virus (14), although 
other enzymes such as pepsin, trypsin, and urease may be in a molecular 
state of the globulin type. As will be stated later, there are other 
enzymes or enzyme-like agents which should be regarded as having a 
particulate nature as do viruses, indicating that the particulate nature 
is never a characteristic feature belonging to viruses only. However, 
on account of the combination of this particulate nature with other 
characteristics which are the very image of microbes, the size of viruses 
is apt to be regarded as being strictly specific to the sort of viruses. 

If virus particles are nothing but the protoplasm protein coagulated 
into particles, it may be impossible for a virus to have always a uniform 
size specific to the virus. It should be noted in this connection that 
even protein molecules are known at present to have no uniform size. 
Thus, recent electrophoretic studies have indicated that 6-lactoglobulin, 
which had been regarded as an excellent example of a protein, pure in 
view of their electrophoretic mobility, sedimentation constant, and solu- 
bility, is polydisperse in a weakly acid solution, although appears 
homogeneous at pH 8 (15). 

Since viruses were generally isolated by the application of centri- 
fugal force, and since, as a rule, particles sedimentable at a given 
revolution rate were separated and regarded as the only virus, it might 
be a matter of course that the preparations thus obtained were composed 
of similar sized particles. Thus in former days and occasionally even now 
each kind of viruses is likely to be regarded as possessing its specific, uni- 
form size, and some workers went so far as to calculate their ‘‘molecular 


10 I. INTRODUCTION TO VIRUSES 


weight’”’. At present, however, their inhomogeneity both in size and 
shape appears to have become generally realized. 

All the particles present, regardless of their different properties, are 
to be isolated by our chemical method and consequently virus prepara- 
tions obtained by our method consist in most cases of particles of a 
variety of sizes. It is, however, worthy of note that the particle size 
seems to be governed mainly by the sort of cells from which the parti- 
cles have been isolated. 

Thus, normal particles trom £. coli were estimated to have an 
average diameter of about 0.1y in the dry state, whereas similar sized 
particles were isolated from the bacterial culture lysed by a phage, that 
is, the phage particles proved to have the size of the normal particles 
(16). Again, vaccinia virus particles, obtained from the rabbit skin in- 
fected with the virus, were found much larger in size than the bacterial 
particles, being a little less than 0.3, whilst the particles isolated from 
the skin tissue rendered inflammatory by toxic agents other than the 
virus possessed a similar large size. On the other hand, when isolated 
from the rabbit testicles infected with the same vaccinia virus, the virus 
particles were found to be of a much smaller diameter, being about 
0.2 2, which was proved in turn to be the size of particles obtained from 
the normal testicles (17). 

In the case of vaccinia, however, some difference appeared to be 
present in sizes between particles isolated from an infected rabbit 
testicle and those from a normal testicle, but to a degree of insignifi- 
cance. Also in the case of coli-phage, the difference due to phage 
strains sometimes was found in particle size, though also usually 
insignificant. 

The presence of the above mentioned remarkable difference in virus 
size due to the difference in host cells seems to be of very importance in 
the consideration of the virus nature. Besides us, some workers appear 
to have already paid attention to this remarkable fact. For instance, 
Bawden and Crook found that the size of potato virus X varies re- 
markably with the kind of tissue extracts from infected plant, although 
no great difference is detected in their infectivity (18). Levaditi also 
stated that the poliomyelitis virus isolated from mice appeared much 
larger than that isolated from monkeys (19). A strain of coli phage was 
reported to exhibit different properties including particle size when 
cultivated in different media (20), suggesting the presence of different 
properties in bacterial protoplasm protein which varies with the media 
in which they were cultivated. Moreover, mouse pneumonitis virus 
preparations were found to show significant difference in size when 
isolated from different sources, 7. e., from mouse or rat lung or from 
the yolk sack of the embryonated hen’s eggs (21). 


II.. THE SIZE OF VIRUS PRATICLES ll 


It is generally believed that vaccinia virus particles are much greater 
in size than influenza virus particles, but according to an electron 
micrographic study by Wyckoff (22) particles similar in diameter to those 
of influenza have been seen developing from certain cells of chick 
embryo inoculated with vaccinia. 

At present, the virus size is usually determined by electron micro- 
graphs, but as we were unable to make use of this method, the particle 
size was measured by us in the following way: The average dry weight 
of a single virus particle was first estimated under the cardiod-ultrami- 
croscope in Beobachtungskammer by counting the number of particles 
in a virus protein solution of a known concentration, and subsequently 
the diameter of the particles was calculated from this dry weight. 

Since the diameter is a function of the cube root of the weight, a 
considerable accuracy should be expected in the values thus obtained. 
By this method the writer estimated vaccinia virus from rabbit or calf 
skin tissue to be about 0.254% (17); this appears agreed well with the 
value estimated by electron microscope and reported by other workers. 

At any rate, it seems probable that the particle size is in the main 
subjected to the host cell in which the virus was produced. If viruses 
are to be produced from the protoplasm, it should naturally be expected 
that not only particle size but also many other properties are more or 
less governed by the host cell. In fact, it is actually shown that even 
the chemical composition of a virus appears sometimes to vary with the 
source from which it was isolated. 

Thus, the work of Knight on influenza virus indicated that the 
virus particles isolated from mouse lungs or from embryonated hen’s 
eggs possess an antigenic moiety characteristic of the respective host, 
although they seem to be homogeneous, electrochemically and in sedi- 
mentation behaviours (23). It was also confirmed that chicken tumour 
virus bears a resemblance in its chemical composition as well as in the 
immunological property to normal particles isolated from the chicken 
tissue (24) (25). With equine encephalomyelitis virus a similar fact is 
known: The virus isolated from embryonated hen’s eggs resembles 
remarkably in the immunochemical behaviour to the normal component 
(26) (27). Polson and Wyckoff, investigating 17 kinds of amino acids, 
found that there was no significant difference in the chemical composi- 
tion between E. coli and its phage (28). 


2. Difference in the Resistance of Virus due to Particle Size 


Virus preparations obtained by our method appear in the main to 
consist of homogeneous particles under the microscope, but if examined 
carefully it will be revealed that this is not the case. We were able to 


12 I. INTRODUCTION TO VIRUSES 


show that the activity for a unit mass of phage or vaccinia virus pro- 
tein varies with the particle size and that larger sized particles are 
more active than smaller ones. Using collodion membranes having 
various pore diameters, we demonstrated that particles capable of 
passing through a membrane of a smaller pore diameter have a much 


Table 1 
Filtrability of Virus Proteins 


| Virus protein! Virus protein 
isolated from | isolated from 
vaccine pulp | rabbit testicle 


Coli- Coli- 
phage, phage, 
Now) Norsk 


i 


through | total _ total 
Berke y.J Virus action | 1/100-1/1,000 1/10 oe ne 


Filtrability Protein 1/100 1/5 almost almost 
Filtrability 
through 
collodion 
membrane of 
0.9 uw average 
pore diame- 
ter 


Protein 12% almost total —— — 
Virus action 1/10 5 = == 


Filtrability 
through 


ape ae of Protein less than 1% 16% 61% 84% 


0.58 » ave: Virus action |1/1,000-1/10,000 1/10 49% 45% 
rage pore 
diameter 


Filtrability 
through 


Bien ik ule Protein —  |iess than 1% | 7.196 9.596 


0.344 average Virus action — 1/1,000-1/10,000 2% 3% 


pore diame- 
ter 
ater ee eee 


About 0.126 virus protein water solution (pH 7.5) was used for the filtra- 
tion. The protein quantity was measured nephelometrically. 


less activity as shown in Table 1. This can be clearly shown with 
phage as its activity can be precisely expressed in numerical values. 
Some workers were liable to consider that viruses were regularly pro- 
vided with extremelly small sizes, and occasionally they even insisted 
that larger particles were only a concomitance, but this fact indicates 
that rather the reverse is the case (16). 

The difference in the activity attributable to the difference in the 
particle size was found with phage to be very striking. The ratio 
of the activity of phage protein for a single particle, which could not 
pass through 0.58 membrane, to the activity of the protein which could > 


II. THE SIZE OF VIRUS PARTICLES 13 


pass through 0.34 membrane was calculated from the experimental 
results indicated in Table 1, and estimated to be about 50-100:1. On 
the assumption that the average size was proportional to the pore 
diameter of the membrane, and that the larger particles were larger 
than the smaller ones by 1.51.5, the calculation was made as follows: 

The activity of the larger-particle phage for a single particle: 

the activity of the smaller-particle phage for a single particle 

= (100-45) x (1.5 x 1.5)3/(100-84) : 3/9.5=100: 1. 

This was obtained from the data of phage II in the Table, while 
from the data of phage I it was calculated as 50:1. This showed 
that the ratio of the number of the active large particles to the total 
number of the large-sized particles was about 50-100 times as great as 
the radio calculated with the small-sized particles. This may be at- 
tributed to the labile property of the small sized particles; as shown in 


Table 2 


Relation between the Size of Phage Protein Partice and Its Resi- 
stance to Heat 


Number of 

active phage 
particles per 
cc. after hea- 


Rate of decrease 
in number of 
active phage par- 


Number of 
active phage 


particles per 


cc. before : ticles after hea- 
: ting (50°C. : 
heating fee 1 howe) ting 
: Phage I 2.6 X10? 2.4107 jee al 

Before heating | Phage |} 46x10? 4.2107 1:1 
Protein capable 
of passing Phage I Te SeOe 5.6 x 108 ho 
through 0.54 w [Phage II 2.2107 9.4x 108 We, 
membrane 
Protein capable 
of passing Phage I 8.2 105 7.8 x 104 Vs hl 
through 0.34 w {Phage II 1.1106 4.8x 104 LOGE 
membrane 


Table 2, the resistance of phage to heat varied remarkably with the 
particle size, and the larger the size the greater the resistance; in 
other words, phage protein existing in a larger particle was more stable 
to heat than that in a smaller particle, showing that the property of 
the phage protein varied with particle size. 

As described already, viruses are considered to have the faculty to 
cause a structural change in the protoplasm protein, thereby the pro- 
tein can acquire the virus function. If the protein coagulates into too 
small particles on such a change, active structures in the particle may 
be labile and readily inactivated. There is a good reason, as will be 
described in a later chapter, to assume that virus protein has to exist 


14 I. INTRODUCTION TO VIRUSES 


in a state of the particle of over a certain size in order to exhibit the virus 
action and that the action is stronger as the particle becomes larger. 

The unstability of smaller sized particles could be proved by the 
writer also with vaccinia virus protein. Galloway and Elford found 
that foot-and-mouth disease virus became more unstable after ‘‘purifi- 
cation”’’ through a collodion membrane having a small pore diameter 
(29). This fact indicates that the small sized particles of this virus are 
also more unstable than the larger ones. Tobacco mosaic virus parti- 
cles appear likewise to have various sizes. Bawden and Pirie (30) have 
suggested that the small particles may be virus that has become non- 
infective without losing serological activity or that they are incomple- 
tely formed or mal-formed virus particles, although it has been claimed 
by a number of workers that particles are 280 my long, and that such 
are only the virus. Crook and Sheffield (81) found that a virus prepa- 
ration containing only particles shorter than 280 my had even some infec- 
tivity. Takahashi and Rawlings (32) also stated that particles shorter 
than 225 mz could be infective. 

Before the universal application of electron microscope, the size of 
virus was usually measured by collodion membranes; the size was 
estimated from the average pore diameter of the most porous mem- 
brane that prevents the passage of the virus particles. Thus, it should 
naturally follow that the virus will show the smallest diameter when 
measured by this method. In short, there seems little doubt that the 
particle size estimated by collodion membranes is of the smallest 
particle in which the virus protein can preserve its activity; in other 
words, when the protoplasm protein is coagulated into particles smaller 
than those of this size the virus activity may fail to be revealed. 

By electron micrographs the particle size of various phage strains 
has been estimated and reported to be a little less than 0.1 yu, while 
by the filtration method some phage was previously reported to be so 
small as having a diameter of the order of 0.01 yw. Also from our ex- 
perimental data, phage appeared sometimes to retain its activity even 
when the protein existed in so small a particle as of this order (16). 

Of the smallest viruses known are those of mouth-and-foot disease 
and of poliomyelitis, and both of which are accepted to be also of the 
order of 0.01. This may be ascribed to their stable nature, capable 
of retaining their activity even when decomposed to such a small size. 
According to Andrewes and Horstmann (33) the viruses that are patho- 
genic to animals and known to have very small sizes show a high resi- 
stance against a variety of chemicals. 

At present, the sizes of many viruses are mostly measured by 
electron miroscope, and it has been recognized that the sizes thus 
measured are larger than those estimated from the filtrability as is 


II. THE SIZE OF VIRUS PARTICLES 15 


expected from the view point above mentioned. However, it should be 
borne in mind that the particles shown in electron micrographs are in 
the desiccated state, while those treated with the membrane are in a 
hydrated state combining with a great quantity of water as will be 
described in the next section, and as a consequence the membrane 
method must give a much greater value if the experiment is carried 
out with the same particle. But the results obtained were entirely 
reverse, indicating the presence of the striking difference in the parti- 
cle size between the average sized particle and the smallest that narrowly 
retains the activity. 


3. Water Quantity Combining with Virus Particles 


Virus particles shown in electron micrographs are in the dry 
state, not in the hydrated state in which they are naturally existing, 
and hence, at least in this respect, they fail to reveal the true feature 
of virus particles. According to the writer’s findings, when suspended 
in water, virus particles are combined with the quantity of water ten 
times as much as its dry weight, and always being accompanied by this 
combined water (34). The evidences found by the writer from which 
the conclusion was derived are as follows: 

Firstly, when the virus particles agglutinated in a weakly acid 
solution were centrifuged and sedimented by an ordinary centrifuge 
for a long period of time and the water content of the precipitate thus 
sedimented was measured at intervals during the course of centrifuga- 
tion, then it was found that the water content of the sediment dimini- 
shed rapidly at the beginning of the centrifugation but that at later 


Water content of the precipitate, %. 


70 20 30 40 50 &0 70 80 90 100 710 120 
Time of centrifugation in minutes. 


Fig. 1. Cenrifugal precipitation of coli-phage protein particles. I: 
Phage-protein, sample No. 1. II: Phage-protein, sample No. II. 


16 I. INTRODUCTION TO VIRUSES 


periods the diminution rate became constant and the water content 
would decrease rectilinearly. If we assume that the decrease in the 
water content with a constant rate is due to the squeezing out of the 
water contained in the particles and that the rapid decrease at the 
beginning of the centrifugation with an inconstant rate is due to the 
pressing out of the water interposing among particles, the water con- 
tent of the particles can be estimated by measuring the content at the 
period when the decreasing rate will become constant. In this manner, 
with both vaccinia and phage, it was estimated to be about 90 per cent. 
In Fig. 1 was shown the experimental result obtained with phage 
particles. Such a result was obtained without any concern with the 
virus activity, no differences being found between virus particles and 
the normal ones. In the case of plant particles, after a certain period 
of centrifugation the water content of the precipitate tended to become 
constant as shown in Fig. 2. Accordingly, in such a case the water 
content can be estimated more accurately than in other cases. 


Water content of the precipitate, %. 


20 40 60 &0 700 720 740 160 
Time of centrifugation in minutes. 


Fig. 2. Centrifugal precipitation of vegetable protein particles from 
the leaves of B. chinensis L. var. oleifera Makino. 


Secondly, it was calculated from the sugar-insoluble space of the 
particle that approximately 10 cc. of water was combining with 1g. of 
dry particles; the sugar-insoluble space of the particle was calculated 
from the concentration of a sugar in the solution in which the virus 
particles were suspended. The sugar concentration was measured 
after a known quantity of the sugar had been fully dissolved in a 
known volume of the virus suspension from which the particles were 
subsequently centrifuged off. Experimental data obtained with vaccci- 
nia virus particles are shown in Fig. 3. 

Thirdly, by counting the particle number under the ultramicroscope 
the average diameter of dry particle was calculated to be about 0.25 4 


II. THE SIZE OF VIRUS PARTICLES 17 


with vaccinia virus isolated form calf skin, and about 0.1 y with coli- 
phage, whereas by measuring the filtrability through collodion mem- 
branes the average sized particle of the vaccinia virus and of phage 
was estimated to be respectively in diameter about 0.54, and about 
0.24. If the particles are always associated with the quantity of water 
10 times as great as its dry weight, the diameter will be estimated to 
be a little more than two times greater. 


Sugar-impenetrable space (cc) per gr. 


0.125 9% 0.25 % 05% 


Concentration of sugars, %. 


Fig. 3. Sugar-impenetrable space of vaccinia virus particles. 
I: Glucose, II: Fructose. 


For these reasons it was concluded that the virus particles in a 
water solution would combine with the water about 10 times as much 
as the dry weight. This appears to hold true generally for protoplasm 
protein particles without reference to virus action. 

On investigating the space of vaccinia particles non-penetrable by 
sugar or protein, McFarlane ef al. (35) concluded that the particles 
are surrounded by a considerably thick layer of water molecules which 
fail to act as the solvent and which combine with the particles firmly 
enough to sediment with them. At present, the writer believes that 
their conclusion is proper, though at first the writer assumed that the 
particles were in an extremely swelled state on absorbing all the esti- 
mated quantity of water, an assumption which must be unreasonable. 
As will be mentioned later there are many evidences to suggest that 
the majority of the estimated water quantity is present outside the 
particles forming a thick layer, and only a small portion of it being 
absorbed by them as claimed by the above authors. If the water 
quantity combines firmly with the particle also in the case of filtration 
through collodion membrane, the diameter will naturally appear to be 
approximately two times as great as that of the dry particle. 


18 I. INTRODUCTION TO VIRUSES 


The decrease in the sugar-insoluble space, as indicated in Fig. 3, 
when the concentrations of the sugar are higher, may be due to the 
penetration of the sugar into the water layer. On the thickness of 
this water layer as well as on the force which attracts the water mole- 
cules around the particles, a due discussion will be made in a later 
chapter. This force appears to play an important part in the accom- 
plishment of the virus action, a matter which will likewise be fully 
discussed later. 

To estimate the water quantity in virus particles usually the spe- 
cific gravity is at first to be measured. For this purpose a certain 
amount of a substance is dissolved into a virus suspension, and the 
change raised thereby in the sedimentation rate of the particles is 
investigated. Sugars or inorganic salts dissolved for such a purpose, 
however, will penetrate into the particle when the concentrations 
are high as indicated in the experiment in Fig. 3. Consequently, the 
specific gravity estimated by such a method may be erroneous; pre- 
sumably the obtainable values are too large, and accordingly the water 
contents are too small. 

A linear relation is expected between the sedimentation rates and 
' the concentrations of a dissolved substance, if the dissolved substance 
do not penetrate into the water which is associating with the particle. 
However, this is not the case, and the sedimentation rate is not a 
linear function of the density of the medium (36). In additon, a shrink- 
age may occur in the particle when the density of the medium is high 
(37). In order to avoid this shrinkage, Sharp ef al. (38) used a protein 
solution of a low osmotic pressure, and estimated the water content of 
influenza virus A and B and also the swine type to be respectively 52, 
34.4, and 43.3 per cent. They claimed that the relation between the 
sedimentation rate and the solvent density fell on a straight line 
when protein solutions were used; but it may be unreasonable to 
assume that the protein, regardless of its concentration, can dissolve 
only into the water layer outside the particle without penetrating 
into the water present inside. Hence, the reported values do not seem 
to the writer to be legitimate. 

According to Schachmann and Lauffer (39), the specific gravity of 
tobacco mosaic virus particles was calculated to be 1.13 in serum 
albumin solutions, while 1.27 in sucrose solutions, indicating, also in the 
case of the plant virus which is known to have peculiar properties 
distinct from usual animal viruses, sugars appear to penetrate more 
readily than do proteins. They calculated the water content of the 
tobacco mosaic virus to be 65 per cent from the specific gravity in 
the protein solutions. From X-ray measurements, Pirie (40) concluded 
that the virus is normally associated with about its own weight of 


II. THE SIZE OF VIRUS PARTICLES 19 


water. It is worthy of note that this water was claimed to exist in 
forming a water layer several molecules thick surrounding the particle 
as in the case of vaccinia virus, since X-ray pattern from the internal 
structure of the particles was found similar for dried virus and for virus 
in solution. 

Not only protoplasm protein particles, but also usual protein mole- 
cules in solution seem to be combined generally with great quantities of 
water. From X-ray analysis Crowfoot (41) estimated the water contents 
of crystalline haemoglobin and lactoglobulin to be respectively 46.6 and 
40.8 per cent. Bull (42) stated that a crystalline protein combined so 
much water as 83 per cent in the saturated water vapor. 

There seems no doubt that the majority of water quantity, proved 
by the writer to exist in the association with the virus particle, is pre- 
sent outside the particle in forming a thick water layer, although the 
true water content, 7. e., the water present inside the particle must 
also be considered. Thus the diameter of the hydrated particle should 
be much greater than that of the desiccated one. Nevertheless, viruses 
were previously called ultramicroscopic pathogens and their invisible 
nature was looked upon even as one of their characteristics. In fact, 
vaccinia virus particles, isolated by our method, whose diameter even 
in the dry state is so large as 0.25 mw are never visible under the 
ordinary microscope if examined in a neutral solution, but usually will 
become visible when the pH of the solution is adjusted to a weakly acid 
pH near the isoelectric point and stained properly with fuchsin water 
solution. 

This remarkable property of the virus may probably be due to the 
thick water layer surrounding the particles as well as the true high 
water content. The fact that the particles become visible at a weakly 
acid pH may be ascribed to the minimum swelling at the isoelec- 
tric point. 

Bacterial cells infected with phage are known to swell up and 
become invisible before bursting into phage particles. We could like- 
wise ascertain this phenomenon (9) which may be attributed to the 
water molecules sttracted by the lyophylic groups which may be libe- 
rated in the course of the coagulation or the denaturation of the pro- 
toplasm by phage infection. 


CHAPTER III 
THE ACTIVITY OF VIRUS PARTICLES 


1. The Difference in Phage Activity due to the Cultivating 
Condition of Host Bacteria 


The demonstration of active phage is usually achieved by using 
an agar plate on which a proper dilution of phage-containing filtrate is 
spread with the host bacteria. When active phage particles are pre- 
sent in the filtrate, there will appear on the agar plate a number of 
small circular areas of clearing in which no bacterial growth occurs. 
These areas are called plaques. A plaque is regarded as produced by 
a single phage particle which affects surrounding bacteria to cause the 
lysis, and therefore the number of active particles can be estimated by 
counting the number of plaques produced by a given quantity of the 
phage-containing solution. 

As mentioned already, the protoplasm protein particles liberated 
from the bacterial cells which have been affected by a phage appear to 
be able to act as the virus. However, some of the particles seem to 
act as such under appropriate conditions only; if all the particles could 
exhibit the phage action, it would occur only in extremely rare occa- 
sions. 

It was found by Ohashi (43) that the ratio of the number of 
particles able to act as active phage on an agar plate to ‘that capable 
of multiplying in a broth varies over a wide range with the filtrate 
examined. He distributed a given quantity of a diluted phage filtrate, 
in which one or less particle can be contained, into 20 tubes containing 
bacterial broth suspension, and incubated them for a certain period of 
time. From the number of the tubes, in which the phage multiplica- 
tion failed to occur, and that of the total, he calculated statistically the 
number of the active particles present. The number of active particles 
thus calculated, however, never agreed with that estimated by counting 
the number of the plaques produced on the agar plate on which a cer- 
tain volume of the same filtrate has been spread with the bacteria. 

Ohashi examined about 40 different filtrates and found that the 
ratio, so far as the examined filtrates were concerned, could vary from 
about 0.1 to 3,000. This ratio was inclined in the majority of cases to 
be smaller than 1.0, with about 80 per cent of the total samples the ratio 
being found to be smaller than 1.0, indicating that the phage particles, 


III. THE ACTIVITY OF VIRUS PARTICLES 21 


in general, exhibited their action easier in a broth than on an agar plate. 

Since the ratio can vary in such a wide range, it seems quite im- 
possible to calculate the absolute number of active phage particles ; we 
can only know the relative number of particles which exhibit their 
action under a definite condition. It is a well known fact that even 
the number of plaques that may appear on an agar plate varies with 
the property of the agar plate. There may exist phage particles which 
can exhibit their action neither in a broth nor on an agar plate. 

This fact must be of the utmost importance, giving warning to 
virus researchers to be very prudent in estimating the activity of some 
virus samples. If all the phage particles, the ratio of which is 3,000 as in 
a sample above cited, can act as the active phage on an agar plate and 
the activity of such particles are measured in a broth, then it will 
appear as if only one of 3,000 particles had the activity; one would 
commit a great mistake, if one concluded from such a result that only 
0.03 per cent of the particles were the true phage, the remaining 99.97 
per cent being ‘“‘impurities’’. 


2. The Effect of the Difference in Bacterial Strains on 
Phage Activity 


The writer discussed above the difference in the phage activity due 
to the conditions under which a certain strain of bacteria are affected 
by phage. If similar observations were made with various bacterial 
strains, much greater differences in the phage activity would be revealed. 

On endeavouring to isolate phage preparations possessing the ac- 
tivity as high as possible, we succeeded in obtaining the phage sample 
whose minimal quantity required for producing a plaque was 6.8107" 
g.; a certain strain of E. coli was used in this experiment (2). On the 
other hand, the average dry weight of a single particle of this pre- 
paration was estimated to be about 1/2x10-%g.; hence almost all the 
produced particles could be considered as possessing the phage activity. 
The average dry weight was estimated by the method already described. 
This indicates that all the particles obtained by our method can reveal, 
under a proper condition, the phage action, a conclusion which is of a 
profound significance from various points of view as will be mentioned 
later. 

The experimental result from which this remarkable conclusion 
was derived was obtained by using a certain strain of E. coli, which 
was highly sensitive to the phage; but if other strains of bacteria were 
used, utterly different results would be obtained. For axample, the 
phage strain used in this experiment could infect a strain of typhoid 


22 I. INTRODUCTION TO VIRUSES 


and of dysentery bacillus as well as the strain of coli, which was very 
sensitive to the phage as above mentioned, from which it had been pro- 
duced, while the activity of the phage sample proved to vary strikingly 
among these strains of bacteria, and the ratio of the number of plaques 
produced by a given quantity of the sample was found to be as follows: 

1: 0.4: 0.001=coli: typhoid : dysentery. 

Therefore, if the typhoid or the dysentery bacillus strain was 
used instead of the coli strain, the minimal quantity required for pro- 
ducing a plaque would be estimated as 10/4X6.8x107"* g. with the ty- 
phoid, and 1,000X6.8x10— g. with the dysentery bacillus. Hence, it 
would be said that the phage sample which could reveal the high acti- 
vity on the coli strain was able to act on the dysentery strain merely 
as a very weak phage, only one particle out of 1,000 being capable of 
acting as a phage (44). 

Plaques would never show uniform properties, but differ strikingly 
in size and shape even when the plaques were produced on an agar 
plate with the same phage sample and with the same strain of bacteria, 
showing that each phage particle had each individuality. The above 
mentioned difference in the plaque number due to the difference in the 
host bacteria may be attributed to this individuality, but it appears 
possible that more than one particle may be required for the produc- 
tion of one plaque if phage is not strong enough or host cells are not 
sensitive enough to the phage so as a single particle can infect a bac- 
terial cell. 

In addition to the function as a virus, some phage particles exhibit 
a faculty of depriving the bacteria of their viability. In a study on 
this faculty, we have found that many particles are necessary, in order 
to deprive the viability, to combine with a single bacterial cell when 
the cell is rather resistant, although a single paricle seems sufficient for 
a sensitive cell (45) (46). It has been reported that also many particles 
are needed to infect a single cell if the phage is inactivated to a proper 
degree by ultraviolet irradiation, wherein the higher the irradiation the 
larger number of particles are required (47) (48). Moreover, the phage 
particles treated by ultraviolet irradiation to be reduced inactive, is 
found to hasten the lysis of the heavily irradiated E. coli, which are 
already inclined to autolysis (49). Similar evidence has been presented 
also with influenza virus; the virus particles irradiated by ultraviolet 
light or heated to 50°C can act as the virus, if great many particles 
affect a small number of host cells (50). Further, it has been claimed 
that the infectivity of mouse-pox virus is enhanced remarkably when 
it is added with a vast number of the virus particles inactivated by 
ultraviolet irradiation (51). 

In view of these facts it may safely be concluded that either when 


III. THE ACTIVITY OF VIRUS PARTICLES 23 


the virus actitity is relatively low or when the resistance of the host 
cell is relatively high, more than one particle are necessary to infect 
a single host cell. In addition, there are many reasons to suppose that 
the degree of protoplasm change is directly proportional to the quantity 
of agents causing the change (46) as will be shown later where a discus- 
sion is made on the mechanism of haemolysis. 


3. The Activity of Vaccinia Virus 


The vaccinia virus, with which we carried out a series of experi- 
ments, did unfortunately not exhibit a high activity upon rabbit skin. 
The number of infectious particles present in a virus sample isolated 
by us was so small that the ratio of the number of infective particles 
to that of the total was found, when examined with rabbit skin, to be 
of the order of 1:1,000. Nevertheless, it may not be proper to conclude 
from this fact that our virus sample was highly impure. The skin of 
the rabbits which we employd showed individually different susceptibi- 
lity to the virus, that is, the susceptibility varied with the individuality 
of the rabbit. Therefore, it might be possible to change the ratio 
if we made experiment with other rabbits more susceptible to the virus 
or with some appropriate tissues other than the rabbit skin. 

Nakamura and Ohfuji (52) found that a minute amount of the vac- 
cinia could much easier be detected when injected into rabbit testicles 
than when injected into its skin; they proved under the microscope the 
existence of the infection in the testicles by the histopathological changes. 
Nakamura and Fukumura (53) have also confirmed that the infective titre 
of the emulsion of cells which had been infected with the vaccinia virus 
which they possessed, as measured by the intracutaneous injection into 
rabbit, was 1x10* to 1x10°, while as measured by the above method 
of the intratesticular injection, 110° to 110°. Consequently, if the 
infective titre of the virus particle, all of which were assumed to be 
active in the testicles, was measured by the intracutaneous injection, 
it would appear, as a matter of course, as if only one particle amongst 
1,000 to 10,000 were the true virus. It is, however, not reasonable to 
consider that all strains of vaccinia are always so highly infective to 
testicles. For example, since the vaccinia isolated by Smadel ef al (54) 
was claimed to be so effective on the skin that almost each particle 
was able to produce the infection, it cannot be expected that such a 
vaccinia is so much more infective to testicles. On the contrary, the 
existence of strains of vaccinia may be possible which is more in- 
fective to skin than to testicles. Moreover, the existence of vaccinia 
particles which can neither exhibit their action in testicles nor in skin 


24 I. INTRODUCTION TO VIRUSES 


tissues though able to infect another proper tissue may also be possible. 

At any rate, as mentioned above, host cells of a high resistance 
may be infected only with a great number of virus particles, a conclu- 
sion which can also be reached with plant viruses. Thus, the minimum 
infective quantity of tobacco mosaic virus particles is said to be usually 
of the order of 10-’°g, which can be reduced to 10-™ to 10-” g, if very 
sensitive host plants are adopted (55). However, even in this latter 
case the mimimum quantity is equivalent to the virus particles more 
than 10°, so that there seems no doubt that an extremely large number 
of virus particles are required for the establishment of the infection. 

The active unit of plant viruses such as tobacco mosaic virus is 
commonly estimated from the number of local lesions produced on the 
leaf on which a given quantity of a virus sample has been rubbed. The 
numbers of lesions, however, are known to vary with each plant, and 
even with one and the same plant various results are obtained with 
different leaves. On this account, in the routine method a test sample 
and the standard are applied symmetrically on the same leaf. It is 
only natural that the discordance becomes greater if the species of 
plants are not the same. For example, potato virus Y can affect 
tobacco plant in.such a high dilution of 1/10,000, but fails to infect 
potato plant unless the dilution is lower than 1/500 (55). 


CHAPTER IV 
THE TRANSMISSION OF PROTEIN CHANGE 


1. Miultiplicable Denaturase 


Living cells are essential for the multiplication of viruses. Viruses 
can multiply only in the living cells. Viruses may cause a change in 
the structure of protoplasm protein of host cells; as a result the protein 
may be endowed autocatalitically with the virus activity. In this respect 
viruses may be looked upon as a kind of denaturase. However, they 
are, if so, by no means the denaturase in a narrow sense. An enzyme 
capable of converting a native protein into a “‘denatured”’ state is usually 
termed denaturase, but the change in the protoplasm protein due to 
viruses never appears to be a ‘‘denaturation’’, and hence viruses, if 
necessary, should be called ‘‘transnaturase’’ instead of denaturase. If it 
was impertinent to regard viruses as an enzyme, it would be considered 
at least as an agent capable of causing a transnaturation in the proto- 
plasm protein, There are, however, many reasons to regard viruses as a 
kind of enzymes, a full account of which will be given in a later chapter. 

Each virus is antigenically specific. Thus, animals infected or 
treated experimentally with a virus give rise to an antibody capable of 
reacting specifically with the virus. The virus is deprived of its acti- 
vity by the antibody. ‘ 

The appearance of virus-antigenicity in the host cell protein follow- 
ing a virus infection may be ascribed to the occurrence of a specific 
structural change in the protoplasm protein by the virus action, and 
owing to the change the cells may fall into a pathological condition. 
Thus viruses can be regarded as multiplicable transnaturase of proto- 
plasm protein. 

Knignt (56) showed the existence of a difference in the amino acid 
composition between the influenza virus particles and those separated 
from the normal hen’s embryo from which the virus particles were 
isolated, indicating that the change of the protoplasm protein due to 
the virus can effect the amino acid composition. According to Andreae 
and Thompson (57) chromatogram of healthy and leaf-roll infected 
potato tubers revealed a striking and consistent difference in the 
occurrence of tryrtophane and tyrosine. Rafelson et al. (58) found 
that the presence of Theiler’s virus stimulated the incorporation of 
radioactive carbon from glucose into most of the amino acids of minced 


26 I. INTRODUCTION TO VIRUSES 


mouse brain, but that it inhibited the incorporation of glucose fragment 
into lysine and histidine, the amounts of these two amino acids being 
reduced in the virus-infected tissues, whereas the amount of the other 
amino acids was unchanged, suggesting that the virus multiplication 
was intimately associated with the change in the amount of these two 
amino acids. 

No difference in the amino acid composition was found between 
particles of phage and its host bacterial cells (28), but it may be 
unreasonable to conclude from this finding that they are entirely iden- 
tical in the chemical composition, since the finding may merely show 
that there was no chemical difference large enough to be detected by 
ordinary chemical methods. If there exists an antigenical difference 
at all, there should also be a chemical difference between a virus and 
the protoplasm protein of host cells. 

The structural change of the protoplasm protein due to a virus 
may spread in the protoplasm as a chain reaction, since the virus does 
proliferate in the protoplasm. This chain reaction may be brought 
about by the protein which has been changed structurally by the virus 
and which may affect in turn, as a newly formed virus, the proto- 
plasm protein surrounding it. 

Fischer (59) claimed that in coagulating blood a coagulating agent 
is produced and that such an agent produced in a blood plasm can cause 
the coagulation of another plasm in which a similar coagulating agent 
will in turn be produced, thus the agent multiplying indefinitely. How- 
ever, the agent cannot be detected in the blood plasm already having 
finished the coagulation. Fischer interpreted this fact as based upon 
the mutual saturation of the liberated active groups with the comple- 
tion of the coagulation. It may not be impossible to regard a virus as 
such a coagulant, by which the protoplams protein is coagulated into 
minute particles, in which active groups capable of acting as the virus 
liberated during the coagulation remain unchanged. 

The coagulation of blood may occur by a physico-chemical change 
in the plasm protein. In a similar way the coagulation of protoplasm 
protein into virus particles may be a result of the structural change by 
the virus, and hence it may be said that minute body formation is only 
a result of the virus multiplication, never the essential feature of the 
virus. 


2. Similarity between Rennin and Viruses 


Many phenomena concerning viruses appear to be readily explained, 
as we have seen above, if viruses are regarded as denaturase or trans- 


IV. THE TRANSMISSION OF PROTEIN CHANGE 27 


naturase of the protoplasm protein. On the other hand, rennin is an 
enzyme which is known as a typical denaturase, and actually there 
seems to be a close similarity in their nature between this enzyme and 
viruses. 

Casein from milk is coagulated by this enzyme, and its coagulating 
action has been described as a conversion of caseinogen into casein or 
of casein into paracasein. Rennin seems to cause in casein molecules 
a physico-chemical change leading to the coagulation. Haurowitz (60) 
considered that rennin causes a slight unfolding of the peptide chains 
of casein, by which polar or ionic groups of the casein molecules are 
rendered mutually accessible to one another and are thereby able to 
form intramolecular salt like bonds. A similar argument may be made 
with viruses, that is, the action of a virus consists in the conversion 
of virogen or provirus into the virus; the precursor, of course, must 
be the normal protoplasm protein, in which polar groups may be liberated 
by the unfolding of the peptide chains, thereby protoplasm may be 
coagulated into minute particles and the rearrangement of the peptide 
chains following this unfolding may lead to the appearance of the virus 
activity. 

According to the writer’s study (61) rennin is similar to viruses 
in its physical and chemical nature, and its activity is associated with 
virus-like particles composed of a protein and lipids like viruses. So 
far as his study confirmed, rennin was stable in such virus-like parti- 
cles, which become extremely labile when decomposed into smaller par- 
ticles just as in viruses. Moreover, the enzyme particles isolated by 
the writer proved to bear a striking resemblance to viruses such as 
vaccinia and phage in many other respects, that is, in the behaviour 
towards inorganic salts and pH changes of the solution, and also in the 
agglutinability at a weakly acid pH; its isolation was achieved by 
applying this latter property as with viruses. It is a well known fact 
that some plant viruses can be crystallized, while rennin was likewise 
prepared in a crystalline form (62). Such a crystallizable character is 
never in discordant with its particulate nature, as will be discussed 
later in another chapter. 

Between the enzyme and viruses, however, there exists an essen- 
tial difference; namely the protoplasm protein coagulated by a virus 
into minute particles can exhibit the virus action, whereas coagulated 
casein fails to act as the enzyme. Nevertheless, an evidence can be 
presented that casein molecules that are being coagulated by rennin 
can promote the coagulation of other molecules. Thus, the length of 
time for the coagulation of milk by rennin was found to be governed 
not only by the concentration of rennin, but also by that of milk. The 
milk solutions of a series of concentrations, to each of which was 


28 I. INTRODUCTION TO VIRUSES 


added the same quantity of rennin, were expected to coagulate the 
sooner, the lesser the concentration, but the result was entirely reverse ; 
for example, of the two solutions of milk powder having different con- 
centrations, z. e. 5 per cent and 0.15 per cent, the former coagulated 
approximately 10 times as faster as did the latter if the rennin was not 
added too much. , This phenomenon can be best explained by assuming 
that the coagulation is a chain reaction, that is, the casein mole- 
cules having been changed to some extent by rennin can affect other 
intact molecules to accelerate the change, which should occur the more 
readily the higher the concentration of casein. 


3. The Infection of Denaturation 


The inactivation change of phage particles by its antiserum appears 
likewise to infect other intact particles to some extent as shown in 
Table 3 (63). Namely, the same paradox phenomenon was observed as in 
the case of rennin and milk, showing that the inactivation occurred the 
more markedly the higher the concentration of phage particles. Thus, 


Table 3 


Activity of Antiserum and of Tannin upon Phage 


Antiserum (1: 100), cc. 1 1 1 1 1 1 
Phage solution (0.01 %)cc.| 1 1/2 | 1/4 1/8 +) 1/16") wy32 
Water, cc. 0 1/2) | 3/4) 778 SS ee | 31/32 
of remaining active 


particles to the total 

particle number mixed, 0.51 1.6 4.3 
3 hours after the mixing 

of above 3 components 


9:0) aoe} ae 


Antiserum | Ratio (%) of the number 


Tannin (0.016 %), cc. 1 1 1 1 1 1 
Phage solution (0.01 9)cc.| 1 1/2° 4" 1/4 + 1/8 | 1/16) 2/32 
Water, cc. 0 1/2 | 3/4>) 7{8, | 15/16 | 31/32 


Tannin | Ratio (%) of the number 
of remaining active 
particles to the total 

particle number mixed, 5.1 6.7 8.4 8.4 8.0 5.1 
3 hours after the mixing 
of above 3 components 


when the concentration of phage particles is high enough to make the 
particles come into contact with each other, the denaturation of a par- 
ticle by the antiserum may successively be transmitted to other particles. 


IV. THE TRANSMISSION OF PROTEIN CHANGE 29 


The inactivation of phage by its antibody can be regarded as a 
result of a kind of denaturation (46). When tannic acid was used 
instead of the antiserum a similar phenomenon was likewise observed, 
although not so conspicuous as in the antiserum as indicated in Table 
3; thus, the denaturation by tannic acid seemed to be less infectious. 
Also with HgCl, the same. could occur to a certain extent as seen in 
Table 4 (64) (65). Again, heat denaturation of a protein was believed by 


Table 4 
Infection of Inactivation of Phage 
I. Infection of phage inactivation due to HgCl, 


Phage concentration (%) | 0.01 | 0.005 | 0.0025 | 0.00125 | 0.000625 


Ratio (%) of the number of remaining 
active particles to the total particles 


added, 30 min. at 37°C, after the 3.9 
addition of HgCl, at the concentration 
of N/200. 
II. Infection of phage inactivation due to heat 
Phage concentration (9%) | 0.05 | 0.025 | 0.0125 | 0.00625 | 0.003125 


Ratio (%) of the number of remaining 

active particles to the total particles 0.7 3 4 5 12 
present at first, after 30 min. of ; 

heating at 65°C. 


some workers to be infectious (66) (67), whereas according to the study 
of the writer heat denaturation of phage also appeared to be so as indi- 
cated in Table 4, where the higher the phage concentration, the more 
striking the inactivation (65). 

Bawden and Pirie (68) showed that the rate of inactivation of 
tomato bushy stunt virus by freezing is increased by the increase in 
the concentration of the virus; this might also be caused by the infec- 
tion. Furthermore, according to Kassanis and Kleczkowski (69) the 
ratio of inhibitor, such as ribonuclease, to tobacco mosaic virus to neut- 
ralize the infectivity decreased as the concentration of the virus in- 
creased ; that is, the higher the concentration of the virus the smaller 
is the amount of inhibitor needed to neutralize a given weight of the 
virus. 

All these evidences suggest that the protein denaturation is gene- 
rally infectious, and there seems little doubt that this infection occurs 
most distinctly in the protoplasm, where an elaborate mechanism is 
probably provided for the occurrence of the infection. As already dis- 
cussed, in the blood plasm the infection seems to occur readily, whilst, 


30 I. INTRODUCTION TO VIRUSES 


since the blood may be looked upon as a kind of free protoplasm or at 
least as a physiological fluid similar to the protoplasm, there should 
also exist in it a sort of the mechanism in favour of the infection. 

Lepeschkin (13) found that the granule coagulation of the protoplasm 
of a spirogyra cell caused by a pressing of the cover glass spread 
successively to another part of the protoplasm until the total mass 
would be coagulated into minute particles. Such a coagulation was 
found occasionally to be transmitted to another cell, never being con- 
fined to the single cell. According to Biinning (70) a cutting of an 
allium cell caused the coagulation of the protoplasm at the cutting site 
and after the coagulation was spread throughout the whole protoplasm, 
it was transmitted to the surrounding cells. The spreading velocity 
was 0.09 to 0.13mm. per min. according to him, and about 0.12mm. in 
spirogyra cell according to Lepeschkin. Thus in the protoplasm does 
occur the infection of the coagulation or denaturation in such a striking 
manner, and this must be one of the most important characteristics of 
the protoplasm. Viruses may be unable to multiply without this cha- 
racter of protoplasm. Presumably, not only viruses but also all the life 
phenomena may depend upon this character, a concept which will be 
considered in great detail in the next Part. 


CHAPTER V 
SUMMARY OF PART I 


1 


There are a great number of infectious diseases caused by agents 
evidently different from ordinary microorganisms. Such pathogens are 
known as viruses. Viruses are usually of sizes much smaller than 
ordinary pathogenic microbes and can pass through bacteria-proof 
filters, and accordingly sometimes called ‘‘filtrable’’ viruses. Like usual 
pathogenic microorganisms, viruses multiply in the body of organisms 
they affected, but for their multiplication living cells are always neces- 
sary. Generally, a virus tends to proliferate solely in the cells of a 
certain kind of a certain organism. 

A virus which affects bacteria is named phage. Phage was formerly 
regarded as a special type of viruses or a virus-like agent, but we 
could not find any essential differences between phage and vaccinia 
virus, one of the typical viruses. Our studies were made chiefly with 
these two viruses, and a theory as regards the nature of viruses has 
been put forwards. 

The activity of both phage and vaccinia virus is carried by parti- 
_ culate protein which can be agglutinated at an isoelectric point of a 
weak acid. Separation of the activity from the particles is impossible 
and therefore the particles are regarded as viruses themselves. No 
essential differences seem present between the particles prepared by our 
isoelectric precipitation method and those isolated by other workers by 
means of usual ultracentrifuge; the latter particles appear in most 
cases to be a portion of the former. Since we can prove that almost 
all the phage particles isolated by our method occasionally exhibit the 
virus action, it is unreasonable to regard our sample as having always 
a great quantity of ‘‘impure’’ particles. 

Such particles are not peculiar to viruses, but can be isolated from 
normal cells without any association with virus. In general, the pro- 
toplasm appears to have the property to be coagulated or disintegrated 
into minute particles by physical or chemical effects, but only those which 
have been produced by a virus are endowed with the virus action. 

The yield of virus particles by our method is so great that almost 
the total protoplasm of the cell affected by a virus is considered to 


32 I. INTRODUCTION TO VIRUSES 


be converted into the particles. This is also the case with normal 
particles having no connection with virus. 

The size of virus particles is mainly determined by the kind of cells 
from which they have been derived. Vaccinia virus particles isolated 
from the calf or rabbit skin is evidently different in size from those 
isolated from the rabbit testicle, the former being much larger than the 
latter. Again, phage particles are even much smaller than the vaccinia 
virus isolated from the testicle, while the normal bacteria are disinte- 
grated into particles of a small size entirely similar to that of phage. 
It seems probable that viruses, not only in size but also in immunolo- 
gical and even in chemical properties, are sujected to the cells from 
which they were produced. 

The water quantity combining with the protoplams particle is 
estimated to be so great as approximately ten fold of its dry weight, 
no matter whether the particle is produced by a virus or not. 


Pe 


Usually virus particles consist of a protein and lipids like the 
normal protoplasm. ‘This may only be a natural result if virus particles 
are nothing but the particles produced by coagulation of the protoplasm. 

The number of active particles in a phage sample is revealed to be 
different with the condition under which the number is measured even 
when the same strain of host bacteria is used. If various kinds of 
host bacteria are used the difference becomes very striking. This may 
be attributed to the different phage-susceptibility of | the bacteria, 
varying with the environmental condition as well as with the strain. 

A strain of bacteria with an extremely high susceptibility for a 
phage may be infected under a very suitable condition by a single 
particle of the phage, though usually more than one particle seem to 
be required for the infection. This holds true also for other viruses 
such as vaccinia and some plant viruses. 

In addition, each virus particle may have each individuality and 
accordingly even in one and the same sample, particles with various 
properties may be involved. Therefore, the estimation of the absolute 
titre of a virus sample seems’ to be extremely difficult, if not entirely 
impossible. 

It is worthy of note that a cell generally tends to be disintegrated 
into similar sized particles whose size seems to be determined by the 
kind of the cell. However, there are usually some differences in size 
even among the virus particles produced from the same kind of cells, 
and smaller particles prove to be much more unstable than larger ones. 


V. SUMMARY OF PART 1. 33 


This may partially account for the fact that viruses exist commonly in 
particles having sizes larger than a certain value; if their particle 
size are too small, viruses may be unable to exist as such because of 
their extremely unstable property. 


3 


Virus particles are immunologically distinct from the normal parti- 
cles isolated from the healthy cells. The antibody against a virus can 
specifically react with the virus particles, whereas the antibody against 
the protoplasm protein isolated from the normal host cells usually 
exhibit no action upon the virus particles isolated from the correspond- 
ing cells. Thus there seems no doubt that a virus is provided with a 
chemical group or groups capable of acting as an antigen different from 
the normal protoplasm protein of the host cells. Actually, certain 
differences are occasionally proved between them even in the amino acid 
composition. 

Viruses appear to provide to the protoplams protein of the cell they 
affected, in changing the property of the protoplasm, with a chemical 
group or groups which are present in their own configuration. Hence, 
viruses can be regarded as a kind of denaturase, or more adequately 
‘“‘transnaturase’’, of the protoplasm protein. When the protoplasm 
protein of host cells is furnished with such chemical groups through 
‘‘transnaturation” by a virus, the protein may acquire the ability to 
act as the virus. 

Protein denaturation is considered, in general, to be infectious toa 
certain extent, that is, a denaturating change occurring in a protein 
molecule tends to infect other intact molecules, and thus the denatura- 
tion spreads as a chain reaction. Such an infection seems to occur 
always strikingly in the protoplasm. Denaturation or coagulation arising 
in a portion of the protoplasm can spread promptly to the total proto- 
plasm and occasionally even to that of surrounding cells. 

Similar chain reaction may occur also in the protoplasm of the 
host cells infected with a virus resulting in the appearance of a chemical 
group or groups specific to the virus; thus the virus may multiply. 

The probable manner in which a virus structure is replicated in 
the protoplasm protein and the mechanism by which the replicated 
structure is transmitted successively in the protoplasm will be described 
in detail in the next Part. 


w w 


tw t Wt Ww 
SEAS 


I. INTRODUCTION €O VIRUSES 


REFERENCES 


Moriyama, H. and Ohashi, S.: J. Shanghai Sci. Inst., (4), 63, 1939. 
Moriyama, H. and Ohashi, S.: Archiv Virusforsch., (1), 571, 1940. 
Moriyama, H. and Ohashi, S.: J. Shanghai Sci. Inst., (5), 189, 1941. 
Brumfield, H. P., e¢ al.: Proc. Soc. Exp. Biol. & Med., (68), 410, 1948. 
Pollard, M. and Finegold, M. S.: Texas Repts. Biol. & Med., (6), 200, 1948. 
Cox, H. R., et /al.: J: Imm), (56), 149) 1947. 

Wahl, R. and Blum-Emerique, L.: Ann. Inst. Pasteur, (76), 103, 1949. 
Moriyama, H. and Ohashi, S.: J. Shanghai Sci. Inst., (3), 155, 161, 1937. 
Moriyama, H. and Ohashi, S.: J. Shanghai Sci. Inst., (3), 329, 1938. 
Delbriick, M.: J. Bact., (50), 131, 1945. 

Moriyama, H.: J. Shanghai Sci. Inst., (3), 135, 141, 1937. 

Wyckoff, R. W. G.: Advances in Protein Chem., (6), 1, 1951. 
Lepeschkin, W. W.: Zellnekrobiosis u. Protoplasmatod, Berlin, 1937. 
Hogeboom, G. H., é¢ al.: J. Biol. Chem., (165), 615, 1946. 

Tristram, G. R.: Advances in Proteinchem., (5), 1949. 

Moriyama, H. and Ohashi, S.: J. Shanghai Sci. Inst:, (3), 317, 1938. 
Moriyama, H.: J. Shanghai Sci. Inst., (3), 239, 1938. 

Bawden, F. C. and Crook, E. M.: Brit. J. Exp. Path., (28), 403, 1947. 
Levaditi, C.: Compt. Rend. Soc. Biol., (136), 96, 1942. 

Hook, A. E., e¢ al.: J. Biol. Chem.,- (165), 241, 1946. 

Cosslett, V. E., ef al.: Brit. J. Exp. Path., (31), 454, 1950. 

Wyckoff, R. W. G.: J. Imm., (70), 187, 1953. 

Knight, C. A.: J. Exp. Med., (83), 11, 281, 1946. 

Kabat, 6. Aj and Furth) Ja: Jc Exp-.Med 5 (7), .50.01940: 

Claude, A.: Science, (91), 77, 1940. 

Engel, L. L. and Randall, R.: J. Imm., (55), 331, 1947. 

Colowick, S. P., ef al.: J. Biol. Chem., (168), 583, 1947. 

Polson, A., and Wyckoff, W. G.: Science, (108), 501, 1948. 

Galloway, I. A. and Elford, W. J.: Brit. J. Exp. Path., (17), 187, 1936. 
Bawden, F. C. and Pirie, N. W.: Brit. J. Exp. Path., (26), 277, 1945. 
Crook, E. M. and Sheffield, F. M. L.: Brit. J. Exp. Path., (27), 328, 1946. 
Takahashi, W. N. and Rawlings, T. E.: Phytopath., (38), 297, 1948. 
Andrewes, G. H. and Horstmann, D. M.: J. Gen. Microbiol., (3), 297, 1949. 
Moriyama, H.: Archiv Virusforsch., (1), 273, 1939. 

McFarlane, A. S., ef al.: Brit. J. Exp. Path., (20), 485, 1939. 

Lauffer, M. A., e¢ al.: Advances in Enzymology, (9), 171, 1949. 
Moriyama, H.: Archiv Virusforsch., (1), 422, 1940. 

Sharp, D. G., ef al.: J. Biol. Chem., (159), 29, 1945. 

Schachmann, H. K. and Lauffer, M. A.: J. Amer. Chem. Soc., (71), 536, 1949. 
Pirie, N. W.: Advances in Enzymology, (1), 5, 1945. 

Crowfoot, D.: Chem. Revs., (28), 215, 1941. 

Bull, H. B.: J. Amer, Chem. Soc., (66), 1499, 1944. 

Ohashi, S.: J. Shanghai Sci. Inst., (4), 259, 1939. 

Moriyama, H. and Ohashi, S.: J. Shanghai Sci. Inst., (5), 189, 1941. 
Moriyama, H. and Ohashi, S.: J. Shanghai Sci. Inst., (4), 39, 1938. 


AGCaAaaaiaiaa&akeRE 
Z Aha ness 


pt es 
D> 


REFERENCES 35 


Moriyama, H.: Immunity (sister volume of this book), in press. 

Lunia S)h.: Proc: Nat. Acad: Sci:, (33), 253, 1947. 

Luria, S. E. and Dulbecco, R.: Genetics, (34), 93, 1949. 

Anderson, T. F.: J. Cellular Comp. Physiol., (25), 1, 1945. 

Henle, W. and Liu, O. C.: J. Exp. Med., (94), 305, 1951. 

Andrewes, C. H. and Elford, W. J.: Brit. J. Exp. Path., (28), 278, 1947. 
Nakamura, Y. and Ohfuji, M.: Med. J. Hokkaido Imp. Univ., (2), 475, 1925. 
Nakamura, Y. and Fukumura, M.: Ditto, (16), 329, 1938. 

Smadel, J. E., e¢ al.: J. Exp. Med., (70), 379, 1939. 

Bawden, F. C.: Plant Viruses and Virus Diseases, 1950. 

Knight, C. A.: J. Exp. Med., (86), 125, 1947. 

Andreae, W. A. and Thompson, K. L.: Nature, (166), 72, 1950. 
Rafelson, M. E., et al.: J. Biol. Chem., (193), 205, 1951. 

Fischer, A.: Bioch. Z., (279), 108, 1935. 

Haurowitz, F.: Chemistry and Biology of Proteins, New York, 203, 1950. 
Moriyama, H.: Arch. Virusforsch., (1) 510, 1940; (2), 71, 1941. 
Berridge, N. J.: Biochem. J., (39), 179, 1943. 

Moriyama, H. and Ohashi, S.: Z. Imm., (99), 282, 1941. 

Moriyama, H. and Ohashi, S.: Arch. Virusforsch., (2), 205, 1941. 
Moriyama, H. and Ohashi, S.: Z. Imm., (99), 419, 1941. 

Fischer, A.: Z. physik. Chem., A, (176), 260, 1936. 

Rondoni, P.: Z. physiol. Chem., (254), 207, 1938. 

Bawden, F. C., and Pirie, N. W.: Bioch. J., (37), 70, 1943. 

Kassanis, B. and Kleczkowski, A.: J. Gen. Microbiol., (2), 143, 1948. 
Bunning, E.: Bot. Arch., (15), 4. 1926. 


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PART II 


THE FUNDAMENTAL STRUCTURE OF 
PROTOPLASM AND OF VIRUSES 


CHAPTER I 


THE STRUCTURE AND FUNCTION 
OF PROTOPLASM 


1. The Structure of Protoplasm. 


Life phenomena are always associated with the protoplasm; indeed, 
where there is no protoplasm there is no life. The protoplasm is the 
principal unit of life. As discussed in the former Part the protoplasm 
is likewise indispensable for virus multiplication. Accordingly, in order 
to solve the riddle of life as well as to study the nature of viruses, 
detailed knowledges regarding the structure of protoplasm are of the 
utmost importance. 

Bensley (1) proposed the opinion that the basic constituents of pro- 
toplasm are of a fibrous structure, and at present this view is generally 
held by the majority of workers. Thus, the predominant opinion held 
by authors in this field seems essentially as follows: The protoplasm 
is composed of parallel alignment of fibrillar elements made of bundles 
of thread-like molecules, and the parallel fibres are joined by lateral 
bonds. Wyckoff has actually observed such fibrillar structures in proto- 
plasm by electron micrographs, and suggested that fibrillar macromole- 
cules may be the essential components of protoplasm (2). 

The writer has reached a similar conclusion as to the protoplasm 
structure through detailed studies on the nature of viruses and on vari- 
ous fundamental phenomena of life as will be descrided later. So far 
as our studies have reached, minute particles produced by the disinte- 
gration of protoplasm are composed of proteins and lipids without 
reference to the association or non-association of virus activity; the 
proteins are demonstrated to have the characters of euglobulin. In the 
writer’s opinion, in the protoplasm the molecules of these proteins are 
made into parallel fibres in their stretched thread-like form with lipids 
interposed among them. 

It is an established fact that the protoplasm mainly consists of 
proteins and lipids, which the latter usually constitute about one third 
of the total cell mass. The lipid contents of mitochondria and micro- 
somes, both of which are the important granular elements of proto- 
plasm, have been reported to be about 24 and 40 per cent, respectively. 
It has been found that the lipids in these cell inclusions occur in a 
firm chemical association with the rest of the structure, since they 


40 FUNDAMENTAL STRUCTURE OF PROTOPLASM 


cannot be removed even by prolonged extraction with organic solvents, 
such as ether or benzene, unless the complex is severely disrupted (3). 

There are, on the other hand, a great number of evidences that 
the protoplasm is composed of granules. As described in the previous 
part, protoplasm is readily decomposed into virus-like particles. On 
the administration of a proper stimulus it will begin to coagulate into 
minute particles at the site of stimulation. The coagulation thus in- 
duced will be successively transmitted to other parts. 

Thus, on the one hand, protoplasm appears to be composed of 
fibrillar elements, while on the other hand, it appears to be granules in 
the nature. Actually many granular elements such as mitochondria and 
microsomes are known to be present in abundance in the protoplasm, 
and hence a series of workers postulated the corpuscular theories of 
protoplasm, insisting on its granular nature; among them Biitschli’s 
foam structure or honeycomb theory is well known. Frey-Wyssling 
insisted, however, that all hypothesis as regards plasm structure which 
postulate distinct submicroscopic particles, such as granules, droplets, 
alveoles, and ultramicrons, must be discarded as being corpuscular 
theories (4). 

Although these two concepts regarding protoplasm structure may 
seem incompatible with each other, in the opinion of the writer they 
are never contradictory. According to the writer’s theory the struc- 
ture of protoplasm is as follows: In protoplasm, extended thread-like 
protein molecules of globulin nature make up bundles, and such bun- 
dles or corpuscles composed of parallel alignment of thread-like protein 
molecules represent the unit component of protoplasm. The writer 
has proposed the name ‘‘elementary body of protoplasm”’ to this unit. 
Lipids may be interposed among the protein threads as indicated by 
I in Fig. 4. If protein molecules combine directly with one another, a 
solid crystal may result; but owing to this lipid interposition, the ele- 
mentary body may be able to exist in a liquid crystalline state. 

Such elementary bodies or bundles of protein threads constitute the 
protoplams in forming in tern a parallel array joined by a loose junc- 
tion end to end as well as side by side, so that, protopasm is on the 
whole also a kind of liquid crystal. The elementary body may be lia- 
ble to be decomposed into several thinner bundles, 7. e., ‘‘elementary 
bundles’’ as shown in Fig. 5; in other words, several elementary bun- 
dles form a thicker bundle, 7. e. an elementary body. 

The extended protoplasm proteins tend to be contracted when 
certain physical or chemical effects are given as stimuli. Elementary 
bodies may be coagulated into separate particles following such con- 
traction of protoplasm proteins. 

On account of the orderly association of protein molecules just 


I. THE STRUCTURE AND FUNCTION OF PROTOPLASM 41 


I Il 
LIPID LIPID 
fon 
LIPID LIPID gk -p o 2. ~ . 
oO R 0 R Hy c «BN c Hy 
{ | H | | N i | | | | | 
c CH - N Cc GH 48 Cea Co ugh Cc 
CNG hi yp ONG IE NEI Y ONAN PLAS Neo Cpe ae 
N Cc cn ow ec CH cH oO cH oO 
H | | H | | | | 
0 R o R R R 
LIPID LPip =) LIPID LIPID 
fe) R fe) R R R 
| H | | H | | | 
Cc N CH Cc N CH oO CH Oo cH 
RR NG ke Nite Nc NBN ee 8 See TN Ng ee es 
N cH Cc N GH ‘Cc N N Cc N Cc N 
H t ] H | H H | | | | | 
R fe) R O Cc Ny C Ny  C 
: : Ge Na ZL SST Fon 
LIPID LIPID re) cH fe) cH fe) 
R R 
LIPID LIPID 
CLL Protein 
ANSANA'N/N//N/N«-- Protei 
| Polen WOOOTUSTOD) =-- Lipid 
TOOOTTTSOSUIOVUN =-- Lipid 
EB POV OO erg 


as 
COOTTTTOTTIOD COOOTTSTON 
commen eel) 
VOOOOTTIID- 
VN AANA 
SICIRO'S. 


Fig. 4. Reversible coagulation of protoplasm 


Elementary bodies 


i 


See — SS ————— ey = Elementary bundles 


Fig. 5. Structure of protoplasm. Hundreds of stretched protein molecules 
form a bundle (elementary bundle) in parallel alignment. Each several 
of such elementary bundles in a parallel association in turn form an 

_ elementary body. Between the protein molecules lipids are inserted as 
shown in Fig. 4. 


i 


2 FUNDAMENTAL STRUCTURE OF PROTOPLASM 


described, the coagulation may spread successively in the protoplasm 
as a chain reaction, a contraction of the proteins at a site of proto- 
plasm becoming in turn a stimulus on the other proteins or on the ele- 
mentary bodies surrounding them (Fig. 6). Such coagulation or con- 
traction of the elementary bodies is, as a rule, reversible, and when 
agents having caused the change, 7. e., stimuli, are removed, they can 
resume their former state. 


Stimulus Stimulus 
ee el t 
CE eae UNS COO- FN -——-C00-—| 1 HIN ——en0— 
wee Ee -—OOC——NH;+ —OOC-——NH; + —OOC — NH; + 
SS) ee + H;N-—— COO- + H;N—— COO— + H,N —-~COO— 
ae iad _ 
——— EERIE O 
——ae + H,N—CoOo- sent + H,N—CcoO- 
i ———— SS 
ee —OOC —— NH; + —0O0C —— NH; + —OOC —— NH; + 
eS SS + H;N—— COO-— + H,sN — COO-— + H;N — COO— 
8 1D aaa) fo) O fa) 
orn am TOP 390 B90 napa 6 Ie 
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——— FO — ; oO 
Seeereereat i) | Fore) eae —OOC — NH, + (eg ae —OOC — NH; + 
__ THR oe ~~ 
ee SS + H,N —- COO- + H;N —COO-— + H;N—COO- 
OO 15EN aM 395 390 Yo 
ee eee ae Fe Te 
TH TT rr 
Gio ua Bie OOH, OOF, 9 OX, 
aon mn en oF ee, ree 
are 70m am $300 9 06 
ane Fin aR Pause pte a he o 
Fig. 6. Transmissible coagulation of protoplasm. In the left, the manner 


in which elementary bodies are coagulated into minute bodies, and in 
the right, the manner in which polar groups of protein molecules 
undergo mutual association are illustrated. 


This may be the principal feature of the irritability of protoplasm. 
The contraction of elementary bodies joined with one another along 
the long axis may account for the various functions of protoplasm, 
such as changes in cell shape, the activity and direction of cytoplasmic 


I. THE STRUCTURE AND FUNCTION OF PROTOPLASM 43 


current, and the formations of spindle fibres which pull daughter chro- 
mosomes. Detailed discussions on these phemomena will be made in 
part IV. 

Many particles known to be present physiologically, such as mito- 
chondria and microsomes referred to above, may be a kind of elemen- 
tary body itself or its combined products, existing normally in a coag- 
ulated state. It may, however, be unreasonable to consider that such 
particles are always present in a coagulated state. According to Ris 
and Mirsky (5), the living interphase nucleus is optically homogeneous, 
chromosomal structures appearing after injury and after treatment with 
most histological fixations. In the living nucleus the chromosomes are 
present in a greatly extended state, filling the entire nucleus homogene- 
ously ; upon injury the chromosomes condensed and become visible, 
suggesting that the visible chromosomes are the conglomerates of co- 
agulated elementary bodies. 

The reasons wherefore the writer has formed the above mentioned 
opinion as regards the protoplasm structure may become comprehended 
gradually as the description proceeds. 


2. The Action of Protoplasm 


Analogy present between the protein synthesis and crystal growth 
has for a long time arrested attention of a number of workers. Hau- 
rowitz considered protein synthesis or protoplasm growth as follows (6): 
The template is formed in protoplasm by a protein molecule identical 
with the protein to be formed, but present in an expanded state, the 
amino acids which form the building stones of the replica are adsorbed 
tyrosine to the tyrosyl residues of the template, arginine to arginy]l, 
and the other amino acids likewise to the corresponding residues, in 
analogy to the process of crystallization, only molecules which are the 
same as the molecules forming the crystal are adsorbed to the surface 
of the crystal. 

As above mentioned, according to the writer’s theory the proto- 
plasm is in itself a crystal, but usually protoplasm fails to show the 
double refraction, a characteristic of crystals, although occasionally it 
shows this property especially when it is streaming. This may be at- 
tributed to a partial contraction of proteins resulting in some irregu- 
larity in the alignment of elementary bodies. There are good many 
reasons to suppose, as will be mentioned later, that protoplasm pro- 
tein molecules in the normal protoplasm are not completely stretched, 
but exist in a partially contracted state. Anyhow, the protoplasm can 
be looked upon as a type of liquid crystals and protein synthesis can 


a4 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


be regarded as the growth of this crystal, wherein each amino acid 
may be adsorbed to the corresponding site to produce the perfect pro- 
toplasm proteins as suggested by Haurowitz. Even a complete protein 
molecule having a configuration slightly different from that of the pro- 
toplasm protein may sometimes be adsorbed, and after being changed 
into the configuration same as that of the protoplasm protein by the 
spatial rearrangement of its polar groups, may be fused into the pro- 
toplasm as in the manner shown in Fig. 7. 


] 
Protein to be 
assimilized 


Fig. 7. Diagrammatic illustration of assimilase action of protoplasm. 
I; The arrangement of polar groups in the protein to be assimilized 

is different from that of the protoplasm in the sites shown by arrows. 
II: The protein is fused into. the protoplasm following the. rearrange- 
ment of polar groups. 


In this respect, the writer regards protoplasm as a kind of en- 
zymes, designated as ‘“‘assimilase’’. As will be discussed in the next 
chapter, the action of this assimilase is considered as arising from the 
polymerization of the same protein molecules. A powerful physico- 
chemical force may be generated in the assimilase by the spatial 
arrangement of polar groups piled up by the polymerization of proteins, 
a force which can cause the rearrangement of the polar forces in the 
adsorbed protein. 


CHAPTER II 


POWERFUL FORCE GENERATED BY 
PROTEIN POLYMERIZATION. 


1. Water-Molecule Layer Surrounding Virus Particles 


As previously mentioned (Part 1, Chapter III), protoplasm parti- 
cles including viruses combine with so much water amounts as appro- 
ximately ten times of their dry weight, and its greater part seems to 
exist in forming a thick layer of molecules around the particle. Next 
we shall consider as regards the thickness of the water ayes as well 
as the cause by which the layer is produced. 

The relation between the sugar-insoluble space of the protein particle 
and the sugar concentration of the solution in which the particle is 
suspended was already shown in Fig. 3 in Part I. As clearly indicated 
in this Fig. the sugar-insoluble space, as a rule, increases remarkably 
as the sugar concentration decreases, and it may be deduced from the 
curve in this figure, that the space will become nearly 20 as the sugar 
concentration approaches 0. In fact, the greatest value obtained in 
the writer’s various experimental studies on the space was about 20. 
If the space was 20 and the total water amount was contained within 
the particle, the water content of the particle would be 95 per cent; 
but it is unreasonable to consider that the particle could swell to such 
an unusual extent. 

Moreover, the fact that the curve in Fig. 3 ascends remarkably as 
the sugar concentration decreases can most reasonably be explained by 
assuming that the water layer cannot, as a rule, act as a solvent, but 
that the,combined water molecules pass gradually into the ordinary 
water molecules as they exist in more outer layer, so that the suger can 
penetrate furthur into the layer as the concentration increases. Water 
molecules may likewise possibly be absorbed into the particle itself, 
and the absorbed water molecules existing inside the particle may be 
similar in their physicochemical character to those existing in inner 
layer, although it may be possible that the former molecules are 
more marked in their inability to act as a solvent. Hence the sugar 
may even penetrate into the particle when its concentration is ex- 
tremely high. 2 

It seems very difficult, if not impossible, to estimate the water 
amount inside the particle. Since sugar may penetrate into the particle 


46 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


when its concentration is high, the estimation is impossible by measur- 
ing the space incapable of dissolving a sugar. 

The routine method of determination of the water content by mea- 
suring the particle density, which is estimated from the sedimentation 
rate of the particle in various concentrations of substances, such as 
sugar, protein, or inorganic salt, also may fail to give any indubitable 
value, since the substances may penetrate into the particle when the 
concentration is high as just pointed out. 

For such reasons the writer has been unable to estimate the water 
amount actually contained in the particle. However, since the ordinary 
virus particles can be regarded as the elementary bodies of protoplasm, 
their water content should be similar to that of protoplasm itself 
whose water content is generally accepted to be about 75 per cent. 
Now, therefore, argument will be advanced on assuming the water con- 
tent to be 70 per cent, a little less than that of protoplasm. 

As a particle should increase in diameter approximately by 1.6 
times when a dry particle absorbes water until its water content be- 
comes 70 per cent, vaccinia virus particle whose diameter is 0.25 y» in 
dry state will become 0.4 on water absorption to this extent. If water 
is further adsorbed on this swelled particle and the total amount of 
combined water reaches as great as 10 times its dry weight, the dia- 
meter must increase again by 1.5 times. In this state, the thickness of 
the water layer is calculated to be 0.09%, or 900 A: in this calculation, 
the density of the dry particle is taken as 1.33, a value which was pre- 
viously obtained by the writer (7). 

According to the writer’s estimation, the combined water amount 
was similar without reference to the kind of virus. For example, the 
amount of combined water was estimated to be similar both with vac- 
cinia vicus and phage, whereas the particle of the former was found to 
be greater in diameter as much as 3 times than that of the latter (8). 
This must be a noteworthy fact, because, if the thickness of the water 
layer was the same, the combined water amount of phage particle 
should be far greater than that of vaccinia virus particle, but the fact 
is that the amount is found to be the same, showing that the layer 
becomes thinner as the particle becomes smaller. 

In the same way as with the vaccinia virus particle the thickness 
of the water layer of phage particle is calculated to be 300A on the 
assumption that the diameter of the dry particle is 80 my and its den- 
sity is 1.45; also this value of density was previously estimated by 
us (9). 

The water molecules forming the layer with such a thickness are 
considered to behave themselves always in association with the par- 
ticle. The sugar-insoluble space, as above mentioned, was sometimes 


II. POWERFUL FORCE GENERATED BY PROTEIN POLYMERIZATION 47 


estimated to be so great as 20. In such a case the layer must have a 
thickness much greater than that estimated above, and hence it seems 
possible that even the water molecules existing at a distance even 
greater than 900 A apart from the particle surface have a property 
somewhat different from that of the ordinary water. 

Estimations are made above on the assumption that the water con- 
tent inside the particle is 70 per cent, but there are no great alterations 
if it is assumed to be either 65 or 75 per cent: with vaccinia virus 
particle it is calculated to be 1,100 and 800 A, respectively; with 
phage 350 and 270 A. 

The thickness of the water layer thus appears to vary with the 
size of the particle in the direct proportion to the diameter. This 
must result in the fact that the quantity of the combined water is 
identical regardless of the particle size. 

Rothen (10) reached to a similar conclusion in his studies on the 
“long range force’’ acting between an antigen and its antibody. Accord- 
ing to him, the force becomes greater and can reach longer distances 
as the thickness of the antigen molecule layer increases. This long 
range force may cause the attraction of the water molecules around the 
virus particle. Since this force is specific as claimed by Rothen, it 
seems highly probable that viruses can act upon the host cells through 
this long range force. If so, the action is expected to become greater 
with the increasing particle size. This, as is argued in the following, 
seems actually the case. 

Long range interaction seems to occur also in a solution of tobacco 
mosaic virus, where the molecules assume regular positions at intervals 
as great as 1,000 A (11), a finding which should be expected from the 
existence of the thick water layer. It has been reported that even in 
haemoglobin crystals molecular layers of the protein seem to be sepa- 
rated by water toa distance of 65 A; 30 per cent of the water of 
haemoglobin crystals is so firmly bound that it is not available as a 
solvent for small ions (12). 

Rothen (13) insists upon :the existence of the long range force act- 
ing several hundreds A between an antigen and antibody and between 
an enzyme and its substrate, while a number of workers are disin- 
clined to accept his view. It is, however, never an unusual pheno- 
menon that various particles or granules in physiologically active cells 
exert their influence on each other even when the distance between 
them is considerably great and can take positions in a mutual connec- 
tion. Since this seems also true iz vitro observation, the writer is like- 
wise firmly convinced of its existence. 


48 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


2. The Force Generated by Polymerization 


It is probably true to say that the thick water layer is attracted 
onto the surface of virus particles by Rothen’s long range force. But 
it must be an important question: what is the nature of the force ? 

Prior to answering this question, Rothen’s experiment referred to 
above should be discussed in detail. He used bovine serum albumin as 
the antigen and homologous rabbit antiserum as the antibody, and found 
that the thickness of molecules of the antibody attracted by the layer 
of the antigen molecules became greater as the number of the antigen 
monolayers increased. When the antigén was a single monolayer the 
thickness of the antibody was found to be about 30 A, while when anti- 
gen was piled up in 8 layers it became 230 A. 

A possible explanation of this phenomenon is that the antibody- 
attracting force of the antigen may result from its polymerization 
and that the force may consist of electrostatic force arising from the polar 
groups: of the protein. If a molecule can be compared to an electric 
cell, the force may be expected to become greater on the polymerization 
or on piling up. The difference in the pattern of arrangement of polar 
groups may account for the specificity of the force. 

Water molecules may be attracted by such a pattern of polar groups, 
positive pole of the molecule being attracted by the negative pattern, 
and negative pole by the positive pattern, so that the water layer thus 
formed should have in itself the pattern of polar forces. If water 
molecules are thus successively attracted in accordance with the pattern, 
forming a thikness of, say, 1,000 A, the outside of the most outer layer 
also may have the pattern. In sucha case, it may be said that Rothen’s 
long range force can reach a distance of 1,000 A. However, as illus- 
trated in Fig. 8, since the arrangement of water molecules may become 
the more irregular as they exist in the more outer layer, the pattern 
will become more incomplete with the increasing distance. It has been 
reported that the long range force failed to be demonstrated if egg 
albumin was used as an antigen instead of bovine albumin. This might 
be due to the failure of the protein to polymerize regularly. 

Haurowitz (14) studied the reaction between the atoxyl-azo protein, 
containing As in various proportions, and its antibody, and found that 
the antibody-attracting force of the antigen seems to increase with the 
increasing number of As molecules combined with the protein; when 
As quantity is very great a single molecule of the antigen can combine 
with even 50 molecules of the antibody. This result may be expected 
if the polymerization of the protein is enhanced with the increasing As 
content, wherein As-containing groups being piled up through the 


II. POWERFUL FORCE GENERATED BY PROTEIN POLYMERIZATION 49 


polymerization. When antigen molecule increases in size, molecules of 
antibody combining with it seem generally to increase in number ; for 
example, according to Heidelberger and Kendall (15), while a single 


i) 
Soa QA LS *-~_ Free water 
oRstes Cone nae 

EEEEPBWU 
SEVHES RE SB 
SS VVSVI ITY 
SBS SVVOGS 
oS OF S83. oS es 2s 


SIGISISISISIOIOIONS <«~-- Surface of 


a virus particle 


---Long range force--- 


Fig. 8. Diagram of the long range force. 


molecule of serum albumin as antigen can combine only 6 molecules of 
the antibody, thyroglobulin which is known to have molecular weight 
about 10 times as great as that of serum albumin can combine 60 
molecules. The increase in the molecular size of antigen can be looked 
upon as the result of polymerization of unit molecules with the piling 
up of the specific active groups. The accumulation of the monomole- 
cular layers of antigen in the experiment of Rothen may be regarded 
as the increase of the molecular size. 

The fact that virus activities are carried by particles having sizes 
greater than a certain value may come from the requirement of 
polymerization of protein molecules for the activities. If the polymeriza- 
tion degree is low, though sufficient for acting as a virus, the virus 
may be able to exhibit only a weak action and in addition unstable and 
liable to be inactivated. 

According to our study, as described in Part I, Chapter III, small- 
sized phage particles have the activity>much less than that of large- 
sized particles, and 50 or 100 small-sized particles seem to be compatible 
in their activity to a single large-sized particle. This fact may be at- 
tributed to the instability of the small-sized particles, but it seems a 


50 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


more probable explanation that most of the small-sized particles cannot 
act as the virus because of the low polymerization degree. Phage 
particles of extremely small sizes were occasionally found to produce 
only very small plaques showing their weak activity. 

In the studies on the interaction of large and small haemoaggluti- 
nating particles of Newcastle disease virus with red cells, Grenoff and 
Henle (15a) have found that whereas large particles were readily ad- 
sorbed onto red cells, only relatively small amounts of small particles 
appear to combine with red cells under the same conditions, and more- 
over, large particles cause haemolysis whereas small ones lack this 
property. 

It is well known that antigenic substances are always large mole- 
cules, of molecular weight of the order of 10,000 or more, and conse- 
quently being usually colloidal in solution. This shows that, in order 
to act as antigens, antigenic substances must be in molecular state of 
considerable sizes as in viruses, although the sizes required for antigens 
are not so large as those of the viruses. Polysaccharides isolated from 
bacteria are generally non-antigenic, but when adsorbed on particulate 
substances, such as collodion particles or kaolin, they can act as anti- 
gens. Again, albumose, though usually non-antigenic, can produce 
antibody if adsorbed on colloidal aluminium hydroxide. Also some 
lipids extracted from various organisms are not antigenic per se, but 
when injected into an animal after mixed with swine serum, can cause 
the antibody production. 

These non-antigenic substances may acquire the antigen-ability by 
the polymerization on the surface of colloidal particles on which they 
are adsorbed, thereby the physicochemical force capable of acting as 
the active group of antigens may become greater. ‘They may possibly 
act as antigens even when no such polymerization occurs, if non-specific 
colloidal particles injected with them can induce some disturbances in 
the protoplasm configuration of the antibody-forming cell, on which the 
mixtures or complexes are adsorbed, in assisting the formation of the 
replica corresponding to the antigenic pattern in the protoplasm. In 
the opinion of the writer antigens like viruses act as templates to 
produce replicas in the protoplasm (22). There are many evidences 
that a non-specific disturbance in the protoplasm caused by factors 
other than viruses may exert a favourable influence upon a virus to 
infect the cell; a detailed account of this will be given later. 

Enzymes are another agent for whose function a similar large 
molecular state may be necessary. A group of enzymes, known as 
conjugated ones, consists of two components, 7. @. coenzyme and apo- 
enzyme; the former in itself has no enzymatic action and has a low 
molecular weight, but on combination with the latter the activity of the 


II. POWERFUL FORCE GENERATED BY PROTEIN POLYMERIZATION 51 


enzyme develops, and this latter is the protein moiety of a colloidal 
nature, possessing also no enzymatic activity. The development of 
enzymatic activity on the combination of the two components may be 
chiefly due to the high molecular weight of the complex so formed. 
Likewise in this case some disturbances in the substrate configuration 
caused by the non-specific or partially specific physicochemical force of 
the apoenzyme molecule, resulting from the polymerization, may par- 
ticipate in the development of enzymatic action. 

It is known that enzyme molecules or, in general, protein molecules 
in a solution tend to associate or polymerize into larger molecules or 
aggregates. This property may contribute to the exhibition of enzymatic 
action. In spite of their protein nature, viruses are usually not affected 
by proteolytic enzymes. This may be accounted for by the higher and 
firmer polymerization of viruses than enzymes. Even if enzymes func- 
tion as such on polymerization, the degree of polymerization may be 
insignificant as compared with that of viruses. 

From a number of facts mentioned above it may be concluded that 
the increase in molecular weight, or the polymerization of molecules, 
seems to make the physicochemical force arising from the configura- 
tion more effective. Life phenomena may be intimately correlated to 
such forces coming from polymerization. Peculiar properties belonging 
to the so-called colloidal substances may presumably depend upon such 
forces. Since all the living bodies consist of colloids, the life phenome- 
na should be based upon these colloidal substances, and therefore life 
phenomena may be said to be developed by the long range forces. 


CHAPTER III 


THE PROPAGATION OF TRANSNATURATION 
AND THE MULTIPLICATION OF VIRUSES 


1. Propagation of Transnaturation in 
Protoplasm by Viruses 


In the opinion of the writer, virus particles are, either in their con- 
struction or in their character, identical with elementary bodies of pro- 
toplasm and consequently are a kind of assimilase. When protoplasm 
is disintegrated into elementary bodies and in these bodies the original 
structure of the protoplasm is retained, the bodies can act as assimilase 
existing in a state of minute particles. 

Such an elementary body can act upon another assimilase, changing 
the structure of the latter to be identical with it; that is, it can exert 
the ‘‘assimilase’’ action upon another assimilase if the latter is weaker 
in action. An elementary body, however, shares no faculty to synthesize 
from amino acids or from their components a protein which possesses 
the same structure as that of the protein of which the elementary body 
is composed. In other words, viruses can ‘‘assimilize’’ other assimilases 
whose action is weaker than that of their own, but cannot proliferate in 
media containing no cells by synthesizing the protein. 

Thus, when a virus affects cell protoplasm whose assimilase action 
is weaker than that of the virus, the protoplasm will be changed to 
become identical with the virus, as shown in Fig. 9. The change may 
start at the site where the virus contacts with the protoplasm and may 
propagate successively to other parts until finally the spatial arrange- 
ment of polar groups in the whole protoplasm protein is altered to be- 
come identical with that of the virus, being followed occasionally by 
the coagulation of protoplasm into elementary bodies. If the proto- 
plasm is disintegrated into coagulated elementary bodies, these bodies 
may be looked upon as virus particles. This is the principal way in 
which the viruses multiply. 

Thus, since for the protoplasm to become identical in its structure 
with a virus is to make the virus multiply in it, particle formation 
is never essential for the multiplication, and accordingly viruses are 
produced in the protoplasm irrespective of the occurrence or nonoc- 
currence of decomposition of the protoplasm into minute particles. 


III. PROPAGATION OF TRANSNATURATION & MULTIPLICATION 53 


The assimilase action, as discussed in the previous chapter, arises 
from the orderly polymerization of protein molecules of the same struc- 
ture, and hence, when the polymerization is incomplete and loose, a 


Virus combining 
with protoplasm 


: 


’ 
H H 
' 
: I 
' ! 
' t 
' ' . 
' 1 Virus 
a NG Oe 
1 ' 
' ' 
‘ ; 
' ! 
, 


by virus 
V v ‘ 


Fig. 9. The mode of multiplication of virus in protoplasm. 


; i 

’ ' ' 

4 ' 4 

t ' \ : 
' H ei 

‘ vow hays H ! ; 
Ha 
J ' 4 ‘ 
Ny V Nese wo ‘ era nN AD, ul 
‘ t 1 ! 
aR aaa | [AWA Sy | 
' 1 ' H 
Ivava Aw fap OR, 
t ; ' i | Protoplasm rearranged 
H ‘ Protoplasm | 
ig fa 1e 
i ' t H 
' ' ‘ 

rT : 4 

i ; 

\ / 


See eo a eo ee oe oe ee = 


virus particle may fail to act as a typical virus, only being capable of 
disturbing the structure of the protoplasm without inducing exact re- 
plica. Such an incomplete virus particle may sometimes be able to 
exhibit its virus action, if many particles are put together to be 
stabilized in its structure or to be united to cooperate. Luria (16) 
found a peculiar phenomenon that phage is produced when bacterium 
is infected with many particles of phage inactivated by ultraviolet light. 
In addition it has been reported that a great number of mouse pox 
virus having been inactivated and being unable to multiply can enhance 
the virus action of a minute quantity of the intact virus (17). Moreover, 
evidences are known that a virus which has been inactivated to a certain 
degree is capable of developing some effect upon the protoplasm of host 
cells though fails to produce the replica. For example, phage particle 
inactivated by ultraviolet ray can promote the lysis of bacteria due to 
irradiation, thereby no virus multiplication taking place (18), and again 
phage particles inactivated by X-ray irradiation is still able to lyse and 
kill sensitive bacteria (19). 

Physiologically active agents such as enzymes and toxins may be 
similar to viruses in their action, but so perfect a polymerization as in 
viruses, if needed, may not be necessary for their function, as the 
action of a template is never needed in these agents. 


54 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


2. Spread of Denaturation in Red Blood Cells 


There are numerous evidences that various physicochemical changes 
can spread readily in the protoplasm. Almost all the life phenomena 
may presumably depend upon the transmission of the changes, which 
cannot be accomplished unless lipids are interposed among protein 
molecules rendering the protoplasm protein freely movable. On this 
account lipids may be regarded as an essential component of the 
protoplasm. 

In this section some detailed discussion will be made on the 
haemolysis, which can be looked upon as one of the phenomena 
resulting from such a transmission of protoplasm change, in the hope 
that it may throw some light on the mechanism of the transmission. 

Red blood cells consist of haemoglobin and stroma, which the latter 
is a substance sedimentable from water solution of lysed cells by pass- 
ing CO, into the solution. According to our study, stroma separated 
from haemoglobin is a particulate substance similar to viruses consist- 
ing of a protein and lipids (20). When acetic acid is added instead of 
CO, to the lysed solution to adjust its pH to about 5.5, it can be sedi- 
mented like virus particles and isolated by an ordinary centrifuge. 
This shows that the stroma thus obtained is nothing but the coagu- 
lated elementary bodies of the blood cells. Red cells seem, therefore, 
to be composed of such elementary bodies to which a great quantity of 
haemoglobin is adsorbed. Mitchson (21) has claimed that the thickness 
of the membrane of the human red cell is 0.54, and that since this 
would occupy about 55 per cent of the volume of an intact cell, a red 
cell is largely made up of such a thick membrane containing haemo- 
globin. This thick ‘‘membrane’’ may be the stroma, which is the pro- 
toplasm itself, and which can be decomposed into virus like particles, 
1. @., into coagulated elementary bodies. 

When a physicochemical change is induced in this stroma to a 
certain degree, the stroma may become unable to maintain the adsorbed 
haemoglobin and thus haemolysis may follow. If so, the changing de- 
gree of stroma will be traced in detail by the liberated haemoglobin 
amount. 

If the protoplasm structure is just as already described, haemolysis 
may take place in the following way: When a haemolytic agent com- 
bines with a red cell to exert an influence upon the protoplasm, a 
change will occur in the site of the combination of the agent and will 
spread successively in the protoplasm; when the change reaches to a 
certain degree haemoglobin will begin to escape from the protoplasm. 
In fact, experimental results obtained with haemolytic agents, such as 


III. PROPAGATION OF TRANSNATURATION & MULTIPLICATION 55 


saponin, HgCl,, and antiserum with complement, agreed well with this 
reasoning. 

Firstly, it was confirmed that there was a certain incubation period 
between the addition of haemolytic agent and the commencement of 
haemolysis. This incubation period is expected from the above reason- 
ing. Secondarily, it was found that this period is inversely proportional 
to the concentration of the agent, showing that the changing rate of 
stroma is directly proportional to the concentration. This fact may 
indicate that the haemolytic agent acts as a kind of catalyzer; if 
the blood cell protoplasm undergoes a change in a manner described 
above following the combination with the agent, the degee of the 
change should be directly proportional to the amount of the agent 
combining with it, and hence the haemolytic agent may appear in its 
action to be a sort of catalyzer. 

The action of the haemolytic agents accordingly is comparable to 
that of partially inactivated viruses, which fail to produce replica but 
capable of disturbing the protoplasm structure. Thus, the fact that 
weak virus particles cannot act as the virus unless many particles are 
put together may be explained in this respect; namely, the protoplasm 
change caused by the virus is proportional to the virus amount combin- 
ing with it, so that even a weak virus may overcome the protoplasm 
if many particles are present. 

In this connection a more detailed discussion will be made on the 
experimental results obtained with saponin and rabbit blood cells. The 
relationship between saponin concentration and the time required for 
the haemolysis completion was found to be as indicated in Table 5. 


Table 5. \ 
Ralation between the Concentration of Saponin and the Time Required 
for the Completion of Hemolysis by the Saponin Concentration. 


Concentration — x 
|e Nl 
of saponin, %. 0.1 Se = 
(Cc); 4 Es 
oO oO 


Time required for 
the completion of 
hemolysis, sec. - 71 84 109 


(t). 
jt, (K). | 0.0141 | 0.0119 | 0.0092 | 0.0072 | 0.0043 | 0.0024 | 0.0010 


142 230 420 1020 


Saponin haemolysis is very fitted for such observations as the haemo- 
lysis is completed in short periods if once begins to occur. 
In this table the time interval between saponin addition and the 


56 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


lysis completion is shown, but as the lysis completes immediately after 
its commencement, time cited here can be regarded as the time required 
for the lysis commencement. Therefore, incubating time required for 
the lysis commencement can be said to become the longer, the lower 
the saponin concentration. 

Now, consideration will be made as Hscenad the following equation 
representing the first order reaction: 1/t-lna/\(a—x)=k, where a is 
the quantity of reacting substance existing at the outset, and x is the 
reaction product in the timeZ; that is, in the case of haemolysis, @ is 
the protoplasm quantity to. be changed and x is the protoplasm amount 
changed in the time f. 

Since haemolysis is to be completed when a/(a—x) becomes a certain 
value, this should be constant irrespective of the saponin concentration 
and hence can be designated as k’, then we have: 

L/ét-lnk’=k or 1/t=k/Ink'=K. 

Thus, 1/t is shown to be directly proportional to the velocity con- 
stant. On the other hand, the relation between 1/t, or K, and saponin 
concentration is indicated in Fig. 10, where it is shown that K tends to 
be a linear function of the saponin concentration. Since K is directly 


0.014 


0.1 


ae 


Fig. 10. Relation between the concentration of saponin (C) and the 
velocity of hemolytic reaction (K). See Table 5. 


proportional to the velocity constant as just mentioned, this indicates 
that the velocity constant also tends to be a linear function of the 
saponin concentration. However, the effect of the saponin concentra- 
tion becomes the lesser as it becomes the higher as clearly shown in 


III. PROPAGATION OF TRANSNATURATION & MULTIPLICATION 57 


Fig. 10; this may be due to the decrease in the effect of saponin per 
unit mass when its great amounts are adsorbed to a blood cell. 

Experimental results obtained with saponin were thus fairly de- 
monstrable, because haemolysis was completed in a short period ; how- 
ever, with HgCl, the situation was somewhat different because the 
lysis by this salt needed an extremely long period, though similar re- 
sults could be obtained. Likewise with antiserum and complement, the 
same proved to be true. 

So far as our studies concern, at least some bacterial haemolysins 
are of a particulate nature like viruses, and may cause a change in the 
blood cell by a physicochemical force arising from their specific con- 
figuration as in the case of viruses; the changes induced by haemolysin 
may propagate through the protoplasm to cause haemolysis (22). Such 
a change, however, fails to cause the production of replica correspon- 
ding to the specific pattern of the haemolysin, so that the lysins can- 
not multiply in red cells and therefore they are not viruses. 

Bacterial lysis caused by a phage can, in like manner, be explained 
by the assumption that the association among elementary bodies in the 
bacterial cell is loosened by the protoplasm change leading to the liber- 
ation of elementary bodies which are usually endowed with virus action 
by the change. It is known that when a bacterial cell is affected by a 
great excess of phage particles, no active particle is produced. This 
may result from a too rapid decomposition of the protoplasm to be 
endowed with the pattern exactly identical with that of the phage. 
Even in the case of lysis by a proper number of phage particles, the 
virus cannot multiply in the absence of certain nutrients or in the 
presence of metabolic ‘‘inhibitors’’ (23) presumably because of a similar 
failure of the replica formation. 


CHAPTER IV 
THE CRYSTALLINITY OF VIRUSES 


1. The Fusion of Particles 


Virus-like particles can be isolated also from healthy plants by 
the same procedure by which the particles are isolated from animal 
tissues. Wecarried out experiments with plant materials such as leaves 
of cucumber (Cucumis sativus, L.), kidney-bean plant (Phaseolus vul- 
garis, L.), tomato plant (Solanum lycopersicum, L.), poplar (Poplulus 
nigra, L.), spindle-tree (Evonynus japonicus, Thumb.), dandelion (Ta- 
vaxacum Melongena, L.) and petals of flowers of bindweed (Catystegia 
subvolubilis, Don.) (24). 

Virus-like particles were recovered without exception from all these 
normal plant materials. In the case of tomato plant, we used leaves 
infected with mosaic virus as well as healthy ones, and were able to 
isolate similar particles from both materials. Berkefeld-filtrates of these 
plant saps diluted with water were added with acetic acid and precipi- 
tates produced thereby were separated by means of ordinary centrifuge. 
In the majority of cases the optimum precipitating point was found at 
a more acid side than in the case of animal materials, and particles 
obtained were proved to be usually smaller than those of animals. 

Plant particles thus obtained shared a peculiar property not present 
in particles of either animal or bacterial origin, and tended to aggre- 
gate into larger particles or bodies. For example, particles of dande- 
lion did form coccoid bodies of about 34 in diameter when left at pH 
4.0 for several days, and particles of cucumber aggregated to bacillary 
bodies which sometimes further developed into large homogeneous pro- 
tozoa-like bodies. It is a noteworthy fact that in such bodies original 
particles entirely disappeared, but on the addition of dilute alkali, the 
particles would reappear and occasionally the homogeneous bodies would 
be broken down into the particles. 

The fusion of particles into such larger masses was never observed 
with animal or bacterial particles. Some of such large masses would 
appear as if they were crystals, but no double refraction was demon- 
strated. Occasionally uniform large bodies were formed immediately 
after the precipitation of particles by acetic acid; in such cases it could 
be clearly seen under the microscope by dark field illumination that 
they were composed of minute particles which, however, when allowed 


IV. THE CRYSTALLINITY OF VIRUSES 59 


to stand for a long period, disappeared gradually in fusing homogene- 
ously. t 

These facts may be interpreted as flollows: Plant elementary bodies 
which have been coagulated in the course of isolating procedure can 
solve the coagulation, when stand at the isoelectric point, in liberating 
the polar groups which subsequently can make the particles associate 
with one another to form large masses which may be looked upon as 
the protoplasm itself. In other words, the coagulated elementary bodies 
can solve the coagulation to unite with one another, thereby they may 
be arranged orderly as in the original protoplasm, thus recovering the 
structure of the protoplasm itself. Therefore, the fusion of particles 
to larger bodies may be regarded as their return to the original proto- 
plasm mass. At the isoelectric point COOH groups of the protein can 
coexist with the NH; groups, probably contributing to the fusion of the 
particles. 

The fact that such fusion can take place with plant particles, 
whereas this is not the case with animal ones, suggests that plant 
particles can readily solve their coagulation and can combine with one 
another while animal particles fail to do so. On the other hand, this 
may account for the difficulty of plant protoplasm to be coagulated into 
minute particles. Whereas animal particles can readily be precipitated 
only by the shift of the pH to its isoelectric point, plant particles oc- 
casionally fail to be obtained by this process; the addition of salts such 
as ammonium sulphate or sodium chloride is required for the complete 
precipitation. Thus, in the case of plant materials, mechanical grind 
may not be sufficient for the coagulation of elementary bodies. It is 
actually known that plant virus particles such as those of tomato bushy 
stunt, tobacco necrosis, southern bean mosaic and turnip yellow mosaic 
are soluble even at the isoelectric point and fail to precipitate (25). 


2. The Expulsion of Lipids 


The property of plant particles to fuse into homogeneous masses 
may be closely related to their crystallinity. Homogeneous masses 
appear sometimes as if they were crystals, but if produced by the 
mere addition of acetic acid, they never proved to be optically aniso- 
tropic when observed through crossed Nicol prisms, whereas precipitates 
with double refraction were sometimes formed when ammonium sulphate 
at 1/3 saturation was added together with acetic acid; that is, crystals 
were formed when ammonium sulphate was added. 

Among the plants serving as our experimental materials, the doubly 
refracting property was most distinctly observed with precipitates ob- 


60 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


tained by this procedure from cucumber, tomato, and egg-plant, while 
from dandelion and poplar were formed precipitates, the greater part 
of which was composed of minute particles without double refraction, 
only a small part presenting the refraction; and from the other plants 
doubly refracting precipitates were never obtained. 

From both tomato and egg-plant were sometimes obtained typically 
crystalline masses with a definite shape and a sheen more intense than 
that of the precipitates without double refraction, whereas in some 
other cases only sediments were yielded appearing as though the mi- 
nute particles themselves were exhibiting birefringency, the doubly 
refracting particles appearing to be so small. 

The nitrogen content of such crystalline masses or particles was 
found to be approximately 16 per cent, the usual N value for ordinary 
proteins, showing that there was no room for the presence of lipids, 
and in fact no lipids could be recovered from such particles. By 
these facts the writer was convinced that the lipids in non-coagulated 
elementary bodies suspended in a plant sap were expelled from the 
bodies by the addition of ammonium sulphate and that consequently 
protein molecules were enabled to combine directly with one another 
in their expanded state to become doubly refracting. 

The absence of doubly refracting property in the homogeneous 
masses produced by the mere addition of acetic acid may depend on 
the presence of lipids which may allow the protein molecules to par- 
tially shrink or contract in an irregular manner. The homogeneous 
masses without double refraction were actually proved to contain large 
amounts of lipids as does usual protoplasm. When once coagulated, 
animal elementary bodies, unlike plant particles, may hardly solve, if 
any, their coagulation. This may account for their inability to form 
larger homogeneous masses not to speak of crystals by the addition of 
ammonium sulphate. 

If the protoplasm protein of plant cells affected by a virus can be 
obtained in such a crystalline form in which the protein structure to 
act as the virus is retained without being damaged by the elimination 
of lipids, the virus may be said to have been isolated in a crystalline 
form. The crystallinity is therefore by no means a characteristic of 
protoplasm protein having virus action. Crystals were actually obtained 
from healthy leaves as above described. 

Since protoplasm protein is to be endowed with a virus action 
through the structural alteration by the virus, some differences may be 
expected in the crystalline form between ‘‘healthy’’ and ‘‘pathologic’”’ 
crystals so that a crystal peculiar to a virus may be obtained from a 
plant affected by the virus. Bawden and Pirie (26) were able to crys- 
tallize tomato bushy stunt virus as rhombic dodecahedra, whereas we 


IV. THE CRYSTALLINITY OF VIRUSES 61 


could isolate a similar crystal from apparently healthy leaves of tomato 
plant. According to our experiments, such a crystal was obtained only 
from the young leaves in spring, in summer no crystalline mass being 
isolated even from infected leaves. 

There should be a physicochemical association between protoplasm 
protein and lipids, so that the expulsion of lipids may have considerable 
influences upon the protein. When treated with ether at laboratory 
temperature, both vaccinia virus and phage were readily inactivated, 
and Japanese encephalitis virus protein isolated by our method was 
deprived even of the complement binding faculty. Ammonium sulphate 
may cause the expulsion of lipids either by damaging the combination 
between the protein and lipids or by enhancing the direct, mutual com- 
bining force of proteins. Whether the cause may be, there seems little 
doubt that the salt can expel the lipids out of some plant elementary 
bodies. Vaccinia virus or phage particles were immediately inactivated 
by the addition of the salt, showing the strong effect of the salt upon 
the virus protein. The fact that some plant viruses can be isolated in 
a crystalline form by the addition of this salt may therefore indicate 
the remarkable stability of the virus protein. There are, however, 
’ ample evidences, as will be described later, to show that the expulsion 
of lipids may occur during the infection of plants with a virus prior to 
the treatment with ammonium sulphate, which may only promote the 
aggregation of proteins. 

At any rate, the fact that some plant virus proteins can retain 
their action even in a crystalline form, in which no lipids are contained, 
must be of the utmost importance in the consideration of the nature of 
virus action, since this fact shows that no lipid is necessary for the 
virus action. Accordingly, the virus action must be attributed to the 
purely polymerized protein molecules. The virus inactivation on the 
removal of lipids, as already pointed out, may be, therefore, caused by 
some alteration in the protein structure occurring asa result of the 
lipid elimination. Hence, the presence of lipids may be responsible only 
for the maintenance of the characteristic liquid crystalline state of pro- 
toplasm. If protoplasm consisted of proteins polymerized without in- 
termixing lipids and existed in a sort of solid crystalline state, various 
complicated life phenomena would never be revealed. However, since 
the action of a virus consists in the function as a template, lipids are 
never needed for the action, but necessary for the protoplasm to de- 
velop a change responding to the template. Lipids are indispensable 
for producing a replica but not for acting as the template. 

That lipids have no concern with the virus action may be evident 
also from the inconstancy of the lipid contents. Of animal viruses, 
that of papilloma has been claimed to have so little quantity of lipids 


62 II, FUNDAMENTAL STRUCTURE OF PROTOPLASM 


as 1.5 per cent (27), whereas equine encephalitis virus contains so much 
as 50 per cent (28). Usually a considerable quantity of cholesterol is 
present in the lipids of vaccinia virus particles; however, Hoagland et 
al. (29) have shown that cholesterol can be removed from the particles 
by extraction in the cold with ether without affecting virus activity. 
According to our experiments, as above mentioned, inactivation occurred 
if the viruses were treated with ether at a laboratory temperature, 
whereas Harris succeeded in eliminating lipids at —65°C. from des- 
sicated rabies virus with petroleum ether and found that the virus be- 
came stable by this procedure (30). 

It should naturally be expected that viruses become stable on the 
lipid removal, since lipids may render the protein molecules easily 
changeable ; when lipids are removed the protein molecules may be en- 
abled to combine directly to form a stable structure. The remarkably 
stable nature of crystalline viruses may be explained in this respect. A 
virus may, however, become the more estranged from living matter, 
the more the lipids are eliminated, since life can appear only on the 
stage of instability. 


3. The Length of Crystalline Virus Particles 


Some plant viruses are isolated by ultracentrifugation in a form of 
minute rod-shaped particles containing no lipids. Such particles are 
not artefacts, because they can be demonstrated in infected leaves 
themselves, and the sap itself shows the anisotropy of flow. From 
electron micrographs the length of a particle of tobacco-mosaic virus 
was estimated to be about 0.3, and the width about 15 my. Its “‘mo- 
lecular weight’’ was calculated to be 40,000,000 (31). 

It is a noteworthy fact that the particles of various strains of 
tobacco-mosaic virus are proved in electron micrographs to be similar 
in both size and shape regardless of the presence of the profound 
differences in their chemical composition. In discussing this fact Stanley 
stated that despite the differences in composition some general directive 
force appears to be effective during the construction of these virus 
particles (32). Is it really necessary to postulate such a mysterious 
being as a general directive force ? 

The majority of workers considered that there are striking differ- 
ences between plant and animal viruses, since the plant viruses are iso- 
lated in rod-shaped particles consisting of a protein containing no lipid, 
whereas the latter viruses in particles having complex chemical composi- 
tions containing lipids, never being isolated in rod-shaped particles 
unlike the former. It is, however, known that some animal viruses can 


IV. THE CRYSTALLINITY OF VIRUSES 63 


be separated in the same rod-shaped particles. 

Insects are generally affected by a group of viruses and the disease 
is called polyhedrosis. Viruses causing polyhedrosis are claimed to have 
also rod-like shapes similar to some plant viruses. For instance, ac- 
cording to Bergold (33). the virus particles of silk-worm polyhedrosis 
are 288 my in length (tobacco-mosaic virus is believed to have a length 
of 280 mz), and 40 my in width, and consist of a nucleoprotein having 
a high P content ; tobacco-mosaic virus has a similar chemical composi- 
tion as referred to later. 

Such rod-shaped particles composed of nucleoprotein are not only 
found in silk-worm, but also in many other insects infected with poly- 
hedrosis. For example, the virus particles of polyhedrosis of the gyp- 
symoth caterpillar (Porthetria dispar, L.) are 41360 my; western tent 
caterpillar (Malacosoma pluviale, Dyar.), 40350 my; california oakworm 
(Phryganidia california, Pack.), 30X270 my; western yellow-stripped 
armworm (Prodenia praefica, Grote), 50270 mz; spruce budworm 
(Christoneura fumiferana, Clem.), 28260 my (34). 

The term polyhedrosis arises from the peculiar shape of inclusion 
bodies appearing in cells of infected insects and it is stated that such 
polymorphic inclusion bodies, in which viruses are contained, are them- 
selves doubly refracting. 

For what reasons do some viruses exist in such rod-shaped particles 
of a similar length not only in plants but also in insects? Is it true 
that a certain general directive force as Stanley suggested is operating 
in the course of virus production ? 

This fact, however, can readily be explained from the writer’s 
view previously described as regards the protoplasm structure and the 
mechanism of virus reproduction. According to the writer’s view rod- 
shaped particles are no more than the elementary bodies from which 
lipids have been eliminated. Lipid expulsion may occur because of the 
structural change of protoplasm protein caused by a virus, as the pro- 
tein becomes thereby incapable of retaining the lipids. The length of 
the particles, therefore, must be the length of an expanded elementary 
body itself whose length in turn must be the length of protoplasm 
protein molecule in a stretched form. As will be shown later, this 
length appears to have no relation to the presence of nucleic acids. 

If we assume that the molecular weight of protoplasm, like that 
of the ordinary euglobulin, is 150,000 and that the distance between 
the two amino acid residues is 0.34my, and that molecular weight 
around a single residue is 130, the length of a stretched thread-like 
molecule is calculated to be approximately 0.4. This should be the 
length of an expanded elementary body and, accordingly, the length 
of a rod-shaped virus particle. The value thus estimated, however, is 


64 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


a little longer than 0.3 or 280 my, a value regarded as the length of a 
tobacco mosaic virus particle, although some insect viruses have been 
claimed to have a length near 0.4 as described above. 

Since the virus particles observed in electron micrographs are in 
the dry state, some shrinkage should be expected in them. It has been 
shown that protein crystals such as those of oxyhaemoglobin or’ insulin 
undergo shrinkage on the dessication. In methaemoglobin and (-lacto- 
globulin even so remarkable a shrinkage as up to 50 per cent is proved 
(35) (36). 

‘‘Molecular weight’’ of tobacco mosaic virus has been claimed, as 
mentioned above, to be 40,000,000; such a high molecular weight cannot 
be attained unless approximately 200 globulin molecules with molecular 
weight of 150,000 are put together in a bundle even when a consider- 
able amount of nucleic acids is intermingled. The bundles are considered 
to be originated from ‘‘elementary bundles’’, already described, from 
which lipids have been eliminated, not directly from elementary bodies 
themselves which tend to be decomposed into the elementary bundles. 

As stated above, insect virus particles are reported to be in length 
approximately 0.3 1 like some plant viruses, whilst in width they ap- 
pear generally to be about two times as thick as plant viruses. The 
elementary bundles of insects may be said, therefore, to be thicker 
than those of plants, that is, in insects far greater number of protein 
molecules appear to be united to make an elementary bundle than in 
plants. 

According to the kind of cells, protoplasm protein molecules may 
tend to form very long rods or filaments through end-to-end association. 
In such a case, virus particles may have lengths several times as long 
as 0.34. The virus particles of latent mosaic of tomato have been 
claimed to be 525 my in length (33); this may be an example of end- 
to-end combination of two molecules. According to Takahashi (37) 
Brassica nigra virus is about 0.7 uw long; this may also be two molecu- 
lar long. 

On the contrary, if the molecules split into shorter fragments, the 
particle should naturally become shorter. Whereas the length of to- 
bacco-mosaic virus protein is generally believed to be 280my, some 
workers are disinclined to accept the existence of such definite sized 
particle. For example, Crook and Sheffield (38) concluded from electron 
micrographs of tobacco mosaic virus that the particle size varies 
according to the method of preparation, showing no presence of basis 
for assigning a length. Bawden and Pirie (39) stated likewise that the 
average length of the particles in a virus preparation is determined by 
both the past history and present environment of the preparation, and 
that it is a compromise between the forces leading to end-to-end adhe- 


IV. THE CRYSTALLINITY OF VIRUSES 65 


sion and those, such as thermal agitation, that tend to break this ad- 
hesion, breakable rod, when broken, leaving polar ends which can join 
together again. 

As already mentioned, plant protoplasm proteins show a high 
tendency to unite with one another,while at the same time it is known 
that the process of purification causes aggregation of the particles 
initially present in the fresh infective sap. In connection with this 
fact, Bawden (25) stated that since the available evidence on tobacco- 
mosaic virus and virus X suggests that any process that concentrates 
them or that removes contaminations from them is likely to cause 
agglutination, agglutination may be indispensable from a high degree of 
purity, and claims to have isolated homogeneous, unagglutinated par- 
ticles may be equivalent to claiming incomplete purification. At pre- 
sent it seems generally accepted that tobacco-mosaic virus particles can 
occasionally exist in unusually long threads under the electron micros- 
cope. Stahmann ef al. (40) found that Wisconsin pea-streak virus par- 
ticles, on standing in distilled water for several weeks, associated end- 
to-end and side-to-side to produce bundles in parallel alignment which 
were often coiled about each other. 

Alteration in the particle lengths appears to occur not only 7” vitro 
but also in the plant cells 7” sztuw. On an electron microscope study of 
tobacco-mosaic virus extracted from pulp and juice after various periods 
of infection, Takahashi and Rawling (41) found that when the virus is 
extracted from finely macerated pulp and juice at the pH of plant 
juice, the proportion of short particles increases between the 4th and 
16th days after infection. They claimed that the short particles did 
not result from fracture of longer particles during maceration. More- 
over, according to Black et al. (42), electron micrographs of sections 
through tobacco leaves infected with tobacco-mosaic virus sometimes 
showed in the cytoplasm of the diseased cells thread-like filaments much 
longer than 280 my, indicating the natural occurrence of end-to-end 
association of the rods. 

Joly (43) showed by the method of streaming double refraction, 
that the prolonged cooling of tobacco-mosaic virus produces a partially 
reversible shortning of the virus particles. Furthermore, according 
to. Schramm (44), in alkaline buffers, tobacco-mosaic virus particles 
dissociated into smaller components having molecular weight of about 
360,000 as compared with about 40,000,000 for the original intact protein, 
but readjustment of the solution to pH 5 yielded a material practically 
indistinguishable from the original native protein in molecular weight 
and shape, in crystal form and electrophoretic behaviour. 

Thus, there seems no doubt that at least tobacco-mosaic virus par- 
ticles can on the one hand split into smaller particles and on the other 


66 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


hand can form longer threads through end-to-end association. It seems 
possible, however, that thread-like particles of approximately 0.3 4 in 
length are the most common and stable. Knight and Oster (45) claimed 
that the most majority of virus particles (20 to 40 per cent) fall into a 
group size of 15x280 my and less than 5 per cent being extraordinarily 
short or long. Rawling ef al. (46) also published the opinion that the 
majority of particles are about 0.3 in length, the longer particles 
being aggregations of these and the shorter units being due to breakage 
of some of the rods during the drying of the specimen before examina- 
tion in the electron microscope. 


4. Various Shapes of Plant Viruses 


Many plant virus particles appear, as mentioned above, to be rod- 
shaped, but this never holds true for all the plant viruses. It is gene- 
rally accepted that the particles of plant viruses such as those of 
southern bean mosaic, tomato bushy stunt, tobacco necrosis, and turnip 
yellow mosaic, are of globular form having diameters of approximately 
30 my (32) (47). If virus particles are produced from elementary bodies 
of protoplasm on the alteration in its structure by a virus, there should 
be an intimate correlation between the rod-shaped particles and these 
globular ones. 

In the study on the effect of high frequency sound vibrations on 
tobacco-mosaic virus, Oster (48) found that the vibrations break the 
virus rods, reducing the length first to 140myz, then to 70my until at 
the end of 64 minutes of continuous exposure to the vibrations one 
eighth of the original length being the most common. If the molecular 
weight of the unit protein forming the rod is 150,000, then the particles 
having one eighth of the original length will be composed of protein 
fragments whose molecular weight is approximately 20,000; this value 
appears to be close to the molecular weight of Svedberg’s unit, 17,600. 
If protein of certain cells was liable to break down into such units by 
the disturbance in its structure on the infection with a virus and there- 
by if the virus action was retained in these fragments, then the virus 
would be obtained as such extremely minute particles. 

Oster suggested that only particles of 280 my in length were in- 
fectious since a falling off of infectivity seemed concomitant with the 
breaking down of the original rods. However, as discussed in the 
previous Part, even with tobacco-mosaic virus there are evidences that 
the particles broken to a certain extent can retain some infectivity. If 
protoplasm proteins in some plant cells affected by certain viruses other 
than tobacco-mosaic virus were readily decomposable to smaller mole- 


IV. THE CRYSTALLINITY OF VIRUSES 67 


cules and if disturbances, which might be brought about by the decom- 
position in the protein structure concerning the virus action, was in- 
significant, then minute particles produced by the decomposition would 
have the virus action. 

Purified virus particles of both Rattle disease of tobacco and stem- 
mottle disease of potato were found under the electron microscope to 
show two peaks in the distribution curves for particle length, one at 
about 150 mz and the other at about 300 my (49). This can be 
interpreted as indicating that in such a case decomposition to half was 
liable to take place. 

With certatn viruses whose particles are extremely liable to decom- 
pose into fragments, needle-shaped crystals like those of tobacco-mosaic 
virus may fail to develop on the addition of ammonium sulphate. In 
fact, the above mentioned plant viruses of globular particles, such as 
those of southern bean mosaic and tomato bushy stunt, have never been 
isolated in needles, but yielded rather in spherical crystals such as 
rhombic plate, hexagonal prisms, or octahedra. 

The crystal shape may be effected by the degree or the manner 
of decomposition of the rods. Bawden (25) stated that the causes 
which determine the crystal form are unknown, and that if inac- 
tive sap is divided and the two parts purified separately, one some- 
times crystallized in one form and the second in another, and that if 
dodecahedral crystals are dissolved and then recrystallized, bipyramids 
or circular laminae may be produced. According to Markham and 
Smith (50) when turnip yellow mosaic virus: is crystallized from salt 
octahedra are produced, whereas when crystallized from alcohol long 
prisms are yielded. 


CHAPTER V 


FINER STRUCTURE OF VIRUS PARTICLES 
AND ITS SIGNIFICANCE 


1. Decomposition and Fusion of Elementary Bodies 
of Protoplasm 


As mentioned in the previous Chapter, some plant viruses tend to 
reveal themselves in rod-shaped particles or sometimes in extremely 
small globular particles which can be regarded as the fragments of 
the rod. An important characteristic of these particles is that they 
contain no lipids, unlike many other viruses which exist in spherical 
bodies having never yet been obtained in crystalline forms. 

According to our experiments, phage particles or phage-like parti- 
cles isolated from normal bacteria contain a considerable amount of 
lipids, and their average diameter appears to be approximately 0.14 
(24) (9). In this connection, it should be noted that from many plant 
materials particles similar in size and composition to these bacterial 
particles were obtained. The writer was convinced that such parti- 
cles are the coagulated elementary body of protoplasm itself. It can 
easily be calculated that a single rod of tobacco-mosaic virus having 
the size of 15X280 my cannot make such a particle even if it contains 
some lipids, unless about a dozen of the rods are put together. On 
the other hand, since, according to our estimation, the dry weight of 
a single phage particle is of the order of 1X10-%g, the number of 
protein molecules in a single particle is estimated to be of the order 
of 3X10%, and ‘‘molecular weight’’ of a particle of this size is calcu- 
lated to be about 480,000,000, a value which also indicates that about 
a dozen of tobacco-mosaic virus particles whose molecular weight is 
40,000,000 can only form a single particle of the phage. 

For such a reason the writer has formed the opinion that the 
elementary body of protoplasm of certain cells tend to decompose into 
about a dozen of elementary bundles; rod-shaped viruses as above 
mentioned are considered to be such bundles from which lipids have 
been eliminated. 

Thus, several hundreds of extended protoplasm protein molecules 
may associate in parallel alignment to make a bundle, 7. é., an elemen- 
tary bundle, and a dozen of the bundles, in turn, in parallel associa- 
tion form a unit particle, 7. e., an elementary body. The establishment 


V. FINER STRUCTURE OF VIRUS PARTICLES AND ITS SIGNIFICANCE 69 


of such a structure may be achieved by a basic physicochemical pro- 
perty belonging naturally to the protoplasm protein. The writer was 
actually able to confirm 7” vitro that some plant protein of globulin 
nature does possess the property of forming virus-like particles. 
According to his investigation, no particulate protein was contained in 
Merck’s ricin preparation, but when its water solution was added 
with ethanol at 30 per cent, its pH being adjusted to 5.5 by the addi- 
tion of acetic acid, and left in an ice box for several days, then the 
protein molecules in the preparation together with some lipids sedi- 
mented in forming virus-like particles; these particles would further 
unite with one another to form homogeneous protoplasm-like body, if 
they were left at laboratory temperature after being isolated from the 
Original solution by centrifugation and subsequently suspended in a 
weakly acid solution at the isoelectric point. When pressed mechani- 
cally or added with alkali, the homogeneous protoplasm-like body thus 
formed would be decomposed again into virus-like particles (51). 

This important finding strongly suggests that at least some pro- 
teins with globulin nature possess a basic character to polymerize into 
virus-like particles, which in turn tend of fuse into a protoplasm-like 
body. But since the association among the particles in such a body 
may not be firm enough, the body may be disposed to disintegrate 
again into the particles. Elementary bodies and the protoplasm can 
be regarded as such particles and the body, respectively. Since this 
fact is the utmost important and the most fundamental, on the basis 
of which the writer’s theory as regards the protoplasm structure have 
been developed, we shall have occasion later to discuss more in detail 
on this subject. 

Svedberg and Pederson (52) postulated the term proteon to designate 
a native protein unit incapable of dividing into still smaller units 
having the native protein character. According to them, every native 
protein may be regarded as a system of such proteons. The concep- 
tion, that a protein particle is not a mere conglomerate of proteons but 
an orderly aggregate or a polymerization, arose from their finding that 
in many cases, where ultracentrifugal studies show that a protein is 
uniform in size and shape under well defined external conditions, the 
protein in question is capable of dissociating into subunits when the 
environmental conditions are modified. It may perhaps be permissible 
to say, that the formation of a virus-like particle or its decomposition 
into unit molecules is only the manifestation of one of the basic 
characters of proteins. 

Whereas haemocyanin has long been regarded as one of the best 
examples of dissociable complex, Brohult (53) found that its depolyme- 
rization is a function of both pH and salt concentration, the depolyme- 


lat II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


rization increasing as the latter increases. Insulin was also thought 
as a reversibly dissociable system, its unit molecular weight being 
believed to be 6,000 (54). 

An interesting fact was found with insect viruses that the viruses 
are isolated not only in the form of rod-shaped particles as above 
described but also in much thicker rods each of which appears to be 
composed of a number of the usual rods fusing together in parallel align- 
ment. For example, in the case of polyhedrosis of the gypsy-moth 
caterpillar such extremely thick rods as 160X415my. were found in 
addition to the usual rods of 41X360my. In the case of nun-moth 
caterpillar most particles were proved so thick as appearing almost 
globular (34). Such a thick globular rod may be interpreted as the 
elementary body itself from which only lipids have been eliminated. 
The thinner usual rods or needles should be regarded as produced on 
the dissociation of such thick rods. 

These thick rods have proved in general to be much longer than 
the usual thin rods or needles, a fact which may depend upon lesser 
degrees of the shrinkage due to the desiccation owing to their great 
thickness. Williams and Steere (55) have found in electron micro- 
graphs that also tobacco-mosaic virus particles exist in forming thick 
bundles in the juices from infected tobacco leaves; if the specimens 
are washed with one or two drops of distilled water before drying 


and shadowing with uranium, the bundles disintergrate into single 
needles. 


2. Virus Particles Containing Lipids 


As above discussed, some plant virus particles or rods can be 
regarded as elementary bundles whose structure has been altered by 
a virus and from which subsequently lipids have been expelled. The 
formation of similar particles may not be impossible if adequate physi- 
cal or chemical effects other than viruses, which may be able to cause 
some disturbances in the protoplasm structure to expel the lipids, 
are applied to the protoplasm. 

It is, however, generally believed that no virus-like rods can be 
isolated from normal plant tissues. Nevertheless, according to our in- 
vestigation as shown already minute particles with double refraction 
could be obtained from various normal plant materials when the saps 
were treated with ammonium sulphate. It was uncertain whether or 
not such particles having double refraction were aggregates of the rods, 
but there seemed no doubt that the lipids could be expelled by the 
action of ammonium sulphate, since the particles contained no lipids. 


V. FINER STRUCTURE OF VIRUS PARTICLES AND ITS SIGNIFICANCE 71 


It appears to the writer rather strange that virus-like rods are 
generally believed to be never produced without virus, although we 
are unable to determine whether or not adequate stimuli other than 
a virus can give rise to the production of virus-like rods in plant 
tissues, aS we unfortunately have neither the electron microscope nor 
ultracentrifuge. The above mentioned fact, however, that particles 
or bodies with double refraction are produced by the addition of 
ammonium sulphate suggests the possibility of rod formation without 
virus. 

Yamafuji and his collaborators (56) claimed that they could pro- 
duce tobacco-mosaic and polyhedrosis virus in plant leaves and silk- 
worms respectively by application of such chemicals as H,0, or 
hydroxylamine. It is a question whether or not viruses themselves 
were actually produced, but at least it seems certain that some che- 
micals could cause the expulsion of lipids to form virus-like bodies or 
aggregates. They attempted to connect oxidation process with this 
apparent virus formation, but it should be remembered that oxidizing 
agents, in general, exhibit profound effects upon protoplasm structure 
and that the general mutagenic activity of peroxide in microorganisms 
seems fairly established (57). Mutagenic activity of some agents can 
be considered to be based upon their ability to produce a structural 
change in nucleoproteins in protoplasm as discussed later. Therefore, 
if some agents could raise, like viruses, certain changes in protoplasm 
protein to break the combination of the protein with lipids, virus-like 
rods containing no lipids would be produced. According to a more 
recent report by Yamafuji ef al. (57a) polyhedral virus crystals formed 
chemically in silk-worm larvae exhibit the same electrophoretic pattern 
as those produced naturally by infection. 

It seems to be believed by a number of workers that certain plant 
viruses, at least tobacco-mosaic viruses, are rod-shaped particles, and 
that such reds or particles are the only feature of the viruses. How- 
ever, the writer holds the opinion that the virus particles without 
lipids are rather an exceptional existence even in tobacco-mosaic 
viruses, but that like many animal viruses these plant viruses usually 
exist in particles containing lipids. If the combination with the lipids 
was not broken by the virus infection, elementary bodies would coa- 
gulate in their natural state even in the plant cells and would come 
out as virus particles. 

We failed to obtain any particles or aggregates having double re- 
fraction from infected tomato leaves in summer even when ammonium 
sulphate was applied, but could isolate in abundance only particles 
containing considerable quantities of lipids as in the case of animal 
viruses. Bawden (25) stated that the yield of plant viruses varies 


72 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


remarkably according to the season where the expriments are carried 
out. For example, the yield of tomato busy stunt virus in summer is 
only about one fifteenth of that in winter; with tobacco necrosis this 
is found to be only about one twentieth. This fact may indicate that 
in summer the combination with lipids is so firm that their expulsion 
is hardly possible. : 

According to Nixon and Watson (58) the sap from sugar beat 
plants infected with beat yellow virus contains at least two kinds of 
specific particles, and one of these is the rod which can be seen with 
the electron microscope, the other probably occurring in a much 
’ higher concentration, appearing to be spherical. They stated that pos- 
sibly the rods are an alternative form of this spherical particles, 
which can be found in large quantities in clarified sap from healthy 
plants. This finding is what should be expected from the writer’s 
view just mentioned. Furthermore, on an electron microscopic study 
of tobacco-mosaic virus and some 35 other viruses, Johnson (59) found 
some rod-like particles with tobacco-mosaic virus and with several 
other viruses, but failed to find any rod in another large group of 
virus-infected plants. He suggested that the rod-like particles in 
question may be a result of disease rather than the ultimate causal 
unit. 

It should be noted in this connection that only extremely small 
quantities of virus particles are usually yielded from infected plant 
saps. For example, the yield of tobacco-necrosis virus particles from 
a litre of infective bean’s sap is stated to be only 0.001g.; the yield 
of tobacco-mosaic virus proteins from infective tobacco plant sap 
appears to be the highest, but only 2.0g. (25). ' 

Wildman and Bonner (60), on an electrophoretic study of plant 
virus proteins, found that a distinct fraction appeared in the sap 
following the infection of plants with tobacco-mosaic virus; the frac- 
tion was detectable after 4 days of infection, and after 20 days became 
very conspicuously. If the expulsion of lipids took place in the pro- 
toplasm proteins, there would appear a peculiar fraction detectable 
in the electrophoretic pattern, and therefore if the lipids expulsion 
failed to occur, the detection of virus formation would not be accom- 
plished by this method. Actually in the sap of Turkish tobacco in- 
fected with curly-top virus no distinct fraction was demonstrated in 
the pattern; it should be realized that this virus has not ever been 
isolated in a crystalline form, indicating that lipid-expulsion cannot 
occur with this virus. 

Insect viruses causing polyhedrosis can be isolated on the one hand, 
as described in a previous chapter, in rods similar to those of some plant 
viruses, whereas on the other hand, it has been reported that there 


V. FINER STRUCTURE OF VIRUS PARTICLES AND ITS SIGNIFICANCE 73 


are remarkable differences between the virus isolated from polyhedral 
bodies and that from the blood. For instance, polyhedrosis virus 
having a rod-shape isolated by Bergold (33) from polyhedral bodies 
was sedimented completely by ultracentrifugation at 10,000 r.p.m. and 
its ‘‘molecular weight’’ was calculated to be 916X10°, while the virus 
particles isolated by Glaser and Stanley (61) from the infected insect 
blood was not sedimented at 10,000 r.p.m. but only at 27,000 r.p.m. and 
its ‘‘molecular weight’’ was estimated to be about 3,000,000. Moreover, 
the virus from the blood was readily inactivated in solutions of more 
acid than pH 5, whilst rod-shaped virus from polyhedral bodies was 
so stable as to stand unchanged for 24 hours at pH 2.0; and again no 
rod-shaped particles similar to the virus was found in normal in- 
sects, whilst globular particles similar to those of the virus isolated 
from the blood were obtained from normal ones (62). These facts 
strongly suggest that the particles from the blood are nothing but the 
coagulated elementary bodies containing lipids, just as in usual animal 
viruses. 

The expulsion of lipids appears to occur not only immediately 
after or during the change of the protoplasm protein by virus infec- 
tion, but sometimes may occur gradually even after the change. The 
particles present in plant saps infected with tobacco-mosaic virus were 
‘separated into various fractions by Bawden and Pirie (63). The most 
rapidly sedimenting fractions consisted mostly of rod-shaped nucleo- 
protein, whilst only about a half of the total material in the most 
slowly sedimenting fraction was virus nucleoproteins; these fractions 
showed no anisotropy of flow and had low infectivity. Electron mic- 
rographs of the most slowly sedimenting fractions showed particles 
most of which are little longer than they were wide, while with 
increasing sedimentation rate the numbers of obvious rods increased. 
All the fractions were unstable and rapidly passed into forms that 
sedimented rapidly, showed intense anisotropy of flow, the change 
being most striking in the preparations that previously sedimented 
most slowly. Electron micrographs showed that these changes were 
accompanied by an increase in the number of rods and in their average 
length. 

This fact can be interpreted as the gradual occurrence of lipid 
expulsion 7” vitro. Since the formation of rod-like particles never 
means the generation of viruses, it is only natural that no increase in 
the infectivity was observed despite the increase in the number 
of rods. 

Bawden (26) cited another interesting fact: When concentrated 
salt-free solutions of a tobacco-necrosis virus are kept at 0°C, crystal- 
lization occurs and thick triclinic prisms and the hexagonal, or pseudo- 


74 -. JI. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


hexagonal, plates are produced. These begin to form after a few 
days, but growth continues for months and will give rise to single 
crystals several milimeters long. In this case the preparations lost 
most of their infectivity by the time crystallisation was completed, 
indicating the occurrence of some change in the protein structure, 
during the lipid expulsion, unfavourable for the virus activity. As 
previously discussed, the expulsion of lipids may be caused by changes 
of the protein structure generally unfavorable for the virus acti- 
vity, though the virus may become more stable after the expulsion. 
In the above example of tobacco-mosaic virus, it has also been 
stated that the formation of rods was never followed by the in- 
crease in infectivity, but on the contrary by the reduction by about 
one-half. 

The presence of an intimate correlation between the viruses and 
such rods or crystals could be demonstrated by the fact that both 
rods and crystals had the serological property specific to the respec- 
tive virus. In general, the serological property of a virus is more 
stable than the infectivity, so that in the case above cited only the 
infectitivity seems to be lost, the serological property being left 
infact. 

In the writer’s opinion (22), virus particles having the property of 
the coagulated elementary bodies, if the coagulation is complete, fail 
to show the agglutinability by their antibody, because of the disap- 
pearance of the active polar groups on the folding of the protein 
molecules through the coagulation. However, if the protein molecules 
are unfolded and expanded, the reactive groups will reappear with the 
recovery of the agglutinability. Such an unfolding results from the 
expulsion of lipids, and as a consequence the rod formation is accom- 
panied by the appearance of the agglutinability, whereas since serolo- 
gical property of plant viruses is usually recognized by the presence 
of the agglutinability, the appearance of the property is occasionally 
thought even to be the production of the virus protein itself, thus 
making the matter fall into the most confusion. It is stated that the 
formation of rods, in the above cited case of tomato-mosaic virus pre- 
parations, was actually accompanied by the revelation of the serological 
property. 


3. The Size of Virus Particles as Coagulated : 
Elementary Bodies 


Among the virus particles isolated by our method those of phage 
are found to be the smallest and the dry particles are estimated to 


V. FINER STRUCTURE OF VIRUS PARTICLES AND ITS SIGNIFICANCE 75 


have the diameter of approximately 0.14; normal bacterial particles 
are also of this size. 

Particles obtained from various normal and infected plant material 
appears in the main to be of the same order of size, although their 
sizes seem far more inhomogeneous than those of phage, whilst animal 
particles are generally larger and their diameters appear mostly of the 
order of 0.2. Vaccinia virus recoverable from skin tissues is found 
to be the largest, its diameter being estimated to be about 0.3 y (7). 

From these facts the writer has formed the opinion that the size 
of the coagulated elementary body, corresponding to the plant-virus 
rods having the length of 0.3 4, may be about 0.1y, in diameter. The 
diameter of various phage particles in electron micrographs haye been 
reported by a number of workers to be little less than 0.1y4. This 
may be the size of a single elementary body fully coagulated in the 
dry state. Thus, an elementary body may be considered to undergo a 
considerable shrinkage, thereby being reduced in length to about one 
third to become a body witha diameter of the order of 0.1y, although 
in the expanded state it is about 0.3 in length. 

If the property of elementary bodies varied according to the kind 
of cells, some tending to combine end-to-end with every two bodies 
while the others with every three bodies, and if side-by-side combina- 
tion corresponding to the end-to-end combination occurred on their 
coagulation into cuboidal bodies, then the diameters of the coagulated 
bodies would be about 0.2” and 0.3, respectively; but when side-by- 
side combination failed to occur in response to the end-to-end associa 
tion, various intermediately sized particles would result. 

Molluscum contagiosum virus particles have been found by Rake 
and Black (64) to be present plentifully in the inclusion bodies; under 
the electron microscope they proved to be brick shaped and 389272myz 
in size, consisting of unit particles of 100X83my. According to the 
writer’s view these unit particles must be the coagulated element- 
ary bodies. Many other examples can be cited to show that viruses 
of large particles appear to be composed of smaller units like this 
virus. 

If such a view is legitimate, particles smaller than 0.1 must be 
the decomposition products of the unit particle. When coagulation 
occurred after the elementary body had been disintegrated into ele- 
mentary bundles which could in turn undergo further decomposition, 
various extremely small particles would be yielded. 

’ As already pointed out, phage particles isolated by our method 
are not of a uniform size and when the particles are the smaller, 
they are the more unstable and at the same time the activity is the 
lesser ; this would only be a natural result if the virus activity arose 


76 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


from the polymerization of the protein molecules. Rothen (65) studied 
the influence of a-particles on the antigenic property of bovine albumin 
and found that the albumin monolayer, when subjected to bombard- 
ment of a-particles from a polonium source, completely lost in less 
than 30 minutes its property of combining with homologous antibody, 
while with a double layer 45 minutes were needed, and with three 
double layers or six monolayers even 135 minutes were not sufficient 
to inactivate the property. 


CHAPTER VI 
THE SHAPE OF VIRUS PARTICLES 


1. Tailed and Filamentous Virus Particles 


It is a well known fact that somé phage has a tail like a tadpole. 
If an elementary bundle in an elementary body failed to shrink, while 
the other remaining bundles coagulated into a body, a virus particle 
having a tail would be produced. The inability of shrinking of a 
certain elementary bundle may be attributed to its peculiar chemical 
composition; if such a peculiar composition occurred in a certain 
bundle of the body following the infection with some strain of the 
virus, each virus particle produced would have a tail. 

Since the structural pattern induced in the protoplasm-protein by 
a virus is governed by the kind of virus, it should naturally occur 
that phages of some kind have a tail while others not. Not only 
the production of a tail but also the degree of the shrinkage or that 
of the decomposition or of the association of elementary bodies should 
more or less be influenced by the pattern of the virus, so that particle 
size would be subjected to a certain extent to the kind of viruses even 
when the cells affected are of a similar type, although the size is 
chiefly determined, as already stated, by the cells from which the 
virus is produced. According to our study (66), the particles of vac- 
cinia virus recovered from infected rabbit testicles are a little smaller 
than the normal particles from noninfected testicles. 

Glaser and Wyckoff (62) stated that normal particles from silk- 
worms are rather more inhomogeneous in the sedimentation rate than 
the virus particles isolated from the worms infected with polyhedrosis. 
This may indicate that the elementary bodies of the silk-worm, when 
affected by the virus, tend to decompose more extensively than the 
normal bodies. Isolation of tailed particles from normal bacteria 
seems to have never been reported. This may show that the occur- 
rence of the peculiar structural change causing inability of shrinking 
in a certain elementary bundle leading to the tail production cannot 
be induced without certain phages. 

It has been reported that in the presence of proflavine bacterial 
cells infected with phage underwent lysis without producing active 
phage particles (67). The bacteria which usually produced tailed par- 
ticles would yield, in the presence of proflavine, lysates consisting 


78 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


mostly of tailless elements though often a small number of tailed par- 
ticles were contained. The tailless particles would not be adsorbed 
by phage sensitive bacteria. This shows that in these particles the 
virus pattern was either absent or incomplete. The presence of tail 
may indicate the existence of complete virus pattern. An evidence 
was shown to suggest that nucleic acid content, if any, in these tail- 
less incomplete particles was much lower than that of complete active 
particles. The tail formation may thus be attributed to high nucleic 
acid content which may render some elementary bundle unable to 
shrink. 

As is well known, tailed particles are never confined to phage. 
For example, Newcastle disease viruses sometimes prove to have a 
tail, though more occasionally they are found in filamentous forms, 
which are believed by the writer to be elementary bundles or elemen- 
tary bodies in an unfolded state. If some elementary bundle was left 
in this unfolded state, whilst the others formed a coagulated body on 
folding or shrinking, a tail would be produced as in the case of phage. 
According to Bang (68) Newcastle disease virus becomes filamentous 
when brought into hypertonic solutions of sodium chloride, whilst 
becomes spherical in water or 0.8 per cent salt solution. It is worthy 
of note that this change is reversible; no loss of infectivity is detected 
during the change. 

Poliomyelitis virus may present another example: Bourdillon (69) 
claimed that electron micrographs of this virus show filaments of 
15my wide, purified preparations readily agglutinating lineally and 
showing anisotropy of flow. In these and other respects they resem- 
ble closely some plant virus preparations, though filaments of animal 
viruses seem far more tender than those of plant viruses, a fact which 
may chiefly be attributed to the presence of lipids. There are also 
many reports informing of the filamentous forms of influenza virus. 
Avian erythromyeloblastic leucosis virus is likewise stated to be some- 
times tailed particles (70). Studies of dengue fever virus by electron 
mircoscope has shown that the virus is thread-like particles, 42 to 
46 my in width and 175 to 220 my in length (71). 

Filaments of viruses such as those of Newcastle disease and 
polyomyelitis may be the unfolded elementary bundles or their end-to- 
end association products, since their width is reported to be similar to 
that of tobacco-mosaic virus, whereas, in some cases at least, this does 
not seem to hold for influenza virus, because electron micrographs 
of influenza virus show that the filaments are far thicker in width 
than those of the former viruses, seggesting that the filaments of 
influenza virus are the elementary bodies themselves coagulated in 
forming a thread by end-to-end association. Actually many workers 


VI. THE SHAPE OF VIRUS PARTICLES a2 


believe that the filaments can undergo segmentation into virus par- 
ticles (72). (73). 

If protoplasm has a structure just as the writer postulated, it will 
only naturally follow that viruses have such various forms. Although 
a phage is customarily regarded as having a definite size and shape, 
a variety of irregular and incomplete forms was shown in the 
untreated bacterial cultures lysed by a phage, and even filaments were 
found in a culture obtained at a low temperature (74). Observations 
made by Chapman (75) in electron micrographs of a certain strain of 
phage revealed various shapes of phage or phage-like particles. includ- 
ing not only the familiar type of a body with a single tail, but also 
dumbbell-shaped particles with or without tails, or various shaped 
bodies with one or more tails at each end, as well as the filaments or 
rod-shaped particles. 

Hoyle (76), in his study of chorio-allantoic membrane of chick 
embryo infected with influenza virus by means of dark-field optical 
microscopy, suggested that elementary infectious units of influenza 
may be cellular fragments. On the other hand, Wyckoff (77) showed 
in electron micrographs of the same materials similarly extruded and 
detached bits of cytoplasm mainly consisting of filaments which in 
turn tends to segregate into spheres. These views and observations 
are of course consistent with the writer’s findings and conceptions. 

If host cells are not injured and broken up following virus infec- 
tion, but can remain in their unchanged, complete forms, then the 
coagulated elementary bodies, which may be able to act as separate 
virus particles if freed from the cells, must exist in the protoplasm 
in a regular arrangement, because the protoplasm consists of elemen- 
tary bodies so arranged. It has actually been found in electron mic- 
rographs that virus or virus-like particles are arranged regularly in a 
crystal pattern in bacterial or some plant cells affected by respective 
viruses (78). In a similar way Straus (79) showed that virus-like par- 
ticles in a equally regular arrangement are present in protoplasm of 
papilloma cells. 

Particles of various shapes are found also in normal cells without 
concerning any virus infection (80), indicating that protoplasm protein, 
in general, has the property to change its shape in various ways. 

Wyckoff (81), in his study by electron micrographs on the forma- 
tion of the particles of influenza virus, has confirmed that there is no 
essential morphological difference between particles arising from 
“healthy”’ cells and virus particles coming from cells infected with 
influenza. They both resemble bits of cellular cytoplasm rather than 
extraneous objects growing and multiplying at the expense of the 
cell. He stated that, if influenza virus particles are fragments of a 


80 FUNDAMENTAL STRUCTURE OF PROTOPLASM 


diseased cell, as electron micrographs seem to suggest, it becomes 
hard to view them as organized invaders which multiplied within and 
at the expense of the diseased cell. 

Besides myosin, a second protein, called actin, is extracted from 
muscles; this protein exists in a water solution in globular forms but 
salt in high concentrations converts the particulate actin into a fibrous 
form, which in turn is reverted to the globular form by removing 
the salt by dialysis (82). Entirely the same phenomenon is observed 
with Newcastle disease virus as stated above. Likewise with insulin 
an analogous finding has been reported; namely, this protein can be 
converted into filaments with a width about 15 my like tobacco-mosaic 
virus, and this filaments again can aggregate into globular forms (83). 
Lundgren (84) considered that filaments are produced by the unfolding 
of globular proteins and the aligning of polypeptide chains. In addi- 
tion, electron micrographs of myosin, actomyosin, tropomyosin, and 
fibrin show that all these proteins have the common property to give 
fibrils of varying length and thickness (85). 

The concept of the writer described thus far as regards the shape 
and size of viruses is diagrammatically shown in Fig. 11. 

The ability of globular proteins to spread in the form of fibrils 
or filaments is also fairly shown in the readiness with which proteins 
spread and form the so-called surface films; the spreading is especially 
easily done in concentrated salt solutions and the film can be com- 
pressed immediately after spreading and occupy the same area at the 
point of minimum compressibility. Again, a change of potein called 
denaturation is thought generally as an unfolding of globular proteins 
into polypeptide chains. For example, it is reported that ovalbumin 
when denaturated in the presence of urea becomes 500 to 700A in 
length (86). 

An adequate system may be provided in the protoplasm for the 
readily occurrence of such a reversible change, 7. e. stretching and 
shrinking, of the proteins. The majority of workers seem to pay 
attention mainly to the elementary bodies in a stretching state, forming 
the opinion that the protoplasm consists of fibrils, whilst others may 
attach importance to the elementary bodies in a coagulated state, ad- 
vancing the globular theories of protoplasm. Usually viruses are in 
a coagulated state, but under certain environmental conditions they 
may stretch themselves to form filaments. 

Sharp (87) has made studies on the ‘“‘purified’’ virus of avian 
erythro-myeloblastic leucosis under varying conditions of electron 
micrography, and found that when preparations of the virus were 
allowed to dry in the presence of salt, the particles exhibited extreme 
pleomorphism, varying from filaments to sperm shapes, while the 


VI. THE SHAPE OF VIRUS PARTICLES 81 


particles of the virus dried without salt were uniformly spheroidal, 
though variable in size, the average diameter being about 120my. On 
this finding he suggested that the true form of the virus is spheroidal, 
the others being artefacts. Similar assumptions have been postulated 


+ 0.34 > 


Elementary 
body — 


Large sized virus particles such as 
vaccinia virus isolated from skin 
tissues. 


Middle sized virus particles such as 
vaccinia virus isolated from testi- 
cles. 


Phage and other small size parti- 
cles. 


Tailed particles. 


ae eee! Thick filaments as seen with 
ap ose at ae influenza virus. 


= 0.34 — 


Elementary 


bundle 
— — |Splitted—~- Zw & 
= = = elementary t 
=o S225C5 bundle 
& 
= 
& 
_— 


rare 
— wae, 


Elementary bundle from which lipids 
have been eliminated. Tobacco-mo- 
saic virus, etc. 


Splitted elementary bundles from 
which lipids have been eliminated. 
Southern been mosaic, tomato bushy 
stunt virus, étc. 


Coagulation of elementary bundle or 
splitted elementary bundle without 
lipid elimination. Extremely small 
virus particles. 


Reversibly coagulable virus parti- 
cles, such as Newcastle and polio- 
myelitis virus. 


Fig. 11. Diagram of various shapes and sizes of virus particles. 


82 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


also by a number of other workers with other viruses, but such views 
would of course be improper. On the contrary, Chu et al. (88) consi- 
dered the filamentous forms of influenza virus as a stage in the multi- 
plication of the virus, the spheroid possibly representing the mature 
“‘resting’’ virus, which lengthens in the host cell to a filamentous 
entity and fragments transversely to form the spheroid again, a view 
which may also be unreasonable. 


2. Particles Elusive in Electron Micrography 


Angulo and his collaborators (89) examined various materials from 
a patient with tropical treponematosis, a non-viral disease, with elec- 
tron microscope and found particles of filamentous forms resembling 
the virus of influenza and Newcastle disease as well as various globular 
paticles like those of influenza, poliomeylitis or pox group. Moreover, 
Murphy and Bang (90) have shown in electron micrographs that in a 
normal chick chorio-allantoic membrane cell ‘‘normal’’ filaments are 
seen everywhere. They demonstrated how much normal filaments 
resemble those of influenza. If the nature of virus particles is actually 
as hitherto discussed, such findings will be only natural. Neverthe- 
less, rather strange to say, it seems commonly believed that particles 
peculiar to viruses are proved only with virus-infected tissues, the 


Fig. 12. Virus-like particles in a red blood cell. Blood cell is photo- 
graphed in Ringer’s solution. Some particles appear to have a tail 
like a tadpole. 


VI. THE SHAPE OF VIRUS PARTICLES 83 


main reason for which is descussed in this section. The reason for 
the production of virus particles having peculiar shape and size cor- 
responding to a given virus was considered already. 

According to our dark-field microscopic observation, the proto- 
plasm of red blood cells also consists of virus-like particles, and whose 
diameter was estimated to be about 0.2 (91). These particles, however, 
fail to reveal their true features in electron micrographs unless exa- 
mined under proper conditions. The writer had the fortunate oppor- 
tunity to investigate a great number of electron micrographs of red 
blood cells taken by members of the physiological laboratory in Yoko- 
hama Medical College and was able to confirm that the particles were 
revealed if Ringer’s solution was used as indicated in Fig. 12. But 
when physiological saline solution was used no particles were shown 
in the micrographs as indicated in Fig. 13. Even when Ringer’s solu- 


Fig. 13. A red blood cell photographed in physiological saline. No 
virus-like particles are seen. 


tion was used, if a little deleterious manipulation was added to the 
cells, the particles became much smaller as shown in Fig. 14. 

We noticed that samples of a certain unstable phage were occasion- 
ally severely inactivated during the isolating procedure by our method 
when physiological saline solution or distilled water was used, while 
this was prevented by Ringer’s solution (91). This may be attributed 
to the divalent cations chiefly those of calcium present in Ringer’s 
solution. A.dams (92) stated that the addition of 107° M. concentration 
of divalent cations, such as Ca or Mg, markedly reduces the rate of 


84 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


inactivation of a certain strain of phage. Moreover, according to Sharp 
et al. (93) the equine encephalomyelitis virus, dried from suspensions 
of low salt content or washed on the film with water, appears in 


SAE BLK Ee WR Se 
bahar aes: 7 A 
Sk we 


Pd 


Kee 
Os 


Fig. 14. Virus-like particles of extremely small size in a red blood 
cell. Partially hemolysed cell by hypotonic saline solution is 
photographed in Ringer’s solution. 


electron micrographs as round images with low contrast, whilst the 
contrast increases greatly in the presence of calcium ions. Also with 
images of influenza virus, it was noted that the periphery becomes 
well defined in the presence of small amounts of calcium. 

* Thus it is thought that elementary bodies, whether they have a 
virus action or not, are liable to be disintegrated or become elusive in 
the electron micrographs, a tendency which can be prevented by the 
presence of divalent cations. The divalent ions in these cases may 
stabilize the polymerization of proteins, thereby viruses can be saved 
from inactivation. 

“ However, if it is true that only virus particles can be caught in 
the micrographs, whilst normal particles cannot, then it would be attri- 
buted to some elementary bodies which have become stable enough by 
virus infection to stand the manipulation of the micrography because 
of an alteration in the chemical structure due to the virus. Usually 
cells may multiply more rapidly when affected by a virus, and since 
the enhancement in the multiplicability appears to be accompanied by 
the increase in the nucleic acid content in the cell, some elementary 
bodies would come to contain more nucleic acid following virus infec- 
tion. Presumably this is the main reason for which “‘virus particles”’ 


VI. THE SHAPE OF VIRUS PARTICLE 85 


are commonly caught well in micrographs. 

On the one hand, it seems generally accepted that nucleic acid 
contents are increased when protein synthesis is to develop vigorously 
for the rapid multiplication of the cells; on the other hand, there is 
a good reason to consider, as will be detailed in a later chapter, that 
nucleic acids are capable of stabilizing the structure of protein poly- 
mers and thereby the character of elementary bodies for acting as a 
virus is intensified. The production of a tail in the phage particle 
may also be attributed to the high content of nucleic acid, which may 
cause some proteins to be left in a stretched form. 

As already mentioned, virus-like particles aré actually absent in 
the normal skin tissue, although virus particles may develop in it on 
the infection with a certain virus. Virus-like particles can be produced, 
however, in the skin tissue when a proper stimulus other than viruses 
is applied. Therefore, even when particles never present in normal 
tissues are found in some pathological tissues, it cannot be said that 
the particles have a definite connection with viruses. The success of 
Angulo et al. referred to above, in demonstrating various virus-like 
particles may be attributed to their investigation with cells undergoing 
a change by the infection with the microorganism which may develop 
a change like that induced by a virus. 

Epestein ef al. (94) have reported that between 24 and 72 hours 
after inoculation of the virus of foot-and-mouth disease into guinea 
pigs, within which interval generalization of the disease reached its 
height and the red blood cells were found infective for normal guinea 
pigs, particles with mean diameter of 246 my were found in the red 
cells by the electron microscope. They claimed that the particles were 
absent in normal control material. However, their particles appear 
to be similar in size to those indicated in Fig. 12. If there was no 
mistake in their demonstration of the particles solely during certain 
periods after the infection, it should be interpreted as that the parti- 
cles become during the periods stable enough to reveal their complete 
figures under the condition of their examination. 

According to electron microscopic studies of Wyckoff (2) the pro- 
toplasm of young actively growing cells of E. coli does not contain 
filaments or particles but old cells commonly have the spherical macro- 
molecules embedded in a matrix of filaments. There are many good 
reasons to believe that the structure of proteins becomes, as a rule, 
more stable and rigid as they grow older, a full account of which 
will be given later in Part V. 

Under the electron microscope, materials to be examined must be 
brought into an unusual dried state because of the vacuum, and at the 
same time must be exposed to a high temperature by the bombard- 


86 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


ment of electrons, so that particles have to stand this hardship to 
show their true figure without being decomposed. 

If the particles are disintegrated to fragments of a size beyond 
the resolving power of the electron microscope, nothing will be photo- 
graphed as in the case of blood cells shown in Fig. 13. However, if 
fragments remain within the limit of resolution, their demonstration 
under the electron microscope may be possible as indicated in Fig. 14. 
In such a case virus particles may be thought to be extremely small. 
Furthermore, it may be possible that the particles present in the cell 
surface are sometimes fused into a homogeneous membrane-like mass, 
wherefore the so-called cell-membrane may be photographed (95). 
Some workers claimed that even virus particles occasionally show a 
membrane-like structure on the surface, which may likewise be a 
dissolution product of protein molecules present on the surface. 


CHAPTER VII 
THE MODE OF VIRUS MULTIPLICATION 


1. Multiplication of Phage within Bacterial 
Cell Protoplasm 


Many facts above cited have provided the basis on which the 
writer has formed the theory as regards the structure of protoplasm; 
meanwhile it may be said that these facts are supporting the theory 
strongly, since they can be explained finely by the theory. On the other 
hand, a vast number of facts have also been known concerning the 
multiplication of viruses. Now, it must be mentioned how these facts 
are to be interpreted by the writer’s theory. 

Phage seems to be the virus with which the most profound and 
most numerous investigations have ever been made, whereas some con- 
clusions reached as to its multiplication by a number of workers ap- 
pear to be inconsistent with what is expected from the theory above 
mentioned. 

According to the writer’s opinion, viruses are produced by the 
rearrangement of polar groups in protoplasm protein, and therefore it 
must be impossible that viruses are synthesized directly without con- 
cerning protoplasm protein. Nevertheless, some workers reached a 
remarkable conclusion that phage could be formed directly from 
components in the culture medium. For instance, a series of workers 
in this field, such as Cohen and Putnam (96) (97), concluded that the 
majority of elements in phage, such as phosphorus, were directly ori- 
ginated from those contained in the culture medium. According to 
Kozloff (98) about 75 to 95 per cent of phage protein nitrogen came 
from the medium after infection, while remaining 5 to 25 per cent 
were provided by bacteria. The writer is convinced, however, that 
these conclusions are entirely incorrect ; the reasons are given in the 
following. 

There are many evidences that only young bacteria are affected 
by phage, aged bacteria being commonly insusceptible, and thus phage 
can multiply, as a rule, only in young cells. First, the erroneous 
conclusions might be reached because of neglecting of this fact; namely 
since phage was produced by young growing bacteria which were 
rapidly synthesizing the protoplasm from the components in the 
culture media, the phage would be mistaken as being formed directly 


88 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


from the components. 

The property of preferably affecting young hosts is not confined 
to phage, but it seems common with other viruses. For example, as is 
well known, rapidly developing plants are liable to be readily infected 
by virus diseases, while aged leaves having ceased growing are seldom 
infected. The fact that the chick embryo is used for cultivation of a 
great number of viruses may be mainly due to its juvenile character, 
for as Woodruff and Goodpasture (99) have suggested, although many 
viruses multiply in the chick embryo, they fail to grow in the adult 
chicken. 

In order to obtain effectively large amounts of phage, we made a 
small number of bacteria multiply in the culture medium with a proper 
amount of phage. During the multiplication of bacteria in the pre- 
sence of phage, newly produced protoplasm are imparted with the struc- 
ture of phage. If phage is added to the culture of bacteria which 
have already ceased to grow, usually no phage develops. Again, if 
bacteria are spread with a small number of phage particles on agar 
plate, a number of minute round areas, in which no bacterial growth 
is occurring are produced, which correspond to each particle present 
at first. The phage present at first would multiply by affecting and 
lysing the surrounding bacteria to which it is attached and the newly 
produced phage in turn would affect the surrounding bacteria in a chain 
reaction to increase the clearing area. The area or plaque rapidly in- 
creases in size when bacteria are multiplying, but when their growth 
stops the increase also comes to a halt, indicating that growing cells 
only are affected and lysed by phage. When bacteria cease growing 
and become aged, the phage fails to affect and to lyse them, so that 
no further increase occurs in plaque size (100). 

Fong (101) claimed that the phage-producing ability of streptococci 
is activated when the cocci are cultivated in a tavourable environ- 
ment for a rapid growth. Such activated cells, preserved at 5°C. in 
Lock’s solution, are able to produce phage abundantly and rapidly 
when affected by phage, but this phage-producing capacity is quickly 
lost when the cells are suspended in distilled water and are exposed 
to 44°C. for 15 minutes. The activated state in this example must be 
the ‘‘young’’ state, and this state can be kept unchanged at the low 
temperature in Lock’s solution, but the cells may grow “old”’ rapidly 
when exposed to 44°C. in distilled water. Kopper (102) has actually 
found certain outstanding difference between old and young cells of 
colon bacteria. According to him, the transformation of old into young 
cells takes place during the lag phase of growth and necessarily in- 
volves a great number of physiological changes. The rejuvenating 
effect was most pronounced with glucose, but also was noted with a 


VII. THE MODE OF VIRUS MULTIPLICATION 89 


number of salts of carboxylic acids, peptone, and culture broths. 
Mixtures of equal parts of NaCl and KCL and low concentrations of 
Na azide and 2.4-dinitrophenol, respectively, were found to exert a 
pronounced inhibitory effect in this process (103). 

Thus young growing cells seem to have peculiar protoplasm 
structure which is fitted for phage production, and accordingly if 
phage was present in bacterial cultures growing rapidly, as soon as 
the protoplasm proteins were synthesized from the components in the 
culture medium, the newly formed proteins would be changed into the 
phage proteins by the rearrangement of the polar groups, so that the 
components would seem to be converted directly into phage. 

On the other hand, it is believed that bacteria are deprived of 
their viability by the infection with phage. If this were true, the 
components in the medium would have failed to enter the bacterial 
cells after the infection, and hence phage itself would have to take 
the components if these were found in the phage. However, this 
assumption is not correct, because the loss of the viability occurs only 
when bacteria are affected by a phage on an agar plate. In broth 
culture it seems not to take place at least in a manner so striking as 
on plate and moreover even on an agar plate only a peculiar phage 
can reveal the growth-inhibiting property, which is therefore* not es- 
sential for phage. The phage particles isolated and purified by our 
method always fail to develop the growth-inhibiting property even 
when it can exhibit the property before the purification. This interest- 
ing fact is discussed in detail in the other book (22). 

As described in Chapter VI in Part I, the activity of phage varies 
with the conditions under which the host bacteria are cultivated. The 
viability and the degree of lysis of bacteria after the infection by 
phage vary ina similar way. Thus, bacteria may lose the viability 
when infected with some phage on an agar plate as just mentioned, 
while this is not the case in a broth culture. In additon, on an agar 
plate bacteria affected by phage usually undergo a complete lysis while 
in a broth this does not necessarily follow. For example, according 
to our observation a certain strain of E.coli fails to undergo lysis in 
broth by the infection with a strain of phage. The difference be- 
tween agar and broth culture may be ascribed to the degree of contact 
with air during the phage intection, since in the case of broth cul- 
ture the greater the surface area of the culture, phage multiplies the 
more rapidly and abundantly (91). The plaque-formation would never 
result if the lysis and the growth inhibition failed to occur on an 
agar plate. 

The fact that phage in broth may not only fail to inhibit the 
growth of bacteria but occasionally even can promote it before the 


90 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


occurrence of lysis is shown in Fig. 15 (104). As already mentioned, 
host cells usually multiply more rapidly on the infection with a 
virus; also in this respect phage presents no exception. However, it 
must be emphasized that a certain phage, which can promote in broth 
the bacterial growth in such a manner, can render the bacteria non- 
viable on agar plate. 


Relative turbidity 


Time in hours 


Fig. 15. Clearing of the bacterial suspension by phages of both large 
and small plaques. Strain: B. dysenteriae (KA 1). 

The broth-suspension of the bacteria (number of the viable bacteria:1 x 
10’/cc.), to which the phage filtrate has been added, is placed in a water 
bath at 37°C. and the clearing degrees are observed nephelometrically with 
Pulfrich-photometer. 

I, II and III are the suspensions to which the large plaque phage (average 
plaque diameter ; 2,6mm.) has been added so that the concentrations of the 
active particles become 1x10’, 1x10® and 1x105/cc. 1, 2 and 3 are the sus- 
pensions to which the small plaque phage (average plaque diameter ; 0,5 mm.) 
has been added, particle concentrations being equal to I, II, and III respec- 
tively. C is the control suspension to which no phage has been added. 


VII THE MODE OF VIRUS MULTIPLICATION 91 


Usually the viability of bacteria is examined by plating on agar 
and the bacteria incapable of forming colonies are regarded as having 
no viability or being dead. This might be another main reason for 
which the erroneous conclusion was reached. Cohen attributed the 
rapid increase in the amount of both protein nitrogen and nucleic acid 
in the bacterial culture infected with phage to the multiplication of 
phage itself, but this may show nothing but the rapid growth of bac- 
teria after the infection. 

Kozloff et al. in the above cited chemical studies of virus reproduc- 
tion, stated that the percentage of the phage protein nitrogen derived 
from the bacteria was inversely proportional to the time of incubation 
and to the number of virus particles produced per bacterial cell. This 
may only be a natural result, since the prolonged period of incubation 
and increased number of virus particles per bacterial cell should be 
accompanied by the increased number of newly developed bacterial 
cells. They appeared to take into consideration only the bacteria 
which had been present at the beginning to reach the erroneous con- 
clusion. 

Such unreasonable conclusions seem, however, to be reached only 
with phage. A study on the uptake of radioactive phosphorus by 
influenza virus by Graham and McClelland (105) has shown that there 
is no direct exchange between the virus and phosphorus. It has been 
known for a long time that the increase of a plant virus is accom- 
panied by a decrease in normal protein. For example, Wildman et al 
(106) made electrophoretic analysis of cytoplasmic protein solutions 
isolated from tobacco plants at different intervals after inoculation 
with tobacco-mosaic virus, and found that a predominant normal por- 
tion decreased as the virus protein increased by approximately the 
same amounts, suggesting that the virus protein is synthesized at the 
direct expense of the main protein component. 

In an experiment with P*-labeled host bacteria, Putnam e¢ al (107) 
have reported that from 60 to 90 per cent of the phage P is derived 
from bacterial nucleic acid, and concluded that bacterial nucleic acid 
is the chief precursor of phage nucleic acid. It is remarkable that 
even with phage and bacteria such rather reasonable conclusion was 
reached, although the small portion of P had yet to be ascribed to 
that contained in the culture media. 

According to Stent and Maaloe (108) on the average 58 per cent of 
the phosphorus is host derived in the first 15 per cent of the phage 
particles formed, while the average over the total yield indicates 
only 30 per cent of the phage phosphorus to the host derived. This 
result again suggests the multiplication of the bacteria after the infec- 
tion. Labaw (109) found that the amount of bacteriophage phosphorus 


92 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


apparently derived from the medium after the infection varies from 
nearly zero to twice that originally in the bacterial host, depending 
on the strain of bacteriophage, that is, the bacterial phosphorus com- 
prises on the average from nealy 100 per cent to about 30 per cent of 
the total phosphorus in the bacteriophage. It should be emphasized 
that this difference was attributed to the strain of phage. If a certain 
strain of phage could render the bacteria utterly non-viable after the 
infection, 100 per cent of the phage phosphorus would appear to result 
from the bacterial phosphorus. 

Even if a small portion of phosphorus did appear to come from 
the medium in an experiment carried out with some bacteria actually 
reduced non-viable, it would be impossible to conclude that the small 
portion of the phage nucleoprotein was directly synthesized from the 
materials present in the medium, because the exchange of the elements 
or groups between the medium and the cell is considered to be possible 
without the net synthesis of the protein (110). In the theory of the 
writer, for the multiplication of a virus, the rearrangement of polar 
groups in the protoplasm protein so as to correspond to the pattern of 
the virus is to be raised. This rearrangement should be transmitted 
in the protoplasm as a wave which may consist in the shift of polar 
groups, and thus a type of transpeptidation may take place. It is 
conceivable that the elements or groups in the medium surrounding 
the cell may come to be involved in such transpeptidation and may 
be found in the virus particles produced. 

On studying the propagation of poliomyelitis in monkey testicular 
tissue, Scherer and Syverton (111) have concluded that the amount of 
virus in the tissue was as much as tenfold greater than that in the 
liquid of the same cultures, and that viral production was evidently 
earlier and was detected for a longer period of time in the tissue than 
in the liquid phase. This should only be a natural result if the virus 
is produced in the protoplasm; the virus is to be liberated in the 
fluid after the protoplasm is decomposed. According to a study by 
Ackermann and Kunitz (112) on the intracellular distribution of herpes 
virus in embryonic liver, a significant amount of the virus was found 
bound to the mitochondria by an intimate attachment and the concept 
was advanced that these particles function in the development of 
virus. However, since mictochondria are a sort of granules of proto- 
plasm with a rigid structure, if the protoplasm is decomposed inte 
particles after being changed into virus structure, the virus activity 
should naturally be retained markedly in mitochondria. 


VII. THE MODE OF VIRUS MULTIPLICATION 93 


2. The Difference in Nucleic Acid Content between Virus 
and Host Cell Protoplasm 


There should be, and actually is, an intimate relation between 
virus and its host cell protoplasm in chemical compositions, but, on 
the other hand, there are claims that considerable differences are 
present between them. The differences are believed to be found in the 
main in both the kind and content of nucleic acids. 

According to the chemical analysis of a strain of phage of E. coli 
by Csaky et al. (113) the virus consists of nucleic acids about 40 per 
cent, while the host bacteria only about 5 per cent. Such a striking 
difference may be mainly attributed to the isolation of peculiar parti- 
cles with high nucleic acid content which are regarded as sole phage 
particles, because the particles with high nucleic acid content appear 
to sediment more rapidly than those containing a little or no nucleic 
acids. According to Markham and Smith (50) the purified preparation 
of turnip-yellow mosaic virus protein separates into two parts on the 
application of ultracentrifugation; the bottom component contains 
nucleic acid, while the top is nucleic acid free, although these two 
components have the same electrophoretic mobility and isoelectric 
point, and both crystallizing in the same crystalline forms. 

Phosphorus content in a phage preparation isolated by our method 
was found to be 1.28 per cent, so that nucleic acid content in this 
preparation at most could not be greater than 13 per cent. This value 
is much lower as compared with that of phage particles isolated by 
means of ultracentrifuge by a number of workers. It must be borne in 
mind that all the particles present in the culture filtrate are to be iso- 
lated by our method, while this is not the case with the isolation by 
the ultracentrifugation. Again, the protoplasm, in general, contains a 
considerable amount of lipids, about one third of it being lipids. The 
phage particles obtained by our method likewise show a high content 
of lipids and also its one third was proved to be lipids, whereas 
lipid content of phage obtained by means of ultracentrifugation has 
been reported to be very low; according to Csaky ef al. it was only 
about 1 per cent. The lower is the lipid content, the sedimentation 
rate may be the greater, and the reverse may hold true for nucleic 
acids. 

Extremely small yields of virus particles by means of ultracentri- 
fugation are also indicating that the particles isolated by this method 
are only a portion of the particles produced by the decomposition of 
the protoplasm on the infection. 


94 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


For example, the yield of phage by Csaky et al, from 150 1. of 
culture filtrate, was 60 mg, while by our method the same yield would 
be easily raised from only one 1. Thus the yield by means of ultra- 
centrifugations is of the order of one hundredth of that obtained by 
our method, and only particles containing nucleic acids in rich amount 
and lipids in poor are presumably separated as virus particles by the 
ultracentrifugation. 

However, there is indeed a good reason for supposing that the 
particles with high content of nucleic acid and therefore those with 
high sedimentation rate are the virus particles with high activity, and 
thus when the others have no or little activity such particles only can 
reveal a high virus activity, the reason for which is discussed in 
detail in the next Chapter. 

From what has been mentioned above it may be reasonable to 
conclude that the high content of nucleic acids in virus particles 
isolated by the ultracentrifugation is attributed to the separation of 
particles with high sedimentation rate. There is, however, another 
important reason for the high content of nucleic acids in virus parti- 
cles. Gratia ei al. (114), comparing the composition of the particles 
obtained by the ultracentrifugation from tissue extracts of the silk 
worm, infected with polyhedrosis virus, with those obtained from non- 
infected worms, found that particles isolated from healthy silk worms 
contained only ribonucleic acid, whereas the particles isolated from 
infected worms contained, in addition, desoxyribonucleic acid. 

Moreover, it is generally recognized that bacteria infected with 
phage synthesize much greater desoxyribonucleic acid than do normal 
bacteria. Price (115) stated that normal staphylococci release neither 
ribonuclec acid nor desoxyribonucleic acid into the medium, whereas 
infected cells release both kinds of nucleic acids. Rafelson ef al. (116) 
found that the uptake of P®’ by the minced, young mouse brain is 
markedly stimulated by the zz vitro infection and growth of Theiler’s 
virus in the minced tissue. They believed that the effect of the 
virus on the P®” uptake by the tissue may be explained by the in- 
crease of ribonucleic acid synthesis in the infective tissue. Saenz and 
Taylor (117) found a similar phenomenon with chick embryo and in- 
fluenza virus. 

On the one hand cells infected with viruses are generally increased 
in their propagating power. Even in the case of bacteria affected by 
phage this is sometimes true as above stated. Thus, although bac- 
terial cells are commonly subjected to lysis when affected by phage, a 
proper amount of phage is occasionally capable of promoting the 
proliferation of bacteria. On the other hand, as is generally accepted, 
rapid propagation of cells is generally accompanied by the increase in 


VII. THE MODE OF VIRUS MULTIPLICATION 95 


the nucleic acid content. It has been well established that nucleic 
acid concentration is much higher in rapidly growing embryonic tissue 
than in the corresponding adult tissue. The virus infection, there- 
fore, should lead to the increase in the nucleic acid content of the 
protoplasm. If such protoplasm is coagulated into particles after be- 
ing endowed with virus structure, virus particles containing nucleic 
acid in rich amount must be formed. 

As already described, virus-like particles are never recovered 
from normal, untreated rabbit skin tissue, whereas when the tissue is 
injected with staphylococcus toxin or sapotoxin, the tissue becomes 
inflamed and swelled just as in the case of vaccinia virus injection, 
and particles containing nucleic acids like those of vaccinia virus are 
isolated in large quantities. If the apparent relation of nucleic acids 
to virus multiplication is merely due to the rapid growth of the cells 
there should be no change in the nucleic acid content when virus 
multiplication occurs in the cells which were preliminarily rendered 
nonviable. Actually, Price (115) claimed that when phage proliferates 
in the cells whose viability was lost on penicillin treatment, there 
occurs neither increase in the nucleic acid content nor change in the 
amount ratio of desoxyribonucleic to ribonucleic acid. 


3. The Mode of Production of Virus Structure 


As mentioned in Part I, the relation between a virus and its host 
cell protoplasm is considered to be analogous to that between an 
enzyme and its substrate. Avirus can combine with host cell proto- 
plasm through the complementarily arranged polar groups distributed 
between them just as does an enzyme with it substrate. 

To begin with, virus particles adsorbed to host protoplasm through 
such polar groups should unfold its coiled structure to show its full 
pattern. It is an established fact that globular protein molecules can 
readily spread in monomolecular films on the intermediate surface. 
The spreading of virus particle on the protoplam surface into a state 
corresponding to such a monomolecular film, may be caused by some 
physicochemical influence coming from the protoplasm, in this spread- 
ing certain amino acids or proteins with low molecular weights as well 
as inorganic salts may play an important role. 

The unfolding of globular proteins including virus particles to a 
state of filaments is brought about, as already pointed out, by the 
presence of inorganic salts in proper concentrations. At the same time, 
there are a number of evidences that proteins with low molecular 
weights like albumin can exhibit the same effect upon the spreading 


96 II. FUNDAMENTAL STRUCTURE OF RROTOPLASM 


(22). Thus, the solubility of horse serum globulin is not increased by 
the presence of pseudoglobulin, but increased by the addition of serum 
albumin (118). This may be interpreted as due to the unfolding of 
globulin molecules owing to the presence of albumin. In addition to 
albumin, various amino acids have shown to increase the solubility of 
euglobulin (119). As is well known, globulin is soluble in the presence 
of inorganic salts which as just mentioned have the spreading action. 
Serum globulin becomes sedimentable as the serum is dialyzed. Ina 
similar way phage particles can be rendered easily precipitable by the 
dialysis of the culture filtrate. 

The writer found that serum euglobulin, if isolated by our iso- 
electric precipitation method, exists in forming virus-like particles. Ac- 
cording tO the writer’s opinion (22), the polymerization product of this 
serum euglobulin, if present in the spreading form, can act as com- 
plement in immunological reactions, but not so in the coagulated, 
virus-like particles, which the latter can be expanded on the addition 
of serum protein with an albumin nature termed end-piece or factor 
C’2, while the coagulated euglobulin particle is named mid-piece or 
factor C’l, so that on the addition of end-piece to mid-piece, 7. e. the 
coagulated euglobulin particle, the complement activity may arise. 

A well known virus-like factor of pneumococcus capable of trans- 
mitting the peculiar character of the coccus, from which it was 
separated, to another strain of coccus is needed for its function a 
component present in serum or serous fluids, which can be supplied 
by a fraction of bovine serum albumin (120). 

As detailed already, protoplasm undergoes coagulation into minute 
particles on the application of stimulus. This coagulation spreads as 
a chain reaction in the protoplasm, but reversible in so far as the 
protoplasm remains “‘alive.’’ The unfolding of such coagulated proto- 
plasm may be possible, if certain low-molecular substances are present 
in the cell protoplasm for their unfolding. 

Phage particles can be seen under the microscope by dark-ground 
illumination. However, if adsorbed onto bacterial surface they would 
become always invisible (9), probably due to the unfolding of the 
particles on the cell surface. In order to show the structure acting 
as the template, viruses should be required to unfold the peptide 
chains. If the physicochemical effect resulting from the spatial 
arrangement of polar groups revealed by the unfolding of a virus 
particle is stronger than that of the protoplasm, the spatial distribu- 
tion of polar groups in the protoplasm may be changed by both the 
electrostatic repulsion and attraction so as to correspond to the pat- 
tern of virus. The change thus induced in the protoplasm structure 
must be the virus multiplication itself. 


VII. THE MODE OF VIRUS MULTIPLICATION 97 


On the contrary, if the protoplasm exhibits a stronger effect than 
the virus, the latter will be altered to become idential with the former 
in its structure and will be fused into it. In such a case the virus 
may be said to be assimilized by the cell, while in case of the occur- 
rence of virus multiplication the protoplasm may be said to be assimi- 
lized by the virus. 

In a study of a strain of phage adsorbed to a resistant bacterial 
mutant, Henry and Henry (121) found that the phage disappears with- 
out exerting any detectable effect on the host. Even in the same sample 
of virus particles, some virus particles may get the upper hand of the 
cells and can induce replica in them, while some other particles may fail 
to prevail over them because of their weak structure. The same may 
hold true for host cells, and some cells with high susceptibility may 
be assimilized by a virus, while others with less susceptibility may 
not be effected by the same virus. 

If the polypeptide chains of protoplasm protein are in a stretched 
state and in a protein moleclue thus stretched, which is situated on 
the surface of the protoplasm, the rearrangement of polar groups is 
raised by a virus, which has adsorbed onto the protein, in such a 
manner that the spatial arrangement of polar groups in the protein 
becomes complementary to that of the virus, then the protein thread 
situated in the next place is in turn to rearrange its polar groups, 
thus the transmission of the viral pattern being established. In such 
a transmission, the pattern must be alternately reverse, that is, the 
replica is to be produced in every second chain as shown in Fig. 7 
and 9. However, if the viral template possesses a pattern in which 
every two polar forces having opposite signs are always arranged ina 
symmetrical position as indicated in these Figs., the replica, if turned 
over, must be equal to the template. 

If the template protein has not such a regular structure, the 
replica is naturally different from the template. It is, however, hardly 
conceivable that such two types of patterns are actually induced in 
the protoplasm. 

On the other hand, Bergmann (122) claimed that a regular repeti- 
tion is present in the amino acid sequence of a protein. If polar 
groups are so arranged in a polypeptide chain as to make the replica 
equal to the turned-over template, such a regular repetition in the 
sequence should be expected. It is believed by a number of workers 
that amino acid residues in the protein molecules are not arranged 
in a random fashion (85) (110). If there exists a regular repetition 
the same two protein molecules can combine with each other since a 
turned-over molecule reveals the complementary structure to the other. 
There are indeed many evidences that protein molecules of the same 


98 FUNDAMENTAL STRUCTURE OF PROTOPLASM 


origin and the same kind have a strong tendency to combine with 
one another. 

The combination between virus and the host cell must be raised 
by complementarily arranged polar groups between them, whilst there 
appears to exist a common antigenic structure or structures between 
a virus and the host cell protoplasm as mentioned in Part I, indicating 
that the complementary pattern is attributed to such common struc- 
tures. Thus, a virus may be able to combine readily with a cell if 
the cell and the virus share a structure or structures in common. 

The structures common to a virus and the host give rise to the 
combining force between them, whereas when they are drawn near by 
the combining force, uncommon structures present between them may 
exert a mutual electrostatic repulsion, and if the virus has a structure 
strong enough to rearrange the structure of the host cell protoplasm, 
the virus can assimilize the cell. Viruses must have such strong struc- 
tures not present in the host cell protoplasm. 


CHAPTER VIII 
REPLICATION OF VIRUS PATTERN 


1. Structural Constitution of Viruses 


It is a generally accepted fact that viruses can be inactivated with- 
out being deprived of their specific antigenicity. This suggests that 
more delicate, unstable part of the structure is required for the virus 
action than for the immunological activity. 

Since virus particles even in the coagulated state can combine with 
their host cell, it is evident that the structure concerning the specific 
combination can exhibit its effect even when the virus are in the folded 
state. Such a structure may belong to the backbone structure of the 
protein and may be concerned in the immunological activity of the 
virus, whilst external, unstable groups which are revealed on the un- 
folding may be involved in the action of the virus as the template. 

Such unstable groups may involve the repulsive force of the virus 
against the host cell protoplasm in contrast to the stable structures 
which may give rise to the specific combining force. On account of 
the disappearance of the repulsive force following the folding of the 
particle, thereby only the combining force being left to exhibit its effect, 
the virus may be able to combine easily with the host cell. 

In the writer’s theory (22), antibodies are produced in entirely the 
same way as viruses. However, in antibodies fine structures corres- 
ponding to those of antigens fail to be induced, only gross structures 
complementary to the determinant grougs of antigens being produced, 
so that antigens do not multiply as do viruses. In other words, anti- 
gens and antibodies share in common with the gross structures, but 
not with the fine structures. Accordingly, antigen and antibody can 
combine with one another but the uncommon fine structures may re- 
pulse mutually after the combination; thus immunological reaction such 
as precipitin reaction may follow. On the other hand, since parent 
viruses and newly produced daughter viruses are identical even in fine 
structures, they combine with one another as do antigen and antibody, 
but without mutual repulsion, whereas virus and host cell share in 
common with the gross structures, but not with the fine structures, so 
that the relation between antigen and antibody is comparable -to that 
between virus and host cell. 

In short, there are two kinds of structures in the virus, the one is 


100 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


stable, being involved in the combining force and able to develop the 
force even when the virus is in a folded, coagulated state, while the 
other is unstable, concerned in the repulsive force and revealed only 
when the particle unfolds. The virus action may be determined mainly 
by the latter structure against which the replica is to be produced in 
the host protoplasm, although the former structure also may have a 
great influence upon the protoplasm in which the complementarily 
shaped structure is strengthened and specialized by the combination 
(22), 

According to Anderson (123) phage particles require some amino 
acids especially tryprophan for the combination with the host cell. 
These ‘‘adsorption cofactors’? appear to combine reversibly with the 
virus particles. This indicates that even for the development of the 
combining force are needed some factors or conditions. 

A study by Hoyle (124) on the production of the complement-fixing 
antigens in the various phases of influenza virus infection shows that 
these antigens, which carry virus specificity without virus activity, in- 
crease in amount before virus activity appears. Moreover, it has been 
well acknowledged that at the beginning of the phage infection of bac- 
teria, large amounts of antigenic substance are formed without being 
accompanied by the production of infective phage (125.) Presumably 
the structure formed at the beginning is incomplete, so that at first 
only the antigenic property may appear, but subsequently, when com- 
plete structure is accomplished, virus activity may arise. Thus it may 
be said that the rearrangement of polar groups is established gradually 
step by step, first only gross structures being formed and then finer ones 
added leading to the appearance of virus activity. 

It has been shown that there occurs, after the inoculation of a 
virus, a latent period during which the virus fails to multiply, accord- 
ing to the virus employed the duration of this period ranging from 6 
hours with influenza virus to 24 hours with mumps virus (126) (127). 
After such a latent period, there exists an interval during which the 
amount of virus in infected tissue increases rapidly. During this latent 
period only the formation of gross structures are probably being 
advanced. 

Magnus (128) has reported an interesting study on the ‘incomplete 
virus’: Undiluted allantoic fluid of chick embryo infected by influenza 
virus was used as inoculum and the amount of both agents concerning 
haemoagglutination and egg-infection produced after the inoculation 
were examined, and it was found that an increasing dissociation be- 
tween the two agents occurred in the course of passages, infectivity 
decreasing markedly as compared to haemoagglutinin. Magnus con- 
sidered that this was due to the production of non-infectious haemoag- 


VIII. REPLICATION OF VIRUS PATTERN 101 


glutinins, that is, ‘‘incomplete virus’’, possibly representing an immature 
form of the virus. His calculations indicated that only one of 10,000 
particles was infective in the third passage. In this case, if diluted al- 
lantoic fluids were used instead of undiluted ones and a proper amount 
of virus particles was inoculated, complete virus was produced instead 
of the incomplete one. As previously stated, large sized particles, as a 
rule, involve strong and complete virus, and further decomposition of 
the particles into smaller fragments may result in the loss or decrease 
in the infectivity; on the other hand, it has been reported that this 
‘‘incomplete virus’’ is smaller in size than the normal infective virus. 
particles (126) (130). 

The above finding shows that the complete structure of virus fails 
to be formed even after a prolonged period if environmental conditions 
are not suitable. It has been reported that there occurs no multiplica- 
tion of phage when the number of virus particles applied exceeds great- 
ly the number of bacterial cells exposed, although the lysis of bacteria 
takes place (131). This likewise may indicate the production of incom- 
plete virus. Moreover, Shlesinger (132) showed that when mouse brain 
is inoculated with a non-neurotropic strain of virus, production of com- 
plement fixing antigen and haemoagglutinin occurred but there was no 
production of infective virus. The yield of incomplete virus was pro- 
portional to the amount of infectious virus originally inoculated. 


2. Change of Viruses Following Their Combination 
with Host Cells 


Doermann and Anderson (123) have followed the production of a 
phage in host bacterial cells by disrupting the cells with intense sonic 
vibration at intervals after the infection, and have found that, up to 
12 minutes after the infection, the disruption of the cells destroys ‘their 
ability to form plaques, although free phage particles are highly resist- 
ant to such a treatment. They concluded from this evidence that in 
the freshly formed complex the virus is in a state sensitive to sonic 
vibration and that at the beginning of the infection the infecting virus 
particles has lost through some mysterious transformation its resistance 
to sonic vibration. 

As above stated, viruses when combined with cells should have to 
unfold their structure in order to show their full pattern which is to 
be replicated in the host cells. However, in the unfolded form viruses 
may be labile unlike the virus in the coagulated coiled up state. Thus 
the phage may become extremely sensitive to sonic vibration on the 
combination with bacteria. 


102 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


We were able to demonstrate that phage and vaccinia virus are 
most stable in water containing no salt; under such a condition their 
folding is considered to be most complete (133) (134). Inorganic salts 
except those of divalent cations, such as calcium and magnesium, al- 
ways render the phage more susceptible to heat. As was already dis- 
cussed, divalent cations can cause virus particles to become rigid and 
stable, whereas salts of monovalent cations may cause their unfolding. 

The disappearance of virus in host cells on the infection has been 
confirmed also with influenza virus. According to Isaacs and Edney 
(135), influenza virus injected into allantoic sac is mostly adsorbed by 
the chorio-allantoic membrane, but only less than 1 per cent of the in- 
oculum is recovered from ground supsensions, recovering of the virus 
being reduced further by 10 fold, if membranes are incubated before 
grinding. Such a difficulty of recovering may naturally result, when 
the virus undergoes a change into an unstable stretched form on the 
surface of host celis so as to be inactivated during the isolating ma- 
nipulation. According to Rountree (136) phage particles adsorbed onto 
host cells lose first its infectivity, followed by its antigenicity, and then 
ensues a period, from 12 to 19 minutes after the infection, during which 
neither infective phage nor antigen can be recovered from cells. 

There are, however, evidences suggesting that viruses are destroyed 
on the infection zm situ not during the isolating procedure. For in- 
stance, in biochemical studies of phage reproduction, Kozloff (137) 
claimed that all phage particles are partially broken down upon adsorp- 
tion on the host cells; up to 80 per cent of the desoxyribonucleic acid 
and protein nitrogen of the adsorbed virus are decomposed during virus 
reproduction to a various small fragments. 

This suggests that the adsorbed virus particles because of their un- 
stable, stretched form may be influenced considerably by the assimilase 
action of the host cell protoplasm so as to be decomposed, although 
some of them may be able in the long run assimilize the protoplasm. 
In the writer’s concept, as will be detailed in Part IV, Chapter V, when 
two protein molecules having different structures are held together, 
they exert to one another physicochemical influences due-to the differ- 
ent structure, and when the effect of the one is stronger enough to 
decompose the other, the former is called the proteolytic enzyme of 
the latter. In this respect, the host protoplasm may be said to act as 
an enzyme upon some of the adsorbed virus. Jesaitis and Goebel (138) 
have isolated water soluble, somatic antigen from a strain of dysenteria 
bacillus. This substance was found to inactivate all phages to which 
the bacillus is susceptible, and they (139) held the opinion that this 
substance serves as the receptor for phages which attack this bacillus. 
According to the theory of the writer, such a somatic antigen is noth- 


VIII. REPLICATION OF VIRUS PATTERN 103 


ing but the bacterial protoplasm protein which can combine with the 
phage through structures shared in common between the phage and the 
bacillus ; the shared structures may be called receptors. 

The protoplasm protein of susceptible cells should have weaker 
structural effect than the virus in order that the viral pattern is repli- 
cated in the cells. However, if some of the proteins acquire rigid 
structure through the combination with certain substances, say with 
polysaccharides, which make the protein structures rigid, then the pro- 
teins can act as virus inhibitor having the receptor for the virus. The 
virus which happens to combine with such rigid structures may be 
splitted since the virus has to be changed into labile, stretched form on 
the surface. The above cited claim that all phage particles are broken 
down upon the adsorption on the host cell suggests that the cell mem- 
brane may be mostly composed of protein complexes having such rigid 
structures. Form the point of this view it should be a natural result 
that the demonstration of the initial infecting virus particles is impos- 
sible in the cell macerates in which cell components acting as the in- 
hibitor may be present abundantly. 

Weidel (139a) has shown in his studies with bacterial membrane 
preparations that the interaction of intact bacteriophage with the 
host-cell membranes results in a disintegration of the membranes. 
This shows that the structure of the membrane was weaker than 
that of the virus. On the other hand, according to Barrington and 
Kozloff (1389 b), electron micrographs taken at zero time and after in- 
cubation showed that adsorption of phage was accompanied by the 
disintegration of the virus and the conversion of the membrane to a 
granular residue, indicating clearly that at least some of the phage 
particles combined with the host cell were decomposed together with 
the cell membrane. In studying the interaction of P*-labelled phage 
and host cells, Mackal and Kozloff (189c) have found that a portion 
of the virus P was released into the medium in the process. But this 
release did not occur after adsorption to heat-killed bacteria. This 
may be attributed to the derangement of the host cell structure by 
heating resulting in the loss of rigid structure becoming unable to 
decompose the virus. 

The virus particles may be decomposed on the cell surface into 
nucleic acids and proteins. The nucleic acids or their constituents thus 
formed appear to be incorporated into bacterial protoplasm whilst the 
proteins seem to be never utilized by the host cells. Thus, by infecting 
E. coli with P® labeled phage, several workers have demonstrated that 
30 to 50 per cent of the phage nucleic acid label is incorporated in the 
resulting “‘progeny’’. An equal high per cent to progeny transfer has 
been reported when the parental nucleic acid contained C™ labeled 


104 II. FUNDAMENTAL STRUCTURE. OF PROTOPLASM 


purines (140). However, Hershy and Chase (141) showed that after 
infection with S*® labeled phage most of the sulphur containing phage 
protein could be stripped from the bactrial surface without inter- 
fering with phage production. The resulting phage contained little or 
no S**, indicating that the protein fragments fails to be utilized by the 
bacteria in contrast to the nucleic acid constituent. 

These findings are interpreted by some workers as indicating that 
phage is decomposed on the cell surface into protein and nucleic acid. 
fragments which the latter only penetrate into cells to act as the tem- 
plate. In the opinion of the writer, however, the decomposition of the 
virus should be accompanied by the destruction of the virus activity, 
so that decomposed nucleic acid cannot act as the template, only serv- 
ing as the cell constituents required for the cell growth. The virus 
particles which can achieve their purpose of reproduction should have 
complete structures. _ 

Recently, Mackal and Kozloff (189c) have actually confirmed that 
the material found in the progeny is largely, if not entirely, due to 
the use of fragments of the parent nucleic acid in the synthesis of 
the progeny nucleic acid, and they concluded that most of the transfer 
of parent material of phage to progeny is unessential for the reproduc- 
tion process. 

The viral pattern may be replicated on the cell surface on which 
the extended virus is attached and the replicated pattern may be trans- 
mitted to the inner side of the cell. However, it is conceivable that 
the virus itself can penetrate into the cell to impress its pattern di- 
rectly to genes or gene-like particles, because according to Coons eé al. 
(142) various proteins injected intraveneously into mice were clearly de- 
monstrated in the unaltered form in the nucleus of certain cells, often 
even in higher concentrations than in the cytoplasm. Similar penetra- 
tion of viral nucleoproteins into cells may be possible. 


3. Essential Factors for the Spread of the Structural 
Change Caused by a Virus 


The first replica will be produced only in the portion of protoplasm 
with which a virus directly combines, no matter whether it be cyto- 
plasm or a nuclear mass. The replica thus formed, however, is to be 
transmitted throughout the whole protoplasm. For the occurrence of 
such a transmission the association among the protein molecules in the 
protoplasm should not be too strong, as otherwise each protein molecule 
may be unable to change its structure independently of others even 
for a short period, whilst there must be associations among them in- 


VIII. REPLICATION OF VIRUS PATTERN 105 


timate enough to make them in the end identical with one another 
in their structure. Such an ideal association cannot be established but 
for the interposition of lipids among the protein molecules. This must 
be the main reason for the presence of lipids in the protoplasm. 

On the other hand, as for viruses lipids are not only unnecessary, 
but unfavourable since viruses which may easily change their structure 
as does the usual protoplasm may commonly be unable to act as a 
strong virus. Rigid, unchangeable structure should be necessary for a 
virus to act as the template, since the action consists in the ability of 
changing the structure of the opponent. The great resistance of cry- 
stalline plant viruses to chemicals or heat may be attributed to the 
absence of lipids. 

Even with the same content of lipids, the mode of association or 
constellation of protein molecules may effect the manner of the trans- 
mission of change in the protoplasm. The general susceptibility of 
young cells to viruses may be dependent upon the flexibility of the 
‘young protoplasm protein. There are good reasons to suppose that 
“newly generated proteins: possess a property to be easily altered in the 
structure as will be discussed in Part IV. -The phage susceptibility of 
young bacteria can be retained for long periods at a low temperature, 
although immediately lost at a higher temperature, indicating that the 
flexibility of young proteins’ can be preserved at a low temperature 
-(101). 

If elementary bodies of: protoplasm are: irreversibly coagulated, 
viruses cannot multiply in:the protoplasm, because the protoplasm in 
such a state may surely be‘inadequate for the production of replicas as 
well as for the transmission of the change. The irreversible coagula- 
tion.must mean the death of protoplasm. But if protoplasm is killed 
without such a coagulation, the multiplication of viruses may. not be 
impossible in the dead protoplasm. It has been established by a num- 
‘ber of workers that phage cam multiply in bacteria which have been 
rendered non-viable by chemical or physical agents such as penicillin, 
mustard-gas, formaldehyde, and ultraviolet rays. Similar’ phenomena 
‘have been proved also with other viruses. For example, chick embryos 
killed by prolonged storage at room temperature or at 4°C are capable 
of supporting the multiplication of influenza virus upon inctibation at 
35°C (143). The growth of Shope’s papilloma virus is also possible in 
the cells sterilized by ultraviolet irradiation (144). According to Weiss 
(145), feline pneumonitis virus,’ a typical virus of the psittacosis- 
lymphogranuloma group, was cultivated for 20 passages in the yolk 
sacs of dead chick embryos; the rate of its growth was claimed to be 
almost similar to that in living embryos. 

Multiplication of viruses in such dead cells is expected to be never 


106 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


accompanied by any nucleic acid change, since the multiplication should 
only be the rearrangement of polar groups in the protoplasm. In fact, 
Price (146) has confirmed that, during phage multiplication in bacteria, 
killed by penicillin, no change in nulcleic acids can be found. This 
shows that the nucleic acid change is not indispensable for virus pro- 
liferation. 

The cell multiplication has to be accompanied by the protein syn- 
thesis for which great quantities of energy should be needed. In the 
above examples host cells are regarded as being dead because of their 
inability to multiply, which may be attributed to the destruction of 
energy-supplying system. If so, the fact that virus multiplication is 
achieved in dead cells, suggests that little or no energy, accordingly no 
protein synthesis, is required for the rearrangement of polar groups 
leading to the virus multiplication. 

On the other hand, it has been reported that, in contrast to penicil- 
lin, streptomycin reduces bacteria on killing them incapable of preduc- 
ing phage. (147) (148). In this case, streptomycin may give rise in the 
protoplasm to a change thereby the rearrangement of polar groups due 
to the virus becomes impossible. While penicillin reveals no phage-in- 
activating ability, streptomycin can inactivate the virus, showing its. 
strong action in disturbing the structure of the assimilase. In a similar 
manner the structure of bacterial cell protoplasm may be so disturbed 
as to be rendered incapable of performing the rearrangement. The 
inability of penicillin to inactivate phage indicates that penicillin has no 
such a strong disturbing faculty, although it can ‘‘kill’’ the bacteria 
presumably by destroying the energy-supplying system necessary for 
protein synthesis. If some host cells are ‘‘killed’’ in the same way by 
a certain agent other than penicillin, virus can multiply even in them. 

Bacteria killed by ultraviolet-irradiation can produce phage, but 
the virus grows slower, and yields a smaller number of new particles. 
(149). This suggests that the protoplasm structure is so changed by the 
killing action as to become somewhat unfavourable for the replication 
of the virus pattern. However, in order to suppress completely the 
ability of the cell to support the virus growth, enormous hits of the 
irradiation are required to reduce to half the number of bacteria capable 
of inhibiting phage after infection (150). 

It has been reported that chick embryo tissue cultivated for 13. 
days in Hank’s balanced salt solution prior to infection lost its ability 
to suport the growth of psittacosis virus, but this capacity could be 
restored by the addition of beef embryo extract at the time of in- 
fection (150a). A significant amount of virus was adsorbed to the 
tissue cultivated under this condition without addition of beef embryo. 
extract, indicating that the failure of virus to grow was not due to 


VII. REPLICATION OF VIRUS PATTERN 107 


failure of virus to attack to and invade the cells. Presumably, the 
culture under the peculiar condition reduced the protoplasm structure 
of the cell inadequate to be impressed by the viral pattern. It may 
be said that the tissue cells under this culture condition may fall into 
a state comparable to that of the ‘‘aged’”’ bacteria, while the cells 
may be rejuvenated by the addition of beef embryo extract to become 
capable of achieving the rearrangement of polar groups in response to 
the viral pattern. 

To sum up, a virus can multiply in a cell even when the cell is 
in a “‘dead’’ state, if the rearrangement of polar groups in the cell 
protoplasm responding to the viral pattern is possible, while even 
viable cell cannot support the viral growth if the cell is unable to 
respond the viral pattern. 

The virus activity may be determined by the spatial arrangement 
of polar groups, which in turn may be chiefly dependent upon the 
manner of distribution of amino acid residues, so that certain differ- 
ences in the amino acid composition may be found between host cell 
protoplasm and the virus derived from it. As already pointed out this 
is said sometimes true. If a change like transamination or transpefti- 
dation took place in the protoplasm when its structure was enforced 
to alter in response to the template of the virus, some amino acids 
would appear as if they were newly synthesized. It may be unreason- 
able, therefore, to conclude that virus was newly synthesized from 
amino acids even when the amino acid composition of the virus was 
demonstrated to be evidently different from that of the protoplasm of 
host cells. 


CHAPTER IX 
VIRUSES AND NUCLEIC ACIDS 


1. Nucleic Acids in Virus Particles 


At present it is generally believed that viruses are nucleoproteins, 
and nucleic acids are customarily considered as being indispensable for 
viruses. However, if writer’s opinion so far discussed as regards the 
viruses is legitimate, it may be said that nucleic acids are not always 
necessary for the virus action. Since the template of viruses to be 
replicated in the host protoplasm should be involved in the structure 
of globulin-like proteins, there appears to be no need to assume nucleic 
acids as being indispensable for the template action. 

In his splendid book ‘‘Plant Viruses and Virus Diseases’’ Bawden 
stated as follows: ‘‘In spite of the wide differences between the pro- 
perties of the individual viruses so far purified, it would be rash to 
assume that they form a random and representative sample of the 
whole group. Because these are chemically similar, it can almost 
be taken for granted that some other viruses are also nucleoproteins, 
but to assume that all are would be decidedly premature. The methods 
of isolation so far used may be acting as a Selective agency, succeeding 
only with those that are nucleoproteins.”’ 

If nucleic acids were essential components for viruses, their content 
and kind would be expected to be constant to a certain extent, but the 
fact is that they are unusually variable. Among many viruses studied 
so far phage appears to have the highest content of nucleic acid, and 
by Taylor (151) it has been estimated to be so high as nearly 50 per 
cent, whereas Newcastle disease virus has been reported to contain a 
quantity of nonlipid phosphorus equivalent to only about 1 per cent 
nucleic acid (152). Chemical compositions of various viruses are shown 
in. Table 6; also from this Table it is evident that there are great differ- 
ences in nucleic acids both in kind and content. 

The writer has assumed as already stated that in the usual virus 
partcles as well as in the protoplasm, stretched peptide chains of protein 
molecules having the nature of euglobulin are arranged in parallel 
alignment, and lipids being inserted among the peptide chains. Nucleic 
acids, if contained any, are considered to be inserted, like the lipids, 
among the stretched protein molecules. X-ray analysis of tobacco-mosaic 


XI. VIRUSES AND NUCLEIC ACIDS 109 


virus by Pfankuch (153) did not indicate a concentration of the com- 
paratively dense nucleic acid in any particular part of the virus particle, 
and it was suggested that the virus may consist of either a long 


Table 6. 


Chemical Composition of Viruses. 
After Beard, J. W.: J. Imm., Vol. 58, 49, 1948. 


Whole complex Lipid Nonlipid 
5) Se tai 3) : 
_ a )@|s _ 2 Nucleic 
B§l 3 | 3) S/E| 5] € BE) cia 
fo! ise] ww eS 
CNTF | ewe | 8 ielel ee rape 
° a =) Ay 
ise) a — o S Ga Zi 
Broth Bacterio- 
PHAZE verre reer seen evens 42.0/13.5] 4.84)13.6} 2.6 | 0 0 |2.6/97.4 50.6) 13.1 40.3 6.6 
Synthetic Medium | 
Bacteriophage::---- AVIS) MBps sera lerA| dlasw|| 0) 0 ae 98.2) 52.4) 11.2/44.6) 1.3 
Vaccinia -+++++e+eeeeeeees 33.7|15.3)0.57) 2.8) 5.7 | 2.2 | 1.4)2.2/94.0/89.0) 2.8) 5.6 
Papilloma «+++++++e+eeees 49.6) 15.0)0.94) 6.5} 1.5 98.5) 90.0 8.7) 


Equine Encephalo- 
myelitis (Eastern 


St.) ctreteseeeeeeeeeeeees 62.2) 7.7/2.2 | 4.0/54.1 |35.0 |13.8/9.6)53.0/49.1! 7.2 4.4 
Influenza A (PRS 

Strain) --+++seeseeeeee 53.2) 10.0) 0.97) 12.5) 23.4 |11:3 | 7.0)5.1)77.5)}65:0| -7.3) 1.5) ? 
Influenza B (Lee 

Strain) cersseeeeeeeeees 52.7| 9.7/0.94) 13.1) 22.4 |11.2 | 3.7/7.2) 76.4 63.6) 9.4) 1.2) ? 


Broth Medium 


Swine Influenza-::--- 51.4 ae 10.0) 24.0 {10.7 | 5.7|7:7|77.6 67.6)10.0) + 2 


Bacterium ---++++++++) 49.1] 13.2) 2.72|12°5| 7.75| 7.75| 0 |0 92.3 67.9 1225) Spe Oa 
Synthetic Medium : 
Bacterium -----++++-- 49.0) 13.2) 2.66) 11.6) 9.11) 9.11) 0 |0° |90.9)67.7/11.6) 2.4/20.9 


Normal Chick Em- | | 
bryo Component:::|55.3 9.53.2 7.0|35.0 |23:4 | 6.5/6.9) 66.5'40.7| 9.5 110.6 


All values are per cent dry weight of the whole complex. 


protein chain with nucleic acid side groups, or a regular arrangement 
of alternate nucleic acid and protein residues. The fact that tobacco- 
mosaic virus is readily decomposed into smaller fragments also supports 
the view that nucleic acids are distributed in a homogeneous dissipa- 
tion in the virus particles. 

According to Schramm (154), tobacco-mosaic virus particles dissociate 
in alkaline solutions into two components, one of them free of nucleic 
acid, but having the same molecular weight as the other contaning 
nucleic acid. Readjustment of the solutions containing either one of 
them to pH 5, yielded particles practically indistinguishaeble from the 


110 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


native protein particles in size and shape. Again, the action of 
nuclease leads to the cleavage of the bond between protein and nucleic 
acid and to a loss of biological activity, but the shape of the particles 
is not altered by nuclease. It seems evident, therefore, that the shape 
and size of ‘the virus particle are determined by the protein, nucleic 
acid apparently having no concern with it. On the other hand, it is 
stated that the molecular weight of desoxyribonucleic acid is 1.5 or 
3.7 million; the length of this thread-like molecule is about 500 my. 
(155). Such a macro-molecule might be an artifact, yielded during the 
isolating procedure, as nucleic acids may have a great tendency to 
undergo polymerization. . 

Jordan (156) stated that all values of the molecular weight of 
nucleic acids, which have been determined from isolated sedimentation 
and diffusion constant determinations, must be regarded as unreliable. 
It is by no means certain that solutions of nucleic acids at the con- 
centrations normally studied are molecularly disperse. The presence 
of divalent cations have shown to cause considerable aggregations, 
and it has been shown that the variation of light transmission with 
concentration shows a critical region at a concentration of 0.003 per 
cent which is regarded as an indication that aggregation commences 
at this concentration. - 

Monné (157) expressed the opinion that protoplasm is constructed 
of fibrils which consist of ribonucleic acid and nucleic acid free-sec- 
tions regularly alternating with each other. Pederson (158) claimed 
that conjugated proteins containing non-protein components have a 
structure in which non-protein components are inserted like cement 
among bricks of protein molecules; thus these latter combining 
indirectly with one another through the inserted substances. Schmidt 
(159) considered likewise that in chromosomes nucleic acids are inter- 
posed like cement among the polypeptide chains of proteins. 

Nucleic acids have long been known to possess the property to 
combine preferably with various proteins; owing to this property they 
were’ frequently used as precipitating agent of proteins. Even when 
no precipitate is formed, the formation of compounds between nucleic 
acids and neutral proteins is proved by a drop in the osmotic pressure 
of the dissolved protein and also by the loss of the high viscosity (160). 

If protein molecules are cemented by nucleic acid, the alteration 
of the protein structure may scarcely occur. In fact, Carter and 
Greenstein (151) reported on a protective effect of desoxyribonucleic 
acid upon the heat coagulation of egg albumin. Nucleic acids may 
serve aS an agent to prevent free motions of the peptide chains in the 
protoplasm, in contrast to lipids which may operate as a lubricating 
oil to facilitate the change of polypeptide chains. 


XI. VIRUSES AND NUCLEIC ACIDS 111 


2. The Action of Nucleic Acids 


When the assimilase activity of protoplasm is inferior to that of a 
virus, the virus can multiply in the protoplasm. On the other hand, 
the superiority in this activity on the virus part should arise from its 
rigid structure. Hence, if nucleic acids are inserted among protein 
molecules to make its structure rigid, the virus will become stable 
enough at least not to be assimilized by other assimilases. 

Even in this respect alone, particles containing nucleic acids are 
expected to be an assimilase stronger than those containing no acids. 
Furthermore, the assimilase activity itself will become stronger, if 
more rigidly and more regularly are held together the protein mole- 
cules by nucleic acid, since the assimilase action is considered to be 
raised from the regular array of protein molecules. 

The purified preparation of turnip-yellow mosaic virus has been 
found by Markham and Smith (50) to consist of two parts, about 
80 per cent of particles containing nucleic acid and 20 per cent of a 
nucleic acid-free ones; this latter apparently non-infective, but share 
many properties of the infective ones containg nucleic acid. Thus, 
the both have similar electrophoretic mobility and form mixed crystals, 
and appear to contain the same antigens and to react fully with virus 
antisera. X-ray measurements on crystals of the two kinds of parti- 
cles indicate that those containing nucleic acid are slightly smaller, 
suggesting that nucleic acid may hold the particles in a tighter mass. 
The separation of these two fractions is accomplished by ultracentri- 
fugation, the bottom component being the protein containing nucleic 
acid. This greater sedimentation rate of the bottom component may 
result from the presence of nucleic acid, which holds tightly the pro- 
tein molecules, while in the particles containing no nucleic acid, the 
combination among protein molecules may be loose, thus being not 
easily sedimentable. 

The failure in proving the virus activity in this fraction may 
likewise be attributable to; this slackness of the combination ; however, 
it may be impossible to conclude that this fraction had always no 
virus activity, since it might not entirely be impossible to demonstrate 
its infectivity, if a suitable host with a high susceptibility was used 
under a proper environmental condition. 

According to Heriott (162) a phage preparation which have been 
rendered nucleic acid free by ‘‘osmotical shocking’’ can still be adsor- 
bed onto the host cells, prevent their multiplication and even lyse them, 
although the phage can reveal only less than 1 per cent of its ori- 
ginal infectivity, a fact which may indicate that nucleic acid is not 


112 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


essential for the virus activity. Moreover, on studying the mammary 
cancer of mice, Barnum and Huseby (163) have obtained by high-speed 
centrifugation lipoprotein particles almost devoid of nucleic acid. These 
particles possessed cancer-producing poteintialities which were equal to 
the original nucleic acid containing preparation of the lactating gland. 
Maaloe and Symonds (164) indicated that in both premature and ordinary 
lysates of bacteria infected by phage there are non-infective particles 
containing sulfur but no nucleic acid. These particles adsorb on 
bacteria and precipitate with antiserum in a way similar to typical 
phage. However, they are somewhat lighter and have no appreciable 
killing effect on bacteria. 

Price (165) has found that the activity of a phage preparation was 
increased by 20 to 30 fold by the addition of nucleic acid. This fact 
can be interpreted as indicating that some phage particles incapable 
of developing the virus activity bacause of the ‘looseness in their 
structure acquire the rigidity on the combination with the nucleic 
acid:with the revelation of the virus activity. The nucleic acid used 
in this experiment was ribonucleic acid isolated from host bacteria or 
yeasts, whereas the nucleic acid contained in phage preparations is 
usually not ribonucleic but desoxyribonucleic acid which is regarded 
by some workers as having the special relation to the virus. This 
shows, however, that ribonucleic acid can contribute to the phage 
activity like desoxyribonucleic acid. 

Bacteria infected with a phage are usually disintegrated into 
minute particles which can be isolated by our isoelectric precipitation 
method. It was found that only one out of 100 or 1,000 particles thus 
isolated shared the virus activity, and that as already described most 
of the activity belonged to larger particles, smaller ones being un- 
stable, most of which existing in non-infective state. This fact may 
indicate that, even if nucleic acid content was equal, larger particles 
could develop the activity with more ‘easiness than smaller ones. 
However, since the distribution of nucleic acids in bacterial proto- 
plasm is not uniform, each particle produced should be different in 
nucleic acid content, and hence, even when the particles were equal 
in size, only some particles with higher nucleic acid content would 
be able to develop the virus activity. Furthermore, it may be 
possible that some phage can exist in larger particles because of their 
high content of nucleic acid which prevents the further decomposi- 
tion of the particles into smaller ones. 

Anyhow, particles with high contents of nucleic acid may show 
greater tendéncies to act as viruses, and since such particles have 
greater sedimentation rates, only those sedimentable rapidly by ultra- 
centrifugation would likely be regarded as the sole viruses. 


IX. VIRUSES AND NUCLEIC ACIDS 113 


The coli-phage particles isolated by our method contain nucleic 
acids at the most only 15 per cent and even one third of the particle 
is composed of lipids, whereas those isolated by ultracentrifugation 
have been reported to contain so much nucleic acid as 40 per cent, or 
more, and lipids only about 1 per cent (113). Moreover, the yield by 
means of the ultracentrifuge is only of the order of one hundredth of 
the yield by our method, and accordingly it is evident that particles 
having a peculiar property only are to be obtained by ultracentrifu- 
gation. Such peculiar particles would be able to act as the virus even 
when other particles failed to do so. It must, however, be emphasized 
that almost all the particles isolated by our method can sometimes 
reveal phage activity when examined under most suitable conditions. 

Polyhedrosis virus, as already mentioned, is isolated, from poly- 
hedral body, in needle shaped particles containing a large quantity 
of desoxyribonucleic acid, but it has been reported that such particles 
are contained in the polyhedral body only about 3 to 5 per cent; the 
most part of the body consists of proteins with a low phosphorus 
content having no virus activity (33). 

The particles separated from normal, healthy plant leaves such as 
those of tobacco and tomato have been reported to contain little or no 
nucleic acid. The fact that they are so unstable as to be decomposed 
by trypsin is apparently due to this scantiness of nucleic acid and 
also to the high content of lipids. The probable reasons for the pre- 
sence of nucleic acid in rich amount in the particles isolated from 
infected leaves are already considered. Virus particles are commonly 
not affected by proteolytic enzymes, indicating that the particles are 
stronger in their structure than the enzymes. Living protoplasm is 
likewise usually not affected by enzymes. However, when its regular 
configuration is damaged to become unable to act as the assimilase it 
may be called dead, and at the same time it will become digestable 
by enzymes. Normal plant particles are presumably in this dead state 
as their configuration would be disturbed severely during the prepar- 
ing manipulation on account of their looseness in structure because of 
the lack of nucleic acid and so they may be split by trypsin in contrast 
to usual virus particles. 

Some bacteria produce agents termed haemolysins which act on 
red blood cells to cause lysis. So far as our studies have reached, 
haemolysins appear to exist in virus-like particles and also their action, 
like that of viruses, consists in causing a disturbance in the proto- 
plasm structure of blood cells. However, in contrast to viruses, 
haemolysins fail to multiply. This is possibly due to their inability 
to produce an exact replica in the protoplasm, that is, haemolysins 
may cause only a structural disturbance instead of producing the 


114 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


replica (22). 

An interesting fact has been found by Hosoya ef al. (166) concern- 
ing streptococcus haemolysin. When haemolytic streptococcus col- 
lected by centrifugation were shaken with 10 per cent nucleic acid 
solution for 20 minutes at 37°C, strong haemolytic power was found 
in the solution after the bacteria were removed by centrifugation, 
whereas no haemolysin activity was obtained if the bacteria were 
washed with saline. Also powerful haemolysin was obtained when 
nucleic acid was added to the culture media. This fact can be ex- 
plained in the same way as the fact, above cited, found by Price 
concerning a phage; namely, the virus-like particles produced by the 
bacteria are endowed with rigid structure by the nucleic acid to be- 
come capable of exerting strong disturbing action on the blood cells. 

We were unable to separate haemolytic action of staphylococcus 
haemolysin from virus-like particles containing lipids; haemolytic 
action would readily be inactivated when the particles were precipitat- 
ed at the isoelectric point, whereas Wittler and Pillemer (167) have 
succeeded in the purification of the haemolysin by isolating it from 
the culture filtrate on precipitating at pH 4.0 in 15 per cent me- 
thanol at -5°C. The expulsion of lipids to some extent seems thus 
possible as in the case of some viruses if the treatment is made at 
low temperatures. 

As already discussed, the protoplasm can be regarded as polymeri- 
zation products of protein molecules possessing certain spatial arran- 
gements of polar groups; the pattern of the spatial arrangement may 
differ with the kind of protoplasm or with the kind of cells, and each 
kind of cells may have its specific pattern. In the protoplasm of a 
certain cell, not only protein molecules but also other components, 
such as nucleic acids, lipids, and sugars, have to be arranged so as 
to be subjected to the specific pattern. Since the pattern of a cell is 
to be changed into the viral pattern following the infection with the 
virus, it may be expected that nucleic acid composition in a host cell 
is deviated from the normal when the acid is synthesized in response 
to the pattern of the protoplasm of the cell infected with a virus; the 
viral pattern is to be replicated in the protoplasm so that the pattern 
of the nucleic acid synthesized by the infected protoplasm must have 
the same viral pattern. In agreement with this view it has been 
claimed that nucleotide composition of a viral nucleic acid is character- 
istic of the kind of the kind of virus; in other words, the nucleic 
acid compositions of unrelated viruses are demonstrably different, 
whereas those of strains of a virus are indistinguishable (168). More- 
over, it has been reported that phage DNA can be distinguished by 
its hydroxymethylcytosine content from bacterial DNA which contains 


IX. VIRUSES AND NUCLEIC ACIDS 115 


cytosine; viral DNA increases and bacterial DNA is destroyed in in- 
fected bacteria (169). Again, it has been found that the DNA of cer- 
tain strains of phage contains no cytosine; instead, they contain a 
hitherto unrecognized pyrimidine, while other viruses contain no this 
pyrimidine (170). 

Since the structure of a nucleic acid polymer may be rigid, even 
when some virus-like particles, or elementary bodies of protoplasm, 
consist mostly of nucleic acids, only a small particle being proteins, 
the specific pattern may exist unimpared in the particles. In an ex- 
treme case where all the protein molecules are eliminated, particles 
thus consisting of nucleic acid only may not be impossible still to 
retain the pattern to act as the template. There seems to be an 
evidence that this may be possible as mentioned in the next Part. 

As regards nucleic acids there are still many other problems to be 
discussed, but they will be reserved for Part IV where the nature of 
genes is to be considered in detail. 


CHAPTER X 
THE SUMMARY OF PART II 


1 


The essential component of the protoplasm is a protein of globulin 
nature, molecules of which exist in parallel alignment in a stretched 
thread-like form, associating with one another in a regular array by 
a physicochemical force. Lipids are other important components, which 
are inserted among the protein molecules. Owing to the lipids, pro- 
toplasm can exhibit properties of a liquid, and protein molecules 
existing in it can change freely their structure; at the same time 
protoplasm itself can possess the specific configuration. 

If the protoplasm is such a system consisting of molecules of 
protein and lipid, each of which associating regularly with one an- 
other to form a certain structure, it will be expected that a change 
arising at a site of the protoplasm will spread to other parts. Actually 
this appears to be the case; for example, it is known that, when an 
adeyuate stimulus is applied to the protoplasm, there occurs at the 
site of stimulation a coagulation of protoplasm into minute particles, 
which spreads succesively through the whole protoplasm. 

The protoplasm coagulation into such minute particles can be 
explained on the assumption that the thread-like protein molecules 
exist in forming bundles in parallel alignment, each bundle being 
formed by several hundreds of threads. This bundle is assumed to 
be the unit of protoplasm, and termed ‘‘elementary body of protoplasm.”’ 
Lipids are inserted among protein molecules in this unit body, and the 
protoplasm is composed of these bodies which are arranged in parallel 
array, combining loosely with one another by a physicochemical 
force. 

The thread-like protein molecules forming the body will contract 


or fold when a stimulus is given, so that the protoplasm as a whole we 


coagulated into the unit particles by the stimulus. Since this coa- 
gulation of protoplasm, that is, the folding or contraction of the pro- 
tein molecules, is reversible, the folded or contracted molecules will 
restore the stretched form when the stimulus is removed, whereby the 
protoplasm recovers its original state. 

On the other hand, as regards its function the protoplasm is a 


LAr 


X. THE SUMMARY OF PART II. 117 


i) 


kind of enzymes, which may be designated ‘‘assimilase,’’ and is capable 
of adsorbing certain protein molecule having a structure somewhat 
different from that of its own protein constituent, thereby the structure 
of the protein is changed by the rearrangement in its spatial distri- 
bution of polar groups to be made identical with that of the proto- 
plasm, thus the protein being incorporated into the protoplasm. Not 
only protein molecules but also amino acids are adsorbed onto the pro- 
toplasm surface to be adapted to the specific arrangement of its polar 
groups, and subsequently they are fused into the protoplasm; thus 
the protein is synthesized and the growth of protoplasm is accom- 
plished. Protoplasm growth, therefore, occurs in the same way as a 
crystal growth, and hence cell multiplication can be looked upon as 
the growth of the protoplasm, a type of crystal. In fact, protoplasm 
is considered as a kind of liquid crystal, since it isa liquid with a 
definite structure. 


2 


The assimilase action of protoplasm apparently arises from the 
polymerization of protein molecules of an equal structure, and the 
action consists in the faculty to cause the rearrangement of polar 
groups of the proteins to be fused. Proteins in a separate, single 
molecular state fail to exhibit the effect which may arise from its spe- 
cific arrangement of polar groups, whilst if many molecules with an 
equal arrangement polymerize in a regular way, the effect is consi- 
dered to become stronger and as a result can exert its influence upon 
other proteins. This effect appears to a certain extent to be directly 
proportional to the number of polymerized molecules, and probably 
resulting from a kind of electrostatic force. 

Virus particles are estimated to be combining with so much water 
amounts as 10 times the dry weight of the particles. The greater 
part of the molecules of the water are apparently present in forming 
a thick layer around the surface of the particle, and the thickness 
of the layer is calculated to be directly proportional to the particle 
diameter. 

The force attracting the water molecules onto the surface of a 
virus particle is possibly identical with the force of the virus to pro- 
duce its replica in the host cells. Virus particles may show a tendency 
to act as the stronger virus as the particle increases in size. 

The fact confirmed by Rothen that the thickness of molecules of 
antibody attracted by the monolayers of the antigenic protein is 
directly proportional to the number of the piled-up monolayers of the 


118 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


antigen, provides likewise an evidence that the force arising from the 
specific arrangement of polar groups may increase in the direct pro- 
portion to the degree of polymerization. It is known that the number 
of antibody molecules combining with the antigen increases as the 
latter molecule increases in size, and it is also accepted that both 
enzymes and antigens have to be in colloidal states in order to exert 
their action; in all these facts seems to be involved the force coming 
from the polymerization. The peculiar property of colloid may also 
be closely connected with the same force. 

The fact that viruses are the particles of more than certain 
sizes may indicate the necessity of polymerization of virus proteins 
to a certain degree for the development of the activity. 


3 


If the protoplasm is disintegrated into its elementary bodies, and 
if in the bodies the original structure of the protoplasm is retained, 
the bodies themselves also can act as an assimilase. Viruses are 
regarded as a kind of the assimilase existing in such minute bodies. 

However, the assimilase, when present in the form of free ele- 
mentary bodies, is much less active than the original protoplasm, and 
is unable to incorporate either amino acids or foreign protein molecules, 
only being capable of assimilizing a certain protoplasm having a 
weaker assimilase action. 

If the assimilase action of a virus is stronger than that of the 
protoplasm of a cell to which the virus is adsorbed, the arrangement 
of the polar groups of the protoplasm will be so changed as to become 
identical with that of the virus. Since this change may be transmitted 
throughout the protoplasm, the whole protoplasm protein may be 
changed finally to have the identical arrangement. If the protoplasm 
is disintegrated into coagulated elementary bodies by this change, the 
bodies may be regarded as virus particles. This is the way in which 
viruses multiply. 

The mode of change of the protoplasm of red blood cells can be 
studied by investigating the mechanism of haemolysis. An incubation 
period required for the commencement of haemolysis after addition of 
a haemolytic agent can be regarded as the period during which a 
change, secondarily induced by the haemolytic agent, spreads throughout 
the protoplasm, 7. é., the stroma of the red cell, to reduce the proto- 
plasm incapable of retaining the haemoglobin which is being held by 
the stroma. 

A similar incubation period is found commonly between the addi- 


X. THE SUMMARY OF PART II. 119 


tion of a phage and the disintegration of the bacteria, the lysis, which 
may be caused by a change of the protoplasm due to the virus disrupt- 
ing the mutual association of elementary bodies. 

It is found that the rate of change in the blood cell is roughly 
proportional to the concentration of a haemolytic agent, showing that 
the protoplasm undergoes a change which tends to be directly propor- 
tional to the amount of the haemolytic agent combining with the cell. 
Also in the case of a virus and its host cell, the degree of influence of 
the virus upon the host cell is apparently proportional to the number 
of the virus particles adsorbed to it. Thus a weak virus fails to infect 
the host cell when the number of the particles is small, but can get 
the upper hand of the cell if many particles affect in union. 

Bacterial haemolysins, usually present in virus-like particles, may 
affect the blood cells in the same way as viruses affect the host cells. 
Thus, haemolysins may disturb the structure of the protoplasm of the 
blood cell to cause a change which spreads throughout the cell; as a 
result the association between the haemoglobin and the protoplasm is 
damaged leading to haemolysis. This change, however, cannot give 
rise to so precise a rearrangement as to produce the very replica of 
the haemolysin structure, so that haemolysins are not viruses. 

Viruses, like haemolysins, may sometimes disintegrate the proto- 
plasm of host cells without producing the replica. If phage or influ- 
enza virus particles are added to respective host cells in a great 
excess, little or no virus is produced, although thereby the host cells 
are injured severely. 


4 


Since virus particles are no more than elementary bodies of proto- 
plasm that have been liberated from the cell, their chemical com- 
position should be similar to that of the protoplasm, mainly com- 
posing of proteins and lipids. However, some plant viruses unlike 
protoplasm are composed of proteins only. This presumably results 
from the lipid elimination which might occur on the destruction 
of the combination between protein and lipids due to the disturbance 
by the virus. Such plant viruses are, therefore, isolated usually in 
the form of particles which can be regarded either as lipid-free 
elementary bodies themselves or as their split products, 7. e@., thinner 
bundles or minute globular particles without lipids. 

Since thread-like protein molecules of the protoplasm may fail to 
contract when lipids are eliminated, the plant viruses without lipid 
may be bundles of protein threads. Usually hundreds of threads 
appear to form a single bundle, whose length, therefore, must be the 


i20 II. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


length of a globulin molecule stretching in its polypeptide chain. 

Such lipid-free particles, however, do not represent the only 
feature of the plant viruses, but they may rather be a peculiar feature. 
Thus, also in plant viruses coagulated elementary bodies themselves 
can be recognized as the most common virus particles as in animal 
viruses. In addition, also the elimination of lipid itself is by no 
means peculiar to plant viruses; various polyhedrosis viruses of 
insects are isolated in lipid-free, needle-shaped particles’ similar to 
certain plant viruses both in shape and length. 

Ammonium sulphate can sometimes expel the lipid from the plant 
protoplasm, so that crystals like those of some viruses are occasionally 
obtained from normal plant saps by treating with the salt. 

In healthy plant cells the combination between the protein and 
lipid appears to be so firm that lipid elimination fails to occur of 
itself without the action of a certain virus, and consequently the 
lipid-free needles are customarily believed as the only feature of the 
virus. It seems possible, however, to produce virus-like lipid-free 
particles in plant or insect tissues by the application of some proper 
agents other than viruses. 

The action of viruses may arise from the pattern of the protein 
molecules constituting the particles, and the effect of the pattern may 
be strengthened by the polymerization of the protein. Lipids are not 
necessary for the virus action, but indispensable for the protoplasm to: 
freely change in its configuration when infected by a virus. Owing to 
the lipids, proteins can change their structure in the protoplasm, the 
change being transmitted throughout the cell, thereby the structure 
specific to a virus is given rise to. The free motions of protein mole- 
cules in the protoplasm owing to the presence of lipids must be the 
essential picture of the life itself. 


5 


The bundles of protoplasm-protein threads appear to have the 
property to associate end-to-end to form unusually long filaments. As 
a consequence certain plant viruses may occasionally reveal them- 
selves in long filaments. At the same time, the bundle tends to split 
into shorter fragments, being accompanied, as a rule, by either a loss 
or decrease in the virus activity. It seems probable, however, that 
some plant virus proteins are so brittle that they can split into ex- 
tremely small fragments without great disturbance in their structure 
and consequently without loss of its activity. In such a case, viruses. 
can be obtained in extremely small particles. A number of plant 


X. THE SUMMARY OF PART II 121 


viruses are actually believed to be so small particles as their diameter 
are of the order of 0.03 or less. 

This is probably true also for the virus particles from which 
lipids are not eliminated. Thus, if coagulated fragments yielded by 
the split of elementary body retained the virus activity, the virus in 
question would be said to be very small particles. On the other 
hand, if every several elementary bodies were coagulated into a 
larger body on the combination with one another, the virus would 
reveal itself as a large particle, and if a number of elememtary bodies 
associated end-to-end and subsequently coagulated, thread-like parti- 
cles would be produced. 

Protoplasm proteins may be able to accomplish a reversible con- 
traction not only in the protoplasm but also outside the cell, so that 
certain viruses may be globular under some environments while filaments 
under others. If some proteins in an elementary body were left in 
the stretched state while most molecules were coagulated or folded, 
then the so-called tailed-virus would appear. 

The origin of various shapes and sizes of virus particles can thus 
be explained by the above concept concerning the protoplasm structure. 
At the same time it should be expected from the same concept that 
there may neither be shape nor size essential to virus particles. Vari- 
ous virus-like particles can. be:obtained from cells having no concern 
with viruses. 

Nevertheless, it seems to be -an established fact that particles 
which fail to be found in the normal cells can frequently be demon- 
strated in virus-infected cells by electron micrographs. Considerable 
changes may take place in the metabolic activity of cells when they 
are affected by viruses, resulting in the production of a different pattern 
in their chemical composition, especially in both amount and kind of 
nucleic -acids.. On account of such a change particles may become 
stable enough not to be disintegrated under the electron beam so 
that they are photographed fairly by the electron microscope. Thus 
particles appearing not present in the normal protoplasm will be 
raised on the infection with certain viruses. 

According to our investigation ‘under the microscope by dark-field 
illumination, red blood corpuscles, like other cells, are disintegrated | 
into virus-like particles on the application of adequate injurious 
effects. But such particles cannot be photographed by the electron mic- 
roscope unless Ringer’s solution is used, and even with Ringer’s solu- 
tion the particles are sometimes disintegrated into much smaller ones 
and photographed as such; the use of saline instead of Ringer’s solution 
may result in the failure of catching any particles in the electron 
micographs. Thus certain particles which do exist actually cannot be 


122 IIL. FUNDAMENTAL STRUCTURE OF PROTOPLASM 


photographed unless brought under proper conditions, and occasionally 
even revealed in shapes entirely different from the actual ones. 


6 


Viruses are commonly believed in chemical nature to be nucleo- 
proteins. The writer claims, however, that the pattern of virus-tem- 
plate is originally determined by the structure of protoplasm protein 
of a globulin nature, nucleic acids having nothing ‘to do with the 
pattern. The effect of the template is enhanced by the polymerization 
of the protein molecules, but if nucleic acids are inserted among these 
molecules, the polymerization products, 7. é., the virus particles, 
may become stable and rigid in the configuration so that the template 
action may be strengthened. In the particles without nucleic acid the 
mutual combination of the protein molecules is loose, and consequently 
the structure of particles are so unstable as to be readily destroyed 
and in addition the templating action itself is insignificant. 

On the other hand, when nucleic acids are contained in the particle 
to make its structure rigid, ,the particle may become more easily 
sedimentable, so that by means of the ultracentrifuge particles con- 
taining nucleic acid in rich amount may be readily isolated, and 
again since the virus activity is mainly to be retained in these particles 
for the reason just mentioned, only particles containing nucleic acids 
in rich amount may come, as a natural result, to be considered as 
viruses. 

Lipids, in contrast to nucleic acids, if contained in the particle, 
may render the structure easily changeable, and consequently may 
have an unfavourable influence upon the viruses against their action. 

Since the function of viruses is to act as the template, lipids are 
not only unnecessary but rather deleterious, whereas for the proto- 
plasm in which the replica is to be produced lipids are indispensable. 
In short, the free motion of protein structure in the protoplasm, a 
motion which is considered to be essential for the development of life 
phenomena, can be achieved only by the presence of lipids, whilst the 
motion may be hindered by nucleic acids. Therefore, particles con- 
taining great quantities of nucleic acids but little or no lipids are 
fitted to act as the template, so that such particles are liable to be 
regarded mainly as viruses. 


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Hershey, A.D. et al.: J. Gen. Physiol., (36), 777, 1953. 

Wyatt, G. R. and Cohen, S. S.: Bioch. J., (55), 774, 1953. 


PART, Vr 


THE EVOLUTION OF VIRUSES AND THE 
GENERATION OF THE SECONDARY ORGANISMS 


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CHAPTER I 
THE ORIGIN OF VIRUSES 


1. The Generation of Phage 


The possibility of the spontaneous generation of some viruses has 
long been argued by a number of authors. Among many viruses phage 
appears to have been discussed most frequently in connection with this 
problem. Phage is usually found in abundance in animal feces especially 
in those of chickens. The writer’s colleague, Ohashi (1) (2), has studied 
the origin of phage in chicken feces and has come to the conclusion 
that the phage in chicken feces is originated from the chicken itself. 

It may appear at first sight that the phage present in the feces 
may come from the phage taken by the chicken with food and that the 
phage, if enters undamaged the alimentary tract, may be able to mul- 
tiply by affecting the phage-susceptible bacteria that may thrive there 
normally. However, it has been confirmed by Ohashi that phage experi- 
mentally fed not only failed to multiply in the chicken tract regardless 
of the presence of phage-susceptible bacteria thriving there, but was 
destroyed to such an extent that only its extremely small portion could 
be defected in the feces. The phage amount detectable in the feces 
was only of the order of 1/1,000 to 1/10,0000 of that given by the mouth, 
showing that almost all the phage particles, far from multiplying in 
the tract by affecting the bacteria, had been inactivated before excreted 
with the feces. ; 

Thus, the conjecture that the phage in the feces may be originated 
from the one taken with the food does not appear to be correct, in 
spite of the fact that in the alimentary tract phage-susceptible bacteria 
are present abundantly. It cannot be expected that phage is continuously 
taken by chickens with their food in so large amounts. If phage is 
actually fed in any reasonable quantities, it will either be totally inac- 
tivated or, if its small portion can escape the inactivation, it will never 
reach the amount which can account for the large phage amount 
usually found in the feces. 

In addition it was found by him that phage amount in chicken 
feces was intimately related to the kind of diet; thus, the feeding of 
diet, such as wheat bran, soy-bean, greens, and rice bran, resulted in 
phage release, while the release would cease almost totally on the 


130 III. THE EVOLUTION OF VIRUSES 


administration of polished rice or wheat. Among the former group of 
diet, wheat bran was found the most effective. This effect was not 
destroyed even when the bran was exposed to 120°C. for 15 min., indi- 
cating that the effect was not attributable to phage contained in the 
diet, because no phage is so stable as to stand such high temperature. 

The conclusion that phage present in the feces is produced by the 
chicken itself is supported strongly by the fact that phage was found 
in a variety of organs of the chicken, such as brain, liver, kidney, and 
lung ; especially abundantly in mucous membranes of intestines. It is, 
however, a note-worthy fact that the demonstration of phage in organs 
was possible not only with chickens but also with mice, in whose feces 
phage could scarcely be found. Mackinley (3) likewise isolated phage 
from the organs of man and various other animals, and Tempe and 
Uhlhorn (4) from the blood of guinea-pigs and rabbits. ‘These facts 
indicate that the presence of phage in animal organs is a general 
feature. It is strange to say, however, that usually phage cannot be 
found in the feces of animals such as mice, guinea-pigs, and rabbits, 
unlike in chicken feces. What is the reason then why phage is demo- 
strated so readily in the feces of chickens while not in those of many 
other animals ? 

As above stated, the phages given by the mouth to the chickens 
were mostly inactivated in the alimentary tract, only their extremely 
small portion being excreted with the feces, while, on the other hand, 
it was confirmed that the degree of the inactivation in the alimentary 
tract was much greater in guinea-pigs than in chickens. In guinea-pigs 
usually the complete inactivation occurred and no phage was excreted 
in the feces even if extremely large amounts of phage were fed. This 
suggests that the absence of phage in the feces of the latter animals 
might be attributed to the complete inactivation of the phage which 
might be excreted by the organs into the alimentary tract. 

This deduction is further supported by the following fact: Guinea- 
pigs given with castor-oil excreted phage markedly in their diarrhoeatic 
feces, showing that the phage produced by organs was excreted without 
being inactivated in the intestine owing to the diarrhoea. Besides 
castor oil, some chemicals such as ethyl alcohol and sodium bicarbonate 
could occasionally exhibit the same effect, but the phage demonstration 
in the feces was possible only when diarrhoea occurred. 

It is said that phage was for the first time isolated from human 
feces suffering from dysentery. This might likewise depend upon the 
diarrhoea which prevented the inactivation of the phage. The phage 
isolated from dysentery patients is likely to be thought as being specific 
to the dysentery bacillus, but such appears not the case, generally no 
peculiarity being found in the specificity of the phage isolated from the 


I. THE ORIGIN OF VIRUSES 131 


patient feces (5). 

The existence of an intimate relation between the kind of diet 
and the phage excretion into the chicken feces is apparently brought 
about by the difference in the digestibility or coarseness of the diet. 
Thus, if a diet consists of coarse, indigestible materials, the defecation 
will readily occur in favour of the phage preservation which will further 
be effected by the envelopment of the phage by these coarse, indigesti- 

le materials so as to be protected from the action of destructive agents 
which may be present in the alimentary tract. 

Wheat bran, as mentioned above, was highly effective in the phage 
excretion but its water or alcohol-ether extract had no action, though 
the residue was somewhat effective. Moreover it was confirmed that a 
coarser bran could excrete phage more readily than finely smashed one. 
Further, wheat bran is the crust of wheat grains, but wheat grains 
themselves proved to be inactive. There seems no doubt, therefore, 
that the effectiveness of some diet in phage excretion was chiefly go- 
verned by its physical rather than its chemical properties. 

In the feces of animals such as mice, rats, guinea-pigs, and rabbits, 
the phage detection was generally difficult, whereas in those of dogs 
or donkeys it was usually easy, a fact possibly dependent upon the 
property of feces which varies with the species of animals, the coar- 
seness of the ingredient or the speed with which the food residues pass 
the digestive tract may play an important role. The digestive fluids 
likewise may vary with animals and may have some connection with it. 

Phage seems, however, not always produced by animal organs, 
since the phage detection in chicken feces is only possible in summer 
or at least in warm seasons; in winter no phage is found even when 
the chickens are fed wheat bran or led to diarrhoea by the administra- 
tion of castor oil. The cells of organs, therefore, seem to alter with 
the change of seasons in their character of producing phage; in winter 
may fall into a state unable to produce the phage. Thus the property 
of the cells of producing phage may not be constant, and may possibly 
be altered by proper agents other than the seasonal effect. The above 
mentioned action of castor oil or some diets such as wheat bran might 
be accordingly not only dependent upon their properties to hinder 
phage inactivation but also upon some other properties which may 
cause the cells to produce phage. 

In addition to the phage having thus its origin in animal organs, 
there appears to be another group of phage. It is generally known 
that certain bacteria produce an agent capable of acting as a phage 
upon certain other bacteria. Such bacteria are termed lysogenic. There 
seems therefore two groups of phage, one of which is produced by 
lysogenic bacteria and the other by certain animal celis. 


132 Ill. THE EVOLUTION OF VIRUSES 


That the phage detectable in animal feces is not originated from 
such lysogenic strains cam be shown also in the fact that there are 
distinct differences between the phage found in feces and that produced 
by lysogenic strains. Feces phage is much less resistant to heat than 
the bacteriogenic phage: for example, whereas the former is inactivated 
to such an remarkable degree as 1/1,000 to 1/10,000 on the exposure to 
65°C. for 5 min., the latter undergoes entirely no change even when 
heated at this temperature for 1 hour, and 75°C. for 20 min. is neces- 
sary for its inactivation to this degree; with several samples Ohashi (5) 
has found without exception this distinct difference between both groups. 
Moreover, he has confirmed that the plaques produced by bacteriogenic 
phage are commonly uniform in size, whereas those produced by feces 
phage tend to vary remarkably, indicating that there are much greater 
individual differences among feces phage particles than among bacterio- 
genic phages. 


2. The Reason for the Phage Production from 
Healthy Bacteria 


According to the writer’s theory, a virus is an assimilase similar 
to the protoplasm or a portion of the protoplasm. A virus can act as 
an assimilase upon a certain cell when the cell protoplasm is weaker 
in the assimilase action than that of the virus. A virus exists usually in 
minute particles into which the protoplasm has been disintegrated and 
coagulated following the infection with the parent virus. Through this. 
infection the structure of the protoplasm:is to be so changed as to 
become identical with that of the virus. If the structure of a particle 
is stable enough to retain the changed protoplasm structure the particle 
can behave as the virus towards another cell protoplasm, thus de- 
veloping its template action. 

This theory will naturally lead to the reasoning that normal proto- 
plasm particles or fragments of some healthy cells must likewise be 
able to behave as a virus to some other cells having a weaker assimi- 
lase action, provided the structure of the former cell protoplasm capable. 
of acting as a stronger assimilase is preserved in the fragments or 
particles. In such a case, the healthy cells may be said to have the 
property to produce a virus acting upon certain other cells. Lysogenic 
bacteria above cited should be recognized as such cells. 

Thus, if certain bacteria are disintegrated into minute particles, or 
fragments and if the original protoplasm structure is held unchanged in 
the particles or fragments, then the latter will be able to act as a 
phage upon certain other bacteria which have a weaker assimilase- 


I. THE ORIGIN OF VIRUSES 133 


action than the former. All the particles, however, may not always be 
able to act as the phage, for it is expected that sometimes certain 
particles only, whose structure is especially stable and strong, can 
exert the template action. Virus particles produced by healthy cells, 
therefore, also should have an intimate connection with nucleic acids. 

In this connection it should be noted the important fact that dis- 
ruption of lysogenic bacteria does not liberate infectious particle (6), that 
is, lysogenic bacteria seem to contain no virus, but that if the bacteria 
are treated with certain chemical or physical agents, such as ultraviolet 
ray, phage liberation will occur (7). This may result from the need 
of a certainslight modification in the healthy protoplasm protein struc- 
ture for the exhibition of the virus action. As detailed later, in the 
bacteria and animal cells the protoplasm protein threads appear nor- 
mally to be present in a partially folded state and the particles consis- 
ting of such partially folded peptide chains cannot act as the template. 
Accordingly, for the virus liberation, healthy cell protoplasm proteins 
have to unfold the chains under appropriate conditions. Ultraviolet ray 
may lead the chains to unfold. As we shall have many occasions to 
consider in detail of this important problem later, for the time being it 
may be wise to put aside this problem and make a further advance on 
the question we are now facing. 

Now, another reasoning led by the above theory is that the suscep- 
tible bacteria will become lysogenic when the bacteria are affected by 
the phage produced by lysogenic bacteria, since the structure of the 
susceptible bacteria is to be changed to become similar to that of the 
lysogenic bacteria through the infection, whereby the protoplasm of the 
susceptible bacteria is endowed with the strong structure able to act 
as a strong assimilase. 

As is well known this is actually the case. Bacteria infected with 
a phage derived from a certain lysogenic strain are changed into the 
lysogenic strain which produces in turn the phage by which they have 
affected. Freeman (8) has isolated virulent strain of C. diphtheriae from 
avirulent strain cultures by incubating with a specific phage; the bac- 
teria were found to become resistant to the phage, and even lysogenic. 

_ Freeman was unable to find any immunological difference between 
the original avirulent and derived virulent strains, but since the aviru- 
lent strains got possession of the faculty to produce toxin besides be- 
coming both lysogenic and phage resistant when inoculated with the 
phage, it is evident that the general property of the bacteria was al- 
tered, although the difference failed to be detected immunologically. 

The manner in which phage-susceptible bacteria are changed into 
lysogenic is illustrated diagrammatically in Fig. 16. The faculty to induce 
such a change must be, of course, present also in the intact protoplasm 


134 III. THE EVOLUTION OF VIRUSES 


or in the cell itself, since even in its fragments, 7. e., in the virus-like 
particles, the faculty can be proved. Smith (9) stated that non-lysogenic 
strain can be changed lysogenic through the incubation with the lyso- 


Lysogenic Non-lysogenic Bacterium changed 
bacterium susceptible bacterium into lysogenic 
eee oe Upp ee 
“— _o—— 
Phage particle Phage particle 


Fig. 16. The change of phage-susceptible bacterium into lysogenic 
by the phage produced by a lysogenic strain. 


genic strain; this change may not necessarily be ascribed to the action 
of phage freed from the lysogenic strain, because such a change should 
naturally to be expected in the interaction between two different strains 
without any participation of virus-like particulate agents. 

If the above conception as regards lysogenic strains is correct, 
every strain of bacteria may be lysogenic to strains having weaker 
assimilase actions than it, provided that the mutual combination be- 
tween them is established. Roustree (10) has proved that 27 out of 30 
strains of staphylococci which have been chosen arbitrarily are lyso- 
genic, and she stated that also with the remaining three strains lyso- 
genicity may be proven if suitable indicator strains are found. 

Such a phenomenon may not be confined to bacteria. If particles 
or fragments of the protoplasm of some animal cells can combine with 
certain bacterial protoplasm of weaker assimilase action the particles 
may be able to act as a phage upon the bacteria. The phage found in 
animal feces may belong to this category. 

In this connection, however, a serious question may arise as regards 
the establishment of the combination between the two different cell 
protoplasm, since there should exist in a virus a structure which cor- 
respond to a structure of the host cell protoplasm for the establishment 
of the combination between the virus and the host cell. The relationship 
in the structures between a virus and the host protoplasm may be 
analogous to that existing between an enzyme and the substrate. Viruses 
can combine with host cells through such a structure, and after the 
combination, structures in the virus not in conformity with those of 
the protoplasm exert their action to rearrange the protoplasm structure. 

Since the structure causing the combination distributed between a 
virus and the host protoplasm must be present, in general, between two 
protein molecules of a similar kind as already discussed in detail in 
Part II, a certain strain of bacteria can act as lysogenic upon a certain 


I. THE ORIGIN OF VIRUSES 135 


other strain of the same kind of bacteria, that is, a certain strain of 
colon-bacteria or of staphylococci can be lysogenic to a strain of colon- 
bacteria or of staphylococci, respectively. In this respect it may 
appear impossible that the protoplasm of animal cells share a structure 
which is in conformity with a structure in bacterial cells. Neverthe- 
less, this must be the case in order that the combination between the 
fragments or the particles of the protoplasm and the bacteria upon 
which the fragments may act as a phage is established; and this seems 
actually to be the case. Bacteria are commonly parasitic on some 
animals, and since the correlation between bacteria and the animals is 
considered to be analogous to that between a virus and the host cell, 
there should likewise exist a structure in common between the bacteria 
and the animals. 

If some bacteria combine with certain animal cells whose assimi- 
lase action is weaker than that of the bacteria, then the animal cell 
will be assimilized by the bacteria or, at least, will be disturbed in 
their protoplasm structure, thereby the bacteria may be able to multi- 
ply and the animal cells may fall into pathological conditions. In 
order to avoid such a disturbance the animal cells should be stronger 
than the bacteria in the protoplasm structure. If this were the case, 
also the fragments or particles of the protoplasm of such animal 
cells would also have a stronger assimilase action than the bacteria 
and would be able to act upon the latter as a virus. Such particles 
if excreted with feces would be called a phage. 

As is generally known, various animal organs produce an agent 
known as lysozyme which can affect certain bacteria to lyse them. 
Fleming (11) who found first this agent emphasized the similarity of 
the agent to phage. There is, however, a striking difference between 
these two agents. Namely, lysozyme cannot multiply unlike viruses. 
Thus it seems possible that lysozyme can combine with some bacteria 
and disturb the structure of the bacterial protoplasm to cause disin- 
tegration or lysis as does phage, but without producing exact replica 
and accordingly fail to multiply, presumably because of its structure 
not fitted for acting as a template. Even phage, under some condi- 
tions, only can cause lysis of bacteria without multiplying as pointed 
out already. 

On the other hand, as mentioned above, if the bacteria are stronger 
than some animal cells, the cells may be injured severely by the 
bacteria. In such a case particles of the bacterial protoplasm may 
likewise be able to affect the cells. Bacterial haemolysin may be re- 
garded as one of such agents; haemolysin is apparently protoplasm 
particles of bacteria which can combine with red blood cells; the 
structure of the latter is disturbed by the particles (Part II, Chapter 


136 III. THE EVOLUTION OF VIRUSES 


III), but without producing the replica corresponding to the bacterial 
template, so that haemolysin like lysozyme cannot be called a virus (12). 


3. Non-Pathogenic Viruses 


Bacteria, as a rule, undergo lysis when affected by a phage, but 
lysis does not always follow phage infection. Some phage can infect 
typhoid or coli bacteria without inducing lysis, though the phage can 
multiply through the infection (13) (14). Bacteria infected with a 
phage acquire the property to produce the phage likea genuine lyso- 
genic strains; thus certain bacteria at least can only be altered in 
their character through the infection with a certain phage, without 
being lysed. 

Since the multiplication of a virus consists in the replica formation 
in the host cells, the cell disintegration must be only a result of the 
multiplication, never an issue of necessity. The disintegration of 
bacteria into minute particles in case of the phage infection may be 
due to the destruction or the weakening of the mutual association of 
elementary bodies of bacterial protoplasm. caused by the structural 
disturbance by the virus. Prior to the occurrence of lysis, bacteria 
infected with phage, as is well known, may become indistinct in their 
contour and swell up, a fact which is probably attributed to the ap- 
pearance of lyotropic groups on the surface of elementary bodies as 
a result of the structural disturbance, most probably of the unfolding 
of the peptide chains. The disintegration may be thus caused by the 
accumulation of water molecules among elementary bodies. If some 
cells can endure such a change no lysis may follow. It is also con- 
ceivable that the degree or the rate of the liberation of lyotropic 
groups may vary with the kind of viruses which induce the change; 
thus certain host cells are disintegrated by some viruses while not by 
others. 

It has frequently been noted that the virus multiplication is not 
always accompanied by the manifestation of any pathological disorder. 
The pathological disorder may be manifest when the changes lead to 
the disintegration of the cells. Certain strains of influenza virus 
multiply extensively in the mouse lung without producing pneumonia, 
whereas a closely related variant may, under the same conditions, 
produce pneumonic consolidation. Moreover, some strains of Coxackie 
virus multiply to an equal degree in a number of organs but fail to 
produce lesions except at certain specific sites (15). 

On the contrary in some cases pathological changes are most 
remarkable, while virus multiplication scarcely occurs. Ginsberg (15) 


I. THE ORIGIN OF VIRUSES 137 


found, for example, that infectious Newcastle disease virus particles 
produce extensive pulmonary consolidation in the mouse in the absence 
of demonstrable virus multiplication, the lesion being indistinguishable 
from those of influenza A virus infection. It may be possible to 
regard lysozyme or haemolysin as virus-like agent which, like New- 
castle virus in this example, can cause a remarkable damage in the 
cell without multiplication. 

The injurious action of viruses upon the host cells may probably 
be effective only during the process of the virus multiplication; with 
the finish of the process the host cells will be released from the suffer- 
ing and will be able to behave like apparently healthy cells, unless the 
structure itself which has been provided by the virus is injurious to 
them. A virus as a rule can be detected in abundance in plant cells 
which have been recovered from the infection by the virus. Also in 
the case of animals, certain viruses are reported to be detected in some 
tissues long after the recovery from the virus infections. 

The injurious effect, as above stated, is not necessarily associated 
with the virus multiplication, a fact which is clearly shown in the 
term of “‘inapparent”’ or ‘‘subclinical’’ infection, which is often regar- 
ded as a characteristic manifestation of viruses. Thus it is generally 
accepced that viruses tend to cause infections that do not give rise to 
clinically evident diseases. 

It will, therefore, naturally follow that fragments or particles of 
some protoplasm capable of producing replica in other cells may fail 
to be realized of their existence because of their inability to cause 
any injurious effect, and that, even when their existence was acknow- 
ledged, they would not be called viruses. The well known principle 
found by Avery ef e@! (16) with pneumococci can be regarded as one of 
such “‘viruses.’’ This ‘‘virus’’ is produced from virulent, capsulated 
pneumococci and can convert avirulent, non-capsulated culture to the 


virulent type. The possible mode of action of this agent is illustrated 
in Fig. 17. 


ee A is pneumococcus 


S 66 66 


3S _—S agent * 


Fig. 17. The change of R-type pneumococcus into S-type by the trans- 
forming agent produced by S-type. 


138 Ill. THE EVOLUTION OF VIRUSES 


It is most remarkable that this agent is reported to consist mostly 
of nucleic acid, and according to Hotchkiss (17) the protein content of 
the agent can be, at most, 0.2 per cent and is probably less. If this 
is true, it must be admitted that the template action is sometimes 
accomplished by nucleic acid only without being associated with any 
protein, although this possibility is not entirely inconceivable as dis- 
sussed previously (Part II, Chapter VIII). 

The nucleic acid concerning this reaction is confirmed to be 
desoxyribonucleic acid, whereas this acid isolated in a pure form is 
reported to exist in forming virus-like particles; its molecular weight 
about 8,000,000 and the length of the particle, 3000my (18). This sug- 
gests the possiblity that even the particles composed of nucleic acid 
only can behave like a virus. 

From avirulent strain could be extracted similar nucleic acid 
resembling, in its chemical composition, to the agent isolated from the 
virulent strain, but it was wholly inactive in producing transformation 
(19), a fact which should be naturally expected, since the agent from 
the virulent strain can transform the avirulent only because the 
former structure is more powerful than the avirulent, and accordingly 
this avirulent strain itself or its particles should be unable to exert 
any influence upon the virulent. 

The nucleic acid complex isolated from the avirulent strain (R- 
type), however, can transform a variant (ER-type), which produces 
very rough colonies, to R-type, indicating that ER-type is still weaker 
than R-type. In this connection, it should be noted that transfor- 
mation in the reverse direction, R-ER, can be carried out with a 
transforming agent of the ER variant if anti-R serum is present 
(20). This fact shows that despite its weak structure the agent isolated 
from ER variant can overcome the R-type owing to the assistant 
cooperation of the antiserum capable of disturbing the structure of 
R-type. 

Similar transforming agents have been isolated from various other 
bacteria such as E. coli (21), Shigella (22), and Haemophillus (23). 
Furthermore, it has long been known that certain strains of bacteria, 
grown in the presence of culture filtrates or extracts of related orga- 
nisms, acquire some of the properties of the latter. Thus, it was 
possible to convert colourless and nonproteolytic culture of B. pyocya- 
meus into virulent and pathogenic form, by two consecutive passages 
in the culture filtrate of a virulent, pigmented and proteolytic strain 
of the same species. There are also claims that cultures of strepto- 
cocci or of Gram-negative bacilli exposed to the cells or products of 
serologically different, but biologically related species, can develop the 
specific agglutinability of the latter (24). 


I. THE ORIGIN OF VIRUSES 139 


4. Latent Viruses 


A vast number of examples of virus production by apparently nor- 
mal cells can be shown also in animals. For instance the occurrence 
of Theiler’s mouse encephalitis virus in the intestines of normal albino 
mice has been well established. As reported by Olitzky (25) and ascer- 
tained by other workers, whilst foetal and sucking mice up to 12 days 
of age are free from this intestinal virus, 20 to 25 days old mice har- 
bour it irregularly, 30 days old mice invariably and old mice (6 or 
more months) again irregularly. Again, it is generally known that in 
the submaxillary gland of normal guinea-pigs a virus is contained, 
which can cause a meningeal symptom, when injected into the brain 
of guinea-pigs. It merits attention that this submaxillary-gland virus, 
like that of Theiler, is found only in the gland of adult animals, 
never in the gland of young ones. Furthermore, an extract of normal 
rabbit skin was confirmed by Daneal (26) to be able to induce serially 
transmissible papillomata on the skin of other domestic rabbits. 

A series of neurotropic viruses, like that of poliomyelitis, have 
been isolated from various sources of animals, apparently having no 
association with the disease, such as feces or excretes of animals or 
men, and also from insect juices. A group of viruses having the 
name of Coxackie may be regarded as a type of such viruses. It is a 
note-worthy fact that non-biting flies are regarded as disseminating 
agents of both poliomyelitis and Coxackie virus, both viruses being 
frequently recovered from flies. There are striking similarities in the 
distribution of these fwo groups of viruses in nature. 

A factor in brain tissue which induces acute disseminated encepha- 
lomyelitis, when injected into rhesus monkeys has been found by 
Kabat ef al. (27) in human, monkey, rabbit and chicken brain. It is 
present in the spinal cord of 3 days old rabbits, but does not appear 
in the rabbit brain until about the 12 th day of life. 

An infectious agent, which appears to be a virus has been isolated 
from the liver of a normal wild raccoon which has led to a highly 
fatal type of disease characterized by conjunctivitis and an elevated 
serum bilirubin, frequently accompanied by jaundice on inoculation of 
raccoons. Ferrets also appears to be susceptible to infections with 
this agent (27a). 

Such examples appear to be too numerous to be cited, but are 
commonly regarded as evidences of detection of ‘“‘latent viruses’, 7. é., 
viruses present in host cells causing an inapparent infection. Pre- 
sumably, this may hold for some examples above cited, but it appears 


140 III. THE EVOLUTION OF VIRUSES 


more reasonable to consider that most latent viruses are protoplasm 
particles of healthy cells stronger in the assimilase action than cer- 
tain other cells on which they can act as viruses. 

Similarly in plants the detection of latent or silent viruses is not 
unusual. A well known example of plant viruses, apparently belong- 
ing to this category, is produced by paracrinkle virus, which is found 
in every plant of the potato variety named King Edward, and which 
is actually regarded by some workers as a normal product of the 
metabolism of the plant with the characters of a virus when trans- 
ferred to other potato varieties. Moreover, the so-called latent virus 
of potato mosaic is said to occur in almost all of the potato plants 
grown in America. It causes no damage to the potato plants, and, 
because of this fact and because of its almost universal presence in 
potato plants, it has come to be called the healthy potato virus. This 
has likewise occasionally been regarded as a normal constituent of potato 
plant. According to Smith (28), samples of apparently healthy sugar- 
beet and mangold collected at random from different farms in England 
and Scotland were all found to contain a virus, the presence of which 
can be rapidly demonstrated by inoculation of the sap to the first 
leaves of cowpea seedlings, which results in the production of charac- 
teristic lesions. 

Whereas various plant viruses are, as is well known, transmitted 
by insects, it is believed that viruses are latent in a high percentage 
of apparently healthy caterpillars (29). Also there are many evidences 
that neurotropic viruses are present in normal insects which transmit 
commonly various animal virus diseases. Since the relation between 
insects and animals or plants, upon whose blood or sap they thrive, 
may be analogous to the relation between bacteria and animals, on 
which the bacteria are parasitic, there should be a common protoplasm 
structure between them. Therefore it seems a natural result that the 
protoplasm particles of some normal insects behave as viruses towards 
some animals or plants. The reason why the protoplasm particles of 
animal cells can act as a phage upon some bacteria can be explained 
in a similar way as already discussed. 

The various facts above cited have naturally led a number of 
workers to the theory that viruses are originated from normal cell 
components. For example, Darlington (30) has postulated viruses ori- 
ginating from specified cytoplasmic units such as plasmagenes. Again, 
de Buy and Woods (31) have expressed the opinion that viruses are 
produced from plastids and mitochondria which have been altered in 
the form and function. 

Since these cytoplasmic particles are known to contain nucleic 
acids in large quantities, also from the writer’s theory it appears 


I. THE ORIGIN OF VIRUSES 141 


highly probable that such particles can act as viruses. It may, how- 
ever, be unreasonable to postulate these particles to be only agents 
able to become viruses. Nucleic acid is contained in most abundance 
in genes themselves and there is a good reason to consider that these 
particles, the genes, have the greatest template action in the cell. 
Therefore, genes are most likely to behave as viruses when they are 
liberated from the cell. On the other hand, it has been suggested by 
a number of authors that viruses may be genes that have gone wild, 
acquiring in the process the ability to exist independently in the cell 
and to move fromcell. Thus the ‘‘free gene’’ theory has been advanced 
by Muller (32) and Duggar and Armstrong (33). 


CHAPTER II 
THE GENERATION OF VIRUSES 


1. Changes in the Structure of Protoplasm Protein 
Leading to Virus Generation 


Many evidences cited in the previous chapter may indicate that the 
normal protoplasm particles of certain cells can sometimes exhibit 
their template action upon certain other cells as viruses. Although 
the existence of such normal particles never mean the generation of 
new viruses, it seems highly probable that new viruses can arise in 
some cells if the protoplasm is altered in its structure following the 
change of environmental factors, because the kind of viruses should 
be determined by the structural pattern of the protoplasm. 

Bacteria sensitive to penicillin usually develop resistance to the 
antibiotic when grow in its presence. It is of most interest that the 
extracts of bacteria thus acquired the resistance are able to transmit 
this character to other bacteria (34). The effective agent prepared 
from a resistant strain has been reported to be involved in a nucleic 
acid fraction (35). This can be interpreted as indicating that the bac- 
terial protoplasm undergoes a change in its structure under the influ- 
ence of the antibiotic and that, as the newly formed structure is 
stronger than the original one, some particles of the protoplasm thus 
changed containing nucleic acid in rich amount can exert like a virus 
their structural influence upon the unaltered bacteria. This may, there- 
fore, be regarded as an example of the generation of a new virus due 
to a certain stimulus given to the cell. 

It is, however, unreasonable to expect that a newly formed 
structure is always stronger than the original... Voureka (36) has re- 
corded the interesting observation that some strains of penicillin-re- 
sistant staphylococci and streptococci lost their resistance during 
exposure to a penicillin sensitive microorganisms or to the extracts of 
sensitive bacterial cells. It should be noted that the sensitizing prin- 
ciple was found in the ribonucleic acid fraction isolated from the 
sensitive bacteria (37) (38). This fact indicates that the newly for- 
med structure is weaker than the original and, therefore, the original 
structure can overcome the newly formed one. The sensitizing princi- 
ple must be nucleic acid rich particles, behaving like a virus towards 
the adapted bacteria. 


II. THE GENERATION OF VIRUSES 143 


Virus-like agents seem to be produced occasionally during the 
adaptation process of microorganisms. It cannot be considered, how- 
ever, that the generation of a structure capable of acting as a virus 
is necessarily associated with the adaptation. On the contrary, even 
if a structural change takes place which is unfavourable for the or- 
ganism, a virus-like agent will be found in the fragments of the 
protoplasm thus changed in its structure, provided that the newly 
formed structure is stronger than the original. 

As is generally known, viruses are liable to alter in their charac- 
ters with the change of environmental conditions. For example, some 
viruses undergo variations when exposed to proper amounts of N-mus- 
tard or of ultraviolet light. Such a variation is, as will be mentioned 
later, considered to be based upon a structural change in the virus 
protein caused by the environmental effects. These effects capable of 
giving rise to the change in viruses, 7. é., in particles or fragments 
of protoplasm, can, as a natural result, exert their influence upon the 
protoplasm itself to cause changes in its structure, altering the charac- 
ter of the cell itself. Thus the variation or mutation of organisms 
will be brought about as a result of the alteration in the protoplasm 
protein due to the change in the environmental conditions. 

In fact, microorganisms such as bacteria are known to undergo 
variation or mutation when treated with N-mustard or with X-ray. 
If the newly formed structure of the protoplasm is stronger than the 
original, its particles will be able to act as a virus. In short, it may 
be said that the mutation of organisms is, in general, liable to be 
associated with the generation of a virus. 

However, as will fully be discussed in Part V, there are good 
reasons to suppose that newly produced structures are, as a rule, 
weaker than the original, so that it cannot be expected that mutation 
is always associated with the production of new viruses. On the con- 
trary, it should be considered that the generation of new viruses 
will take place only on rare occasions, since a newly produced virus 
is always provided with a structure weaker than the original. 


2. Environmental Change and Virus Generation 


As stated in the foregoing chapter, there are many evidences sug- 
gesting that normal, healthy cells are producing viruses. The viruses 
appear, however, are not constantly excreted by the cells, but in most 
cases their production seems to be connected intimately with the en- 
vironmental conditions. For example, Theiler’s virus cannot be found 
in the feces of very young mice, though when the mice grow older to 


144 III. THE EVOLUTION OF VIRUSES 


a certain age the virus is constantly excreted. The same is true for 
submaxillary-gland virus of guinea-pigs. These facts suggest that 
the animal cells may undergo a change in their protoplasm structure 
with the growth of the animal to yield the virus at certain periods 
of age. Such physiological change of the protoplasm structure to be- 
come capable of acting as a virus may be regarded as another example 
of virus generation. 

The fact that phage can be detected in chicken feces in warm 
seasons, especially in summer, may also be explained by the assump- 
tion that the protoplasm structure of some chicken cells capable of 
acting as a phage can be provided only in warm seasons, in winter 
the structure being lost. 

As emphasized by Bawden (39) the flowering-stimulating agent 
called florigen, present in the leaves of some plants, is remarkable in 
its virus-like properties; thus, like a virus, it moves from the cells in 
which it occurs initially and can be transmitted between plants by 
grafting. It is a fact worthy of note that this agent can be produced 
in the leaves of some plants when the latter are exposed to day-light 
of an appropriate duration. ‘This may indicate that a certain struc- 
tural change may be brought about in the protoplasm of the leaves on 
the exposure to day-light, the change being transmitted by a virus- 
like agent to another plant to stimulate the flowering. In this con- 
nection, it is of interest to note that Japanese encephalitis is believed 
to occur frequently when one’s head is exposed to the violent day- 
light of summer. Again lysogenic bacteria which usually fail to 
liberate phage may be lysed with phage production on the irradiation 
with ultraviolet light (7). 

These facts have led the writer to consider that the environmental 
effect may cause a change in protoplasm structure of some cells, the 
fragments or the particles of which are subsequéntly enabled to trans- 
mit the changed structure to other cells as a newly produced virus. 

It should, however, be emphasized that for the production of a 
structure capable of acting as viruses, there should exist the cell pro- 
toplasm having the character to be altered into the virus structure by 
the environmental effect. In other words, for the generation of viruses 
certain cells are necessary possessing the character to generate the 
viruses; namely, certain cells with the predisposition to yield the 
virus are needed in addition to appropriate environmental factors. 
Viruses are yielded by such cells when the cells are exposed to certain 
stimuli involving environmental changes. Thus, a certain protoplasm 
having a certain predisposition and a proper simulus are essential for 
the generation of a virus. 

In the case of Theiler’s virus, for instance, the mice having the 


II. THE GENERATION OF VIRUSES 145 


predisposition to produce the virus are necessary. It is known that 
there are virus-free strains of mice which never produce the virus 
throughout their life. 

Cancer is, in a sense, a virus disease. As is well recognized, for 
its production above cited two factors, z. e. predisposition and stimulus, 
are indispensable. The important significance of predisposition has 
been well realized in the case of mammary cancer of mice, in the 
generation of which a virus is believed to be involved. Likewise in 
the case of fowl leucosis, a kind of malignant tumour caused by a 
virus, the predisposition of chickens plays an important role. It has 
been reported that in a certain line of chickens the disease is almost 
completely absent, whereas in some other lines over 50 per cent 
are afflicted by the disease (40). 

Common cold is undoubtedly a virus disease, and as is well known 
the disease is established when a causative factor, chilling, is effected 
to man predisposed to catch cold. It merits attention that herpes 
febriles, known to occur following febrile discases such as pneumonia 
and malaria, may also be induced by artificial application of heat, 
such as infra-red ray or hot water bath. Moreover, poliomyelitis is 
liable to occur after trauma of various sorts especially following ton- 
sillectomy. For the occurrence of these virus diseases it is needless 
to say that predisposition is also an essential factor. But it is com- 
monly supposed that in such cases latent viruses are activated by the 
stimuli to cause manifest infections. This may indeed be an explana- 
tion more reasonable than to assume that viruses are newly generated 
by the stimuli, as will be fully discussed in Part V, but according 
to the writer’s theory latent viruses are not the viruses themselves. 
They are present ina state of ‘‘provirus’’ before activation; “‘provirus”’ 
is the protoplasm structure able to be changed into virus structure by 
adequate stimuli. In short, by the term ‘“‘provirus’’ is meant predis- 
position. In this connection, it should be mentioned that sometimes 
an active virus itself is enhanced in its activity by the presence of 
an adequate stimulus. 

For instance, it is stated that when the leaves of tebacco piant 
are rubbed with carborundum the number of local lesions produced by 
tobacco mosaic virus increases approximately to such an extent as if 
the virus concentration were increased a hundred-fold. Potato virus A 
is sap-inoculable to potato only when carborundum is used (39). It is 
also stated that when the virus of Rous sarcoma is injected into the 
blood of a fowl, the virus seems to become distributed throughout the 
blood and organs in the body but does not produce tumours. If, how- 
ever, a muscle is injured immediately after the intravenous injection 
of the virus, then a tumour develops at the part of injury, but if the 


146 III. THE EVOLUTION OF VIRUSES 


injury is made before injection of the virus, then no tumours are 
developed (41). A similar phenomenon is also observed with papillom 
virus: When a rabbit is injected intravenously with the papillom 
virus after smeared repeatedly with tar on the skin, a papillom de- 
velops only in the skin on which tar has been applied. The growth 
of the papillom is much more vigorous than the case when the virus 
only is applied, occasionally even developing to a cancer. Instead of 
tar, methyl-cholanthrene can be used to cause the same effect. Thus 
the infective faculty of the virus can be increased up to about a 
hundred-fold (42) (43). 

These phenomena should not be compared to the above stated 
change of provirus into virus. These may only suggest that viruses 
can produce their replicas more readily in the cell protoplasm when 
the protoplasm is disturbed in its structure by proper stimuli. As dis- 
cussed already, virus particles which have been inactivated to a 
certain degree can sometimes exert a favourable influence upon the 
intact virus to infect the host cells, showing that the inactive parti- 
cles, though unable to produce their replicas, can disturb the proto- 
plasm structure of the host cells contributing to the infection. 


3. The Seasonal Change in the Virus Infection 


Generation of viruses is apparently connected with the environ- 
mental change as above stated. Especially the connection seems in- 
timate when the seasonal change is involved. The seasonal effect on 
the virus production is seen in the case of chicken feces phage as 
already mentioned. There are many other examples showing the same 
correlation. Poliomyelitis is prevalent in early autumn, common cold 
and influenza in winter, and smallpox from winter to early spring. 
German measles, according to our investigation, occurs preferably in 
early spring (44). Japanese encephalitis is epidemic only in summer. 

In addition, seasonal factors affect the symptom picture of many 
plant virus diseases aS well as the pattern of their occurrence. Ac- 
cording to Smith (45), the bright yellow mottling characteristic of the 
aucuba type of tobacco mosaic does not develop under winter condi- 
tions in the glass-house but affected plants show an indistinct green 
mottle only. If, however, inoculations are made from a plant showing 
this indistinct mottle to other plants which are kept under artificial 
illumination, the characteristic bright yellow mosaic will develop in 
the illuminated plants. 

Such seasonal effects may appear at first sight to be dependent 
upon the seasonal change in the resistance of host cells against the 


II. THE GENERATION OF VIRUSES 147 


viruses not upon the generation of viruses themselves. However, as 
will be mentioned later, there are many good reasons to assume that 
effect is involved rather in the virus generation or in the provirus 
activation than in the alteration of the host resistance. 

The writer made an investigation into the seasonal effect upon the 
incidence of measles, and obtained interesting data. The investigation 
was carried out with pupils of Japanese primary schools in Shanghai, 
when the writer was a member of the Shanghai Science Institute 
(46). 

The seasonal effect upon the occurrence of the disease was found 
to be very striking as shown in Fig. 18. However, the curve shown 


Index of infection 


1 on Se APL Olea on eo pO IR Ne 42 


Months of infection 


Fig. 18. The seasonal change in the incidence of measles in Shanghai. 


in this figure representing the relation between the morbility and 
season is distinctly different from those obtained by other investigators 
at different districts as indicated in Fig. 19, in which it is also shown 
that the season effect varies considerably from district to district. In 
Shanghai and Cleveland the epidemic occurs only once in a year, in 
late spring or in early summer, while in other districts, especially in 
Wien and Ltibeck, in addition to this peak, the second epidemic is 
seen in late autumn or in early winter, although in Shanghai there 
is some indication of this second peak. 

On the other hand, it is of very interest that the seasonal change 
of impregnation of Japanese women bears a striking resemblance to 
that of the morbility as indicated in Fig. 20. The impregnation 
months cited in the figure are calculated by substracting 3 from birth 


148 II. THE EVOLUTION OF VIRUSES 


months. Also with Europeans living in their native countries the 


Same is the case. In most cases with Europeans two peaks are found 
as with Japanese in Aomori. 


Index of infection 


Months of infection 


Fig. 19. The seasonal change in the incidence of measles in various 


regions. I: Shanghai. II: Cleveland. III: Wien. IV: Hamburg. 
V: Liibeck. 


Index of impregnation 


re ee ee ee ee oe eee Ce el ht Sah eee 
Months of impregnation 


Fig. 20. The seasonal change of human impregnation in various parts 
of Japan. I: Nagasaki. II: Iwate. III: Hokkaido. IV. Aomori. 


II. THE GENERATION OF VIRUSES 149 


This peculiar seasonal change in the human impregnation may be 
based upon certain metabolic activities mainly concerning with hor- 
monal secretions, upon which the season may have great influences. 
It may be a reasonable explanation that the same seasonal influences 
are involved in the morbility change of measles, and the change in 
the susceptibility to the disease due to season may account for the 
phenomenon. However, if the susceptibility fluctuate regularly with 
season, measles virus should be present everywhere, always, and in 
abundance, in order that the susceptibility curve was precisely repre- 
sented by the morbility curve, since the elevation of susceptibility 
would not be followed by the increase in the morbility unless the 
virus was present everywhere and in abundance. Can this actually be 
possible? As the time seems not ripe to discuss furhter of this pro- 
blem, it will be left untouched until we have a proper occasion later. 

In order to show how complicated is the problem concerning the 
virus generation, an experiment carried out by us (44) in connection 
with phage production will be discussed below. As above stated, 
phage is excreted from some animal cells, while flies appear likewise 
to excrete phage. 

According to our investigation, common house flies almost con- 
stantly contained large amount of phage. However, phage could never 
be detected in the flies which had been raised from eggs in rearing 
boxes. If large amounts of phage were fed to such flies with foods, 
only small portions of the phage were found in the flies and even the 
detectable phage itself would disappear in a few days. It was quite 
impossible to make them carry phage as in naturally living flies. In 
addition, naturally living flies carry the virus only in summer, a fact 
comparable to the phage detection in chicken feces only in this season, 
suggesting that the virus is also excreted by the insect as by other 
animals. 

Thus it was supposed that some factor or factors capable of stimu- 
lating the phage production might be lacking in the reared flies. We 
were, however, unable to find the supposed factor, although a variety 
of kinds of foods were given under various conditions in taking into 
consideration such physical factors as temperature and sun-light. 

Though we failed to arrive at any definite conclusion on this pro- 
blem, our finding may present some suggestions as to the origin of 
common cold. As stated above, for the occurrence of common cold the 
predisposition and the stimulus, 7. e., chilling, appear to be necessary, 
but according to English workers (48), chilling neither induces nor 
favours common cold. The writer is inclined to think that such an 
unexpected conclusion, obviously contradictory to every man’s personal 
experiences, has been arrived because of the isolated, unnatural living 


150 II. THE EVOLUTION OF VIRUSES 


conditions under which the volunteers were left, for it is a very well 
attested fact that among a group of people isolated on a remote island, 
particularly if the community is small, common cold tends to die out. 
The experiments were reported to have been carried out in a solitary 
environment. The volunteers in this case, therefore, may be comparable 
to the flies bred by us in laboratory. In both cases some factor may 
be lacking against the virus production. According to the English 
workers, although common cold was induced in the volunteers when 
the virus was administered, the symptoms were unusually slight, 
mostly without fever, recovering in two or three days, and about 
50 per cent of the volunteers did not show any symptoms. This may 
remind one of the fact above mentioned that phage harbours only for 
short periods, when the virus is administered to the reared flies. 
Thus the factor necessary for the virus production may be needed also 
for the thriving of the virus in the host cells. 

It should be remembered in this connection that poliomyelitis virus 
and also Coxackie virus, which is closely related to the former, are 
very frequently isolated from flies, suggesting that these viruses, like 
phage, may be produced in flies. A number of evidences can be pre- 
sented to suggest that epidemic poliomyelitis cannot be explained on 
the basis of person to person contact, but that the epidemic seems 
rather to be induced by a common source. Attention should be paid 
on the fact that the seasonal concentration of healthy virus-carriers, 
especially virus-carrying flies, is proved in the area where the disease 
is prevailed (49). 

Like phage, both poliomyelitis and Coxackie virus fed to flies bred 
in laboratory not only fail to multiply in the flies but also fail to be 
retained in the insect body for long periods, suggesting that only 
naturally living flies can produce the viruses (50). 

Rous sarcoma can be induced by the stimulus of tar, and it can be 
sometimes transmitted by a virus-like agent, whereas the action of 
the latter is remarkably enhanced by the simultaneous application of 
tar. A factor or factors like tar in this case may be lacking in the 
above case of common cold, whose virus may be unable without such 
a factor to exhibit its full action and at the same time the virus it- 
self may not be produced. The virus, however, may be incapable of 
thriving indefinitely even when the factor is present, but gradually 
disappearing, and the virus may be generated de novo in succession. 


CHAPTER III 
IMPERFECT VIRUSES 


1. Eczema or Allergic Dermatitis 


There is a large group of skin diseases called eczema, a dermatitis 
usually accompanying itching, erythema, vesicles or pustules. It is 
acknowledged that for the occurrence of this disease both the predis- 
position liable to be affected by this disease and adequate external 
irritants are needed. 

The mechanism of eczema formation may be as follows: Skin 
cells having the predisposition may change in their protoplasm struc- 
ture by the effect of a stimulus, and the change thus occurs may 
subsequently spread successively to surrounding cells followed by 
various pathological symptoms. The symptoms may be attributed in 
the main to the disturbance in autonomous nervous systems. 

An abnormal reactivity of man to some agents is termed allergy, 
and eczema is commonly regarded as a kind of allergy. It should be 
borne in mind that allergy cannot exist without a peculiar predisposi- 
tion which is occasionally hereditary and that the symptoms cannot be 
brought about without a proper stimulus. Common cold, as mentioned 
in the previous chapter, is also raised when a stimulus, chilling, acts 
upon the peculiar predisposition, although some other factor or factors 
appear to be needed, and hence sometimes common cold is also re- 
garded as a kind of allergy. 

If, in the case of allergic dermatitis, the fragments or particles of 
changed protoplasm were stable enough to retain its changed struc- 
ture, they would be able to give rise to the same changed structure 
in other healthy cells as a virus. This is, however, never the case, 
presumably because of the lability or the weakness of the newly in- 
duced structure. The dermatitis cannot be called, therefore, a virus 
disease, although it may not be quite impossible to transmit the dis- 
ease through the protoplasm fragment, if the latter is applied to a 
proper healthy region of the skin of the same individual immediately 
after the isolation. 

The newly induced structure causing the dermatitis seems to be, 
on the one hand, stronger than the original, because the structure 
has to overcome the original structure at least for the period of its 
spreading, while on the other hand its strength seems to be lost 


152 : III. THE EVOLUTION OF VIRUSES 


promptly, as the pathological structure may soon. disappear with the 
revelation of the normal. As a rule pathologically changed skin re- 
covers its normal state in a few days when the causative agent is 
eliminated. Such a transient nature of the change may account for 
the failure of detecting the structure in the protoplasm particles. As 
will be stated later in greater detail, the change is transient because 
it is readily reversible. 


2. Cancers 


There are good many reasons that the structural change in proto- 
plasm is generally reversible. In the case of the dermatitis above 
mentioned the reversibility seems very manifest, whereas there occur 
sometimes changes which appear to be irreversible, never to be expel- 
led by the normal structures. Occasionally such changes may even 
overcome gradually the surrounding normal structure to proliferate 
still further at the cost of the latter. 

Tumours may be involved in such irreversible changes. The high 
persistence of the changed structure may be ascribed to the transmis- 
sion of the change to the genes themselves. The structural change 
in the gene, if occurs, should give rise to new cells which may be 
called tumour cells. The proliferation of the changed structure can 
be achieved by the multiplication of such cells. When the multipli- 
cability is so striknig that the cells can proliferate indefinitely, the 
cells may be called malignant tumours, whose representative is can- 
cer. The cancer cells multiply at the expense of normal cells so vi- 
gorously that the animals themselves affected by cancer are ultimately 
to be killed. 

It is generally known that cancers are induced by a variety of 
physical or chemical agents, and it is also well realized that there is 
the predisposition to cancers. It seems, therefore, probable that the 
protoplasm having the predisposition must be changed in its structure 
by some stimulus to give rise to cancer cells. If cancerous structure 
was preserved in protoplasm particles, the cancer would be said to be 
transmissible by a virus. Cancers are usually readily transmissible by 
cancer cells but the transmission through the particles or a virus 
seems difficult to occur, suggesting the lability or the weakness of the 
structure. 3 

One of the best known tumours proved to be transmissible by a 
virus is Rous tumour, a kind of cancers affecting fowls. The finding 
that this tumour could be induced by a virus might be shocking to. 
those who regarded viruses as microorganisms like bacteria, but in 


III. IMPERFECT VIRUSES 153 


the writer’s concept of the nature of viruses it is never an unex- 
pected result. In addition to this tumour, papilloma which affects cot- 
tontail rabbits is known to be transmitted by a virus, and which is 
commonly called Shope papilloma for it was described first by Shope 
as the fowl tumour is called Rous tumour after its first observer. 
Shope papilloma is characterized by the presence of warty or horn-like 
protuberances on various parts of the rabbit body, whereas human 
wart was also ascertained to be infectious through a cell-free filtrate 
long before the virus nature of Shope papilloma was confirmed. 
Mammary cancer of mice is likewise believed to be caused by a virus. 
This virus has been demonstrated in the milk and also in high con- 
centrations in both the lactating mammae and mammary cancer of in- 
fected mice. The injection of so little as 107° to 10° gm. equivalents 
of lactating breast tissue has been sufficient to infect suitable host 
animals (51). The virus nature has been confirmed also with a kidney 
cancer of a frog, Rana pipiens. ; 

Again, fowl leucaemia can be regarded as a kind of malignant 
tumours, and it was found that the infection could be transmittted by 
0.000,0001 ml. of cell-free plasma, and that the virus could withstand 
drying for at least 54 days (52). Furthermore, both a myxoma and 
~ fibroma of rabbits can be transferred by filtrates of the tumour sus- 
pensions, and it is said that the virus of one of these diseases has a 
remarkable relationship to the virus of the other, and in view of the 
pathological changes caused by the viruses, these two diseases are 
considered to lie between tumours and usual virus diseases (53). 

In these two tumours and also in fowl leucosis natural infections 
are said to be possible. Wound-tumour diseases of plants is likewise 
a tumour in which a virus is involved; this tumour is transmitted 
by an insect as in the case of usual plant-virus diseases (54). Never- 
theless, in many other tumour diseases natural infections may fail to 
occur, although the transmission can sometimes be brought about arti- 
ficially through cell-free filtrates; for instance, with Rous tumour it 
has been definitely confirmed that natural infection is entirely impos- 
sible despite the fact that a virus is involved in the tumour. 

Carr (55) has emphasized the fact that the greatest recovery of 
infective virus from Rous tumour represented a yield of only one in- 
fective particle per 20 tumour cells. This appears to be surprising, 
since even when peculiar cytoplasmic particles only, containing large 
amounts of nucleic acid, could act as the virus, at least scores or 
hundreds of virus particles would be obtained per cell. This might 
be attributed to the requirement of a vast number of particles to 
cause a tumour because of the weakness or the lability of the tumour 
structure. 


154 III. THE EVOLUTION OF VIRUSES 


Mueller (56) has shown that the infectivity of a filtrate of Rous 
tumour may vary from nil to such an effective dose as 0.001 ml., and 
moreover, it has been ascertained that the more slowly growing Rous 
tumours are less filtrable than the rapidly growing ones, and that 
occasionally the growth seems to lose its filtrability entirely, although 
still transmissible by cell inoculation (57). It has been found that 
the tumour of older chickens are tend to be more filtrable than that 
of younger ones. In addition to this effect of host age upon the 
filtrability, the age of the tumour itself, and whether the growth had 
been transferred by cell suspension or filtrate previously, prove to be 
important factors. It has been shown that non-filtrable growth be- 
comes filtrable by the treatment with X-ray. In the case of Shope 
papilloma, although the virus is readily demonstrable with wild cot- 
ton-tail rabbits, if the tumour is transplanted to domestic rabbits, the 
demonstration of the virus becomes very difficult, usually impossible. 

McIntosh (58) has reported that fowl tumours induced by tar can 
be transmitted to normal birds by cell-free filtrates, though this could 
not be ascertained by some other workers. On the other hand, it is 
known that if a fowl with achemically induced tumour which cannot 
be transplanted by cell-free filtrates, is injected with Rous virus, then 
filtrates of this tumour will give rise to Rous tumour in other fowls 
(43), a fact which might suggest independent nature of the virus. 
This fact, however, can be interpreted as indicating the transmission 
of the strong structural pattern of Rous tumour to chemically induced 
one through the protoplasm particles of the former, thereby chemically 
induced tumour is endowed with the strong structure to be held in its 
protoplasm particles. Chemically induced tumours seem generally to 
have structures weaker than those of naturally occurring tumours, 
which may be the cause of the difficult demonstrability of the virus. 
Oberling and Guérin have proved filtrability of the tumour induced 
by methyl-cholanthrene in legs of chickens, but active fiiltrates were 
only obtained in the fifth and sixth passage (59). 


3. The Maintenance of Assimilase Action 
in Protoplasm Fragments 


Considerable damage of the assimilase action of protoplasm seems 
to follow even when the protoplasm is only slightly disintegrated, not 
so completely as to pass through porcelain filters. 

On studying the number of metastases appearing in the lungs 
following the injection of tissue emulsion of a transplantable mouse 
tumour, Zeidman (60) claimed that, although the number of emboli 
produced is effected by such single factor as the initial size of the 


III. IMPERFECT VIRUSES 155 


tumour inoculum, it depends to an even greater extent upon factors 
within the primary tumours as yet unknown. This may indicate that 
the structure of tumour-cell protoplasm varies with conditions under 
which the tumour grows, and that if it becomes stronger, the structure 
will remain intact to a greater extent in the emulsion, meanwhile 
the assimilase action itself becoming more effective, resulting in the 
increase in the number of metastases. If the structure became still 
stronger, it would be retained even in the filtrable particles; thus the 
virus would become demonstrable. 

Gye and his associates (61) (62) have carried out a series of inter- 
esting experiments to demonstrate the virus etiology of animal cancers 
which cannot be transmitted with the filtrates. They have made ex- 
tensive use of freezing and drying technics and have indicated the 
successful transplantation of cancer tissues after treatments which 
are considered sufficient to kill all the cells. Thus, normal embryonic 
tissue could not be successively transplanted, and no normal cells 
could be found by histological examinations after an hour’s exposure 
to —75° C, and normal embryonic tissue was also killed when treated 
with glycerol and stored at —40° C, while fine emulsions of frozen 
tumour tissue made with distilled water, 40 per cent glycerol or 40 
per cent glucose, caused cancer. Three mouse sarcomas, one induced 
chemically and the other spontaneously, were successively propagated 
with tissues dried completely after freezing, some mouse sarcomas 
even retained their activity after drying without preliminary freezing. 

Hirschberg and Rusch (63), however, held the opinion that freezing 
technics alone are not suitable for the demonstration of virus etiology 
of the animal cancer, since there are a number of evidences for the 
survival of normal cells after freezing. Nevertheless, they acknowl- 
edged that the introduction of drying after freezing appears to provide 
a stronger evidence against the presence of intact cells. 

According to the writer’s opinion, it may be not impossible that 
the assimilase action can be retained in the tissue emulsion after the 
above stated treatments whether the tissue be normal or cancerous. 
Since such a treatment as freezing or drying may be less destructive 
upon the structure capable of acting as assimilase than the filtration 
procedure, even when the demonstration of the infectivity in the fil- 
trate failed, tissue emulsions would sometimes stand the treatment 
and retain their infectivity. There are, however, a vast number of 
evidences that the structure of cancer cells are, as a rule, not so 
strong as to be capable of transmitting their structure in the form of 
protoplasm particles. If Shope papilloma is transplanted to domestic 
rabbits, not only do papillomas develop readily, but a considerable 
proportion of them undergo cancerous transformation, and at the same 


156 III. THE EVOLUTION OF VIRUSES 


time the demonstration of the presence of the virus becomes impos- 
sible (64). It is a note-worthy fact that the demonstration of the 
virus always becomes difficult in this way, when tumours undergo 
cancerous transformation. ‘This may indicate that the structure be- 
comes weaker as it changes into cancerous one. 

Such a weakness of cancerous structure may be based upon its 
juvenile and primitive nature. As will be fully discussed in Part V, 
there are sufficient reasons for supposing that non-differentiated, juve- 
nile protoplasm structures are generally weak in their template action, 
so that they may be readily assimilized by other stronger, more dif- 
ferentiated structures. As is well known, chick embryos are exten- 
sively applied to cultivate various viruses, a fact which may be based 
upon their juvenile character to be readily assimilized by other proto- 
plasm structures. In a similar way, it has been ascertained with a 
number of viruses that cancer cells can sustain the multiplication of 
virus like chick embryos (64a) (64b). 

Cancer cells are distinct in their embryonic nature. Although the 
cells having the embryonic nature are generally able to proliferate 
vigorously when existing as intact cells, their specific protoplasm 
structure may be lost because of its unstable character when the cells 
are decomposed into fragments. Even when the structure was re- 
tained in the fragments, the particles would be unable to act as the 
virus because of its inability to act as the template on account of its 
weak pattern. 

The. main reason why cancers are regarded as one of the most 
dreadful diseases: may lie in their faculty to form metastasis in remote 
parts of- the.body. It is generally believed that for the metastasis the 
intact cells~are indispensable. But it may be conceivable that the 
fragments. of. the » ‘cells may also transmit the structure, since the 
fragments ‘can enter the blood-vessels in their fresh state and can 
reach,.-immediately after their liberation, healthy organs or tissues 
where prevails the high predisposition to the cancer. 

Virus diseases are called as such because they are transmitted by 
protoplasm particles of the diseased cells, and hence they are regarded 
as infectious diseases, whereas ordinary tumour diseases, such as Rous 
sarcoma and Shope papilloma, cannot be called infectious, although 
they are sometimes transmissible by virus-like agents. They are not 
infectious because the infection cannot take place in natural ways, a 
fact which may depend upon the unstableness or the weakness of proto- 
plasm structure of the tumour cells. As a rule the structure appears 
to be preserved only in the intact cells. Accordingly the disease can 
be transmitted generally by the intact cells, not by their fragments, 
and hence the structure cannot exist independently of the living intact 


III. IMPERFECT VIRUSES 157 


cells. The structure shares its fate, therefore, with the organism in 
which it has developed. The structure has to cease to exist when the 
organism perishes. 

The writer designates such structures as imperfect viruses. 
Nevertheless, it is impossible to draw definitely a line of demarkation 
between imperfect and perfect viruses. For example, as already re- 
ferred to, natural infections are said to be possible with such tumours 
as fowl leucosis and plant wound-tumour. Moreover, a mutant strain 
of rabbit fibromatosis has been shown to cause acute inflammatory 
lesions similar to those of ordinary virus diseases instead of the usual 
proliferative change (65). Rivers (53) claimed that myxomatosis of 
rabbits, which has the remarkable relationship to this fibromatosis, 
mediates between tumours and ordinary virus diseases. ‘The disease 
is transmissible to normal rabbits naturally by mosquitoes. Labora- 
tory transmission of fibromas in rabbits by means of fleas and mosqui- 
toes is also possible (66). Duran-Reynals (67) has made an investigation 
with a duck variant of Rous sarcoma and found that lesions induced 
in the central nervous system of ducklings by the virus is very simi- 

lar to that observed in the infection by ordinary viruses. 


CHAPTER IV 
THE VARIABILITY OF VIRUSES 


1. The Inheritance of Altered Structure 


Viruses are liable to alter in their properties, this being recog- 
nized as one of their characteristics. The altered property is occasion- 
ally transmitted to newly produced viruses, a phenomenon analogous 
to the mutation of organisms, though appearing to occur more fre- 
quently than the mutation of higher organisms. 

Higher organisms may have acquired the prominent characters 
through their extremely long history of the struggle for existence. 
They seem to have elaborated wise mechanisms to maintain the good 
quality of these prominent characters, whereas, as this is not the case 
with viruses, viruses appear to change their structure abruptly and 
extensively with the change of environmental! conditions. 

For example, the virus of smallpox, normally infecting only man, 
undergoes variation to the virus of vaccinia when repeatedly trans- 
ferred through the skin of rabbits. The virus has lost the capacity 
for causing smallpox, although antigenically it is still related to the 
variola virus, immunizing against it. Another variant of interest is 
a strain of yellow fever developed by passage through tissue cultures. 
This strain gives rise to excellent immunity to yellow fever and, 
since it is devoid of all tendencies to cause yellow fever in man, it 
can be employed as a live virus vaccine of high immunizing potency 
with safety. In a similar way, rabies virus changes its character 
when inoculated subcutaneously into a rabbit and passed in succession 
through a series of rabbits. The virus thus reduced in its virulence 
for man is likewise used as a vaccine. 

For a virus, such a host change must be a great environmental 
change, so that viruses may be altered in their properties on continued 
passages in new host. A phage newly isolated from chicken feces is 
usually non-specific, being capable of affecting simultaneously several 
strains of bacteria, but when repeatedly transferred through a certain 
strain of bacteria, the phage will obtain the property to affect the 
strain specifically with the vigorous phage production. We (13) were 
able to reduce the virulence of phage by removing Ca and Mg from 
culture media of the host bacteria. ‘‘Virulence’’ designates here the 


IV. THE VARIABILITY OF VIRUSES 159 


rate of multiplication of the phage, which can be estimated from the 
plaque sizes. 

For the variation of phage, however, the multiplication under the 
altered conditions is not essential. It can be induced, without any 
multiplication, by a mere addition of such substances as formaldehyde, 
mercuric chloride, or the antiserum against the phage (68) (69). In 
these cases the variation would occur in the reduction of the viru- 
lence, and the altered characters occasionally tended to be transmitted 
to new viruses produced from the altered virus. If these agents were 
added in proper quantities just enough to completely inactivate the 
phage, the antigenic specificity of the latter would be retained unal- 
tered. The change in the virulence appeared to take place when the 
inactivation was imcomplete. 

Similar phenomena have been observed with various viruses other 
than phage. For instance, Salaman (70) found that incubating tobacco 
roots infected with potato virus Y fora week at various temperatures 
above 22° C. produced less virulent forms. By keeping Rous sarcoma 
tissues for a long time at a temperature below 0° C., Gheorghiu (71) 
has succeeded in reducing the virulence of the virus to be unable to 
kill the fowl, although active enough to produce the tumour. Bjork- 
man and Horsfall (72) found that a single treatment of influenza 
virus with lanthanium acetate or with ultraviolet radiation, fol- 
lowed by subsequent passage in chick embryos, altered the virus in 
its behaviour towards red blood cells. This altered property of the 
treated virus persisted through several passages. Miller and Stanley 
(73) have prepared the phenylureido derivatives of tobacco mosaic and 
found that although these derivatives produced as many lesions as 
untreated virus in Nicotiana glutinosa, they were less infective when 
treated in Phaseolus vulgaris. 

Thus certain alterations in the structure of virus proteins appear 
to be accompanied by some change in the virus property, provided 
alterations are not so extensive as to entirely inactivate the virus. 
This should only be natural, since the property of a virus should be 
determined by the structure of the protein and the altered structure 
in turn should be transferred to the new viruses derived from it as 
the multiplication of a virus is nothing but the reproduction of the 
same structure. Newly formed structure of a virus therefore should 
always be inherited together with the altered characters. However, 
frequently newly formed character would soon be lost with the re- 
covery of the original one. This is the most important phenomenon 
arising from the reversibility of protein structure, the detailed ac- 
counts of which will be given later. 


160 III. THE EVOLUTION OF VIRUSES 


2. The Variation of Newly Produced Viruses 


It may be not impossible to regard allergic dermatitis and cancers 
as virus diseases, but, since the pathogenic factors of these diseases, 
as a rule, fail to be carried by protoplasm particles, the pathogenic 
agents or viruses share their fate with the organisms in which they 
have been generated, and therefore become extinct without being 
transmitted to other organisms. 

However, in the case of common cold, the virus of which may be 
generated under the influence of environmental changes, the situation 
is entirely different. If a man of the predisposition to the disease 
was exposed to chilling under some peculiar conditions, some change 
would result presumably in the cells of mucous membrane of the up- 
per respiratory tracts, the change being able to spread successively in 
the protoplasm of the cells, accompanied by inflammation, which 
would in turn lead to the decomposition of the cells into fragments or 
protoplasm particles, and if the newly formed structure generated by 
the change remained intact in some of the particles, these latter, when 
transferred to another man, say, by coughing, would transmit the 
structure to cause common cold. It may be possible that further al- 
terations would occur in the structure of the particles through some 
environmental changes during the periods they would affect succes- 
sively different persons, and thus the virus would be able to undergo 
variations to become a more virulent virus entirely different from the 
one generated at the start. 

This may also hold for viruses released from normal cells, 7. e., 
normal protoplasm particles, capable of acting as a virus upon proper 
other cell. Thus, if a normal protoplasm particle of a certain organ- 
ism capable of acting upon a man as a virus giving rise to symptoms 
like those of influenza undergoes variations during the successive pas- 
sages through man to increase in its virulence for the latter, then it 
may be possible that the virus will acquire the properties entirely 
similar to those of influenza, thus becoming a strain of influenza 
virus itself. 

Presumably each virus particle shows each individuality. This 
may be based upon the degree of precision in the replication which 
may vary with particles, and perhaps also upon the degree of damage 
which each particle may suffer during the course of the protoplasm 
decomposition into the particles or after their liberation from the cells. 
The difference in these degrees resulting from the dissimilarities in 
the structure of particles may cause the divergence in variations. 


IV. THE VARIABILITY OF VIRUSES 161 


In emphasizing of the virus individuality Schutz (74) stated that a 
striking feature of viruses is their heterogeneity when considered as 
a group. They are ‘“‘individualist’’. They are not like in their resist- 
ance to heat, cold, drying, hydrogen ion concentrations, organic or 
inorganic chemical compounds or to the action of individual enzymes. 

The occurrence of such a variation, however, seems to be a com- 
mon feature of protein molecules, not being confined to virus particles 
only. There are a vast number of evidences that each molecule of 
proteins, such as toxins, antibodies, and enzymes, has each individu- 
ality. 

It has been sometimes claimed that living matters are distinct 
from non-living matters in their variability as well as in their multi- 
plicability. The variability, however, is merely a property of pro- 
teins, by no means a characteristic of living matters. 

The individuality can be readily demonstrated with phage, for the 
individual property of this virus is clearly seen in the property of the 
plaques. Since a plaque is formed by a group of phages derived from 
a single particle, the degree or manner of the damage of bacteria due 
to the particle, derived from the single phage particle, is shown in it. 
Thus a large plaque will show the great multiplicability of the parent 
phage while a smaller plaque lesser multiplicability. If some parent 
particle has the property to lyse the bacteria completely a plaque of 
complete clearness will be obtained, whilst only a turbid plaque will 
be raised by a phage group derived from a parent particle causing an 
incomplete lysis. 

Owing to such properties of plaques, we can easily demonstrate 
that even a phage sample, originated from a single particle, never 
consists of uniform particles. Some workers have attempted to divide 
phages affecting E. coli into several types, but it would be very sur- 
prising if there was no gradual transition among them. 

The individuality of viruses is probably analogous to that of 
higher organisms, but presumably because of the easy occurrence of 
variation in viruses individual differences appear to be more distinct 
in them than in higher organisms. Virus particles having such dis- 
tinct individuality may undergo further variations which may vary 
with each particle when exposed to various environmental effects. 
Thus the divergency in the individuality will become still greater, 
leading to the appearance of numberless strains. 

Jensen (75) has isolated over fifty strains of tobacco mosaic virus 
from the yellow spots of the leaves. Some of these are apparently 
similar to tobacco aucuba mosaic virus, a strain commonly found in 
nature, but others differ from any strains previously described. Jensen 
distinguished the various strains by the ease with which they infect, 


162 III. THE EVOLUTION. OF VIRUSES 


and by the symptoms they cause on tobacco and tomato plants. All 
of them at normal temperatures produce only necrotic lesions on the 
latter, but some strains produce smaller lesions than others. All 
cause local lesions on tobacco plants, but some cause necrotic lesions 
and others chlorotic. Also, some give systemic infection and others 
do not. According to Kunkel (76), over 400 different strains of tobacco 
mosaic virus have already been recognized. And yet, still new strains 
are continuously being found to increase the number. 

Of animal viruses the individuality or variability seems to be 
especially distinct in influenza virus. Sigel (77) stated that strains of 
influenza type A virus which originated in the same institutional out- 
break from specimens collected on the same day, treated in the same 
manner, and isolated by individual procedures, exhibited certain bio- 
logical differences; namely, dissimilarities in the capacity to aggluti- 
nate chicken and guinea-pig red cells, speed of elution from the red 
cells, and resistance to ultraviolet light. Even significant differences 
in antigenic composition were found. 

As already mentioned, from various sources of animals have been 
frequently isolated agents that cause pathological symptoms, when 
transferred to proper other animals, like those of influenza or of en- 
cephalitis, suggesting that normal or pathological protoplasm particles 
of some animals tend to give rise to the pathological symptoms in 
other animals. Such agents may vary in their properties with animals 
or organs from which they have been generated, and even with the 
same organ of the same animal, considerable differences should be ex- 
pected as with tobacco mosaic viruses or phages in chicken feces. If 
an agent is forthcoming having a striking affinity and a high viru- 
lence to a man, then a new epidemic of influenza or of encephalitis 
may follow. As is generally acknowledged, from every epidemic of in- 
fluenza can be isolated a strain which differs in some or other aspects 
from any strains ever isolated from other epidemics. It is also known 
that new strains of neurotropic viruses causing symptoms like those 
of encephalitis or of poliomyelitis are continuously being isolated from 
various sources. 

There is arare disease called postvaccinal encephalitis, which may 
arise a short time after vaccination against small pox or rabies. It 
should be expected that agents above considered are sometimes con- 
tained in the vaccines, the emulsions of animal cells. It may be pos- 
sible that by such agents postvaccinal encephalitis is caused in indi- 
viduals with high susceptibility to the agent. 

Following a variety of infectious diseases such as measles similar 
encephalitis is known to occur in which a virus causing the cencepha- 
litis generated in certain cells of the patient by the stimulus of the 


IV. THE VARIABILITY OF VIRUSES 163 


infection may be involved. Margulis ef al. (78) have claimed that 
they were actually able to isolate a virus from a patient of postinfec- 
tion encephalitis. The agent causing postvaccinal encephalitis may 
either be present in normal animal cells cr produced by the stimulus 
of the inoculated virus. 


3. Increase and Decrease in the Virulence of Viruses 


It is generally accepted that when a virus is propagated serially 
in a host different from the customary, it increases gradually in its 
virulence toward the host. For example, an influenza virus isolated 
from a man fails usually to produce no lung lesions in hamsters, 
although it can readily multiply in the animal, but on serial passage 
it will acquire the capacity to cause pulmonary consolidations and 
even the death of the animal (79). If rabies virus isolated from a dog 
is inoculated into a rabbit, the incubation period of the disease is 
about a fortnight, but if the virus is passed in succession through a 
series of rabbits, the incubation period gradually falls, till after 20 to 
25 passages only to 8 days, and after further 20 to 25 passages it is 
no more than 7 days. Such a virus has a greater affinity for the 
nervous system of the rabbit than the virus isolated from the dog. 

In the concept of the writer, such an enhancement in the viru- 
lence should result mainly from the increase in the combining force 
between a virus and the host cell. Namely, the virulence is in- 
creased because the combining force is increased when a virus is 
passed in succession through a series of the same kind of host. The 
reason for the increase in the combining force is considered as follows: 
The protoplasm of the host cell and the virus affecting the host are 
both assimilases, though the latter is stronger since it acts as a virus 
upon the host. However, the host protoplasm, though its assimilase 
action is weaker than that of the virus, will be able to exert some 
influence upon the latter, as the protoplasm is overwhelmingly larger 
in quantity than the virus, so that in the long run the virus itself 
may be assimilized by the host protoplasm to some extent in case 
when the virus is passed successively through the same kind of host, 
and as a result the protoplasm and the virus may come to share a 
structure or structures in common; namely, they may become gradu- 
ally to resemble to each other. On the other hand, as already discus- 
sed, the development of common structure is nothing but the produc- 
tion of combining force between them, and therefore the affinity of 
the virus for the host cell may be enhanced with the increase in the 
virulence on continued passage through the same kind of host. 


164 III. THE EVOLUTION OF VIRUSES 


The reason for the increase in the virulence during the passage 
can thus be elucidated. But in connection with this a more important 
problem has to be considered. 

A virus can assimilize the host protoplasm, so that all the struc- 
tures present in the protoplasm may become identical to the virus. 
Thus, since the parent and offspring viruses share all the structures 
in common, they can completely combine with each other, but without 
exerting any effect on one another because there is no differences in 
the structures. 

In like manner, if a virus propagates continuously in the same 
kind of host until the degree of assimilation by the host protoplasm 
reaches to such an extent that almost all the virus structures become 
identical to those of the protoplasm, then the virus will have little 
influence upon the host in spite of its high affinity for the host. 
Thus, in order that a virus exerts injurious effect upon the host, the 
virus must have a structure or structures foreign to the host proto- 
plasm, which may be lost when the assimilation by the protoplasm is 
advanced to an extreme. On the other hand, in order to combine with 
the host cell a virus must share a structure in common with the host 
protoplasm. This common structure can be increased or strengthened 
when the virus is assimilized by the protoplasm to a certain extent 
through the host passage. 

It will be expected from this reasoning that, although a virus may 
increase gradually in its virulence against a certain host when passed 
successively through the host, the increase will come to cease sooner 
or later, and then a decrease will begin until at last the virus be- 
comes entirely avirulent. In fact this seems actually true: An 
epidemic, for example, of influenza usually begins gradually, at first 
with only a slight indisposition, but in due course of time the viru- 
lence will increase in a striking way, the disease spreading vigorously 
and extensively ; yet, after several months or a year or two, it will 
begin to lose vigour until entirely fades out and disappears. 

In order to avoid the reduction in the activity, vaccinia virus is 
usually passed at times through rabbits apart from the customary 
cattle. We shall have later many occasions to return to this point, 
since this is one of the most important problems concerning life 
phenomena. 


CHAPTER V 


FIXED VIRUSES 


1. Immunity against Viruses 


Usually an effective and lasting immunity is afforded by a single 
attack of a virus, a fact which is sometimes regarded as one of the 
characteristics of viruses. Small pox, yellow fever, varicella, measles, 
mumps, cattle plague, swine fever, and dog distemper, all these are 
common virus disease, and all confer an immunity that, in the majori- 
ty of cases, appears to last throughout life. In some virus diseases, 
however, such as influenza, common cold, and poliomyelitis, the im- 
munity is believed to be only transient. 

As above mentioned, viruses such as those of common cold, in- 
fluenza, and poliomyelitis, appear to be generated de novo. From this 
point of view, it may be able to explain the transient nature of the 
immunity against these viruses, because the immunity against a virus 
formerly produced would be ineffective to a newly produced virus. 
Not long ago foot-and-mouth disease would have been placed in this 
non-immunizing or poorly immunizing class, but we are now informed 
that there are at least 3 immunologically distinct types, and that 
animals that have recovered from infection with one type proves im- 
mune to further infection with the same type, but are readily infected 
by the other types. 

Experimental results with one and the same strain of influenza 
virus showed, however, that the immunity is actually transient. For 
example, ferrets that have recovered from an attack of a strain of the 
virus are, for a time, resistant to the infection, but this persists only 
for about 3 months, and then gradually wanes. Again, the immunity 
which develops following an attack in man is said also not to last for 
long periods, usually becoming insignificant after 6 to 8 months (80). 
It must, therefore, be admitted that the immunity in itself is actually 
transient in some viruses, although sometimes the possibility of their 
successive generation may account for the apparent transient im- 
munity as above pointed out. It is, however, a very remarkable fact 
that immunity appears incomplete in only the viruses that can be con- 
sidered to be generated de movo such as those of influenza, and 
poliomyelitis. 


166 III. THE EVOLUTION OF VIRUSES 


At present, influenza virus is divided into two distinct and im- 
munologically unrelated serological types, termed A and B. However, 
it is of much importance that individual strains of influenza A virus 
or of B virus are not identical immunologically with other strains of 
the same type. It is said that, in occasional instances, the immunolo- 
gical differences between individual strains which belong to one type 
are so great as to cause considerable difficulty in the proper identifica- 
tion and classification of a strain. It may be said that these two 
types may only be an artificial product of classification. 

Various strains of virus causing symptoms like those of encepha- 
litis are also known, and new strains are constantly increasing. 
Normal or pathological structure of the protoplasm of a certain animal 
may be called influenza virus when it is capable of giving rise to 
symptoms like those of influenza because of its special affinity for re- 
spiratory tract of some other animals, whilst when it is endowed with 
special affinity for central nervous systems, it may be called encepha- 
litis virus. It seems that comparatively little is known of the dura- 
bility of the immunity against encephalitis. 

Poliomyelitis may be concerned with agents belonging to this 
category. It is certain that there are a vast number of different 
strains in poliomyelitis virus. Recent observations suggest that there 
are at least three main types (81) (82). As already pointed out, polio- 
myelitis-like viruses have frequently been isolated from various sources 
especially from flies apparently having no connection with the disease. 
It has been known that some immunity is established on the infection 
with this disease, but that it is only transient and inconsiderable (83). 

The so-called Coxackie virus appears to be closely related to this 
virus. Its strains are known to be very numerous, and 16 antigeni- 
cally distinct types have been reported (84) and like poliomyelitis virus 
it has frequently been detected in flies. Within the past decade, it 
has become established that during typical summer epidemics of polio- 
myelitis the causative agents, frequently also Coxackie-like virus, can 
be isolated from several different species of flies. 

Whatever the reason may be for the presence of the temporary 
nature of the immunity, there seems no doubt that spontaneous gen- 
eration is impossible in such viruses as those of small pox, mumps, 
measles, efc., which confer a lifelong immunity, since, if new viruses 
which could cause a similar disease were successively generated, the 
immunity would not appear to last effectively, for new viruses would 
be more or less immunologically different from the previous ones, 
though they could cause a similar disease. 

It appears, therefore, reasonable to conclude that there are at least 
two groups of viruses. Those of influenza, poliomyelitis, efe. belong 


V. FIXED VIRUSES 167 


to the one group which may be produced de movo, and those of mea- 
sles, mumps, small pox, etc. belong to the other; spontaneous gene- 
ration may be impossible in this latter group. 


2. The Fixation of Viruses 


In general, viruses can be changed in their character, sometimes 
even inactivated completely, without losing their immunological spe- 
cificity. For example, vaccinia virus is evidently different in some 
aspect from the variola virus from which it has been derived, but im- 
munologically similar, so that it is capable of immunizing against the 
latter. Phage treated with such agents as formaldehyde, mercuric 
chloride, efc., may be reduced in its virulence, but may retain its im- 
munological specificity even after the complete inactivation, although 
too severe inactivation may lead to the destruction of the antigenicity 
itself (68). This fact indicates that the structure involved in the im- 
munological specificity is more stable than that concerned in virus 
activity. Consequently it is supposed that the variation in the activi- 
ty may develop without difficulty in contrast to the variation in the 
immunological specificity. 

It may, however, be possible that a virus generated de novo can 
be changed in its immunological specificity as well as in other charac- 
ters to become in the long run a virus entirely different from the 
original. It is actually confirmed that also in immunological proper- 
ties variations can occur. For example, evidence has been presented 
that antigenic variations take place in a strain of influenza virus pro- 
pagated through continuous passage in tissue cultures and eggs as 
compared with a line of the same strain maintained in mice (85). 
Through such variations, a virus may acquire the properties differing 
in every respect from any other viruses which can be generated de 
novo. 

In this way, the viruses that can induce lifelong immunity might 
have been developed. There are good reasons to suppose as will be 
detailed later that viruses such as those of measles and small pox, in 
contrast to those of influenza and poliomyelitis, are neither generated 
de novo nor lost in short periods. The writer proposes therefore the 
name “‘fixed viruses’’ to designate the former group of viruses. The 
reason why the viruses have acquired the fixed character and why 
they can produce rigid immunity will be fully discussed in later 
chapters. 

It cannot of course be considered that these fixed viruses are all 
the same in their ages; some may be young, rather fixed recently, 


168 III. THE EVOLUTION OF VIRUSES 


while the other old, generated much longer before. The particle size 
of virus may present some indication; the virus of foot-and-mouth 
disease and of poliomyelitis, and some kinds of plant viruses have been 
reported to be the smallest in size. Such viruses may be regarded as 
possessing comparatively primitive, undifferentiated structures, since 
“‘small size’’ may be interpreted as indicating that the virus or the 
assimilase can maintain its activity even when decomposed to this 
small size. Indeed, such small-sized viruses seem liable to develop de 
novo. Coxackie virus which bears a strinking resemblance to polio- 
myelitis virus has been reported to have the size of 10 to 15 my (86) 
indicating that this virus is also one of the smallest viruses. On the 
other hand, the so-called lymphogranuloma-psittacosis group viruses 
are generally accepted to be most differentiated ones, and they are be- 
lieved to be as large as 250 to 500 mz. Nevertheless, there appears 
not always such correlation between differentiation degree and the 
particle sizes. For instance, influenza virus, spontaneous generation 
of which appears most possible, is generally acknowledged to be in 
size about 100 mz, whereas with yellow fever virus which appears to 
be a fixed virus such small figure as 22 my has been reported (87). 
As already stated, since the size of a virus may be determined by the 
property of the protoplasm which the virus has affected, and also by 
the smallest protoplasm particles, in which the virus activity can re- 
main, no great significance may be expected in the Size. 

However, fixation of the character of a virus as an independent 
entity seems to be accompanied by the establishment of character to 
form uniform particles, and moreover the complexity of the activity 
necessarily associated with the development of differentiation in virus 
may require large sizes because small particles may be impossible to 
contain the complex structure. Consequently, large uniform sizes may 
commonly be associated with old and differentiated viruses. In the 
case of Rickettsiae, as referred to in the next chapter, this seems es- 
pecially the case. 


CHAPTER VI 


DEVELOPMENT FROM VIRUSES 
TO ORGANISMS 


1. Rickettsiae 


Lymphogranuloma-psittacosis group of viruses is believed to be 
largest-sized, whilst there are much more differentiated, advanced 
viruses or virus-like agents, which are commonly called Rickettsiae. 
The Rickettsiae seem to occupy a place intermediate between the smal- 
lest cultivable bacteria and the largest viruses. The diseases caused 
by these agents are transmitted by arthropods, usually louse and ticks. 
They resemble usual viruses since they will multiply only in living 
cells, but differ from the former in that they fail, as a rule, to pass 
through bacterial filters and that their particles are distinctly seen 
under the ordinary microscope when stained properly. 

This property of easily staining with proper dyes is note-worthy, 
because it may indicate the development of structure much different 
from that of the protoplasm of the host cell. With the usual viruses 
no staining technic have ever been found to make the virus particle 
distinct from the protoplasm, showing that the virus is alike in struc- 
ture to the protoplasm. Moreover, specific therapy by chemical 
substances has usually been failed with virus diseases, whilst it proves 
effective in Rickettsiae, indicating also the development of peculiar 
structure in them. It is, however, known that specific chemotherapy 
is likewise effective to some extent in viruses of psittacosis-lympho- 
granuloma group, and that some viruses of this group sometimes stain 
like Rickettsiae. These facts show the presence also in these viruses 
of highly developed structures resembling to those of Rickettsiae. 

The fact that these highly developed viruses as well as Rickettsiae 
are uSually associated with rather uniform and large sized particles, 
suggests that the protoplasm, when changed in its structure by these 
agents, will decompose into large particles. Since the property to 
form large particles should be determined by the structural pattern 
peculiar to these viruses and Rickettsiae, and since this pattern is to 
be transmitted to the protoplasm they affect, newly formed particles 
will be always endowed with large sizes. The inheritance of the 
shape may be thus established. 


170 III. THE EVOLUTION OF VIRUSES 


In order to form a particle larger than an elementary body of the 
protoplasm, a certain number of elementary bodies have to associate 
with one another to form a larger body, since the size of elementary 
body cannot be altered, and hence highly developed viruses including 
Rickettsiae have to be composed of a number of elementary bodies. In 
fact, this has been shown in the electron micrographs of Rickettsiae 
and of some large sized viruses. 


2. Indisputable Organisms 


The tact that Rickettsiae can be resolved microscopically by visi- 
ble light, and that they are held back by membranes which allow 
most of the filtrable viruses to pass through, may bring them into 
line with the bacteria. However, their general failure to grow on or- 
dinary culture media, and their predilection for intracellular multipli- 
cation, show that they are more akin to filtrable viruses. 

It is, however, a fact worthy of note that although none of the 
pathogenic species of Rickettsiae has ever been cultured apart from 
living cells, the commensal species, R. melophagi, found in the sheep- 
ked, is said to be able to grow on blood agar (88). 

The ability to grow without living cells may seem the indication 
of the appearance of mysterious nature, peculiar to living organisms 
and lacking in viruses. Nevertheless, there seems no need to postulate 
the appearance in this Rickettsiae of a mysterious nature, because the 
ability to grow without living cells may be interpreted as to be the 
faculty to produce its replica in blood proteins as well as in proto- 
plasm proteins, whilst viruses and usual Rickettsiae are merely in 
the position to produce their replicas only in the protoplasm. 

In addition, in various respects, blood may be looked upon as a 
large pool of a fluid protoplasm. If acetic acid is added to blood 
serum diluted with water, virus-like particles, composed of euglobulin 
and lipids, will be yielded as in the case of cell protoplasm treated in 
a Similar way as was already stated in Part I. Moreover, intimate 
mutual association seems to exist among protein molecules in blood as 
in the protoplasm. In the protoplasm, owing to this mutual associa- 
tion among protein molecules, various changes, including coagulation 
process, can spread readily. In like manner, in blood the coagulation 
process can spread successively as will be mentioned later. 

The mechanism, therefore, by which the Rickettsiae can accom- 
plish the multiplication in blood agar can be regarded as being similar 
to that whereby viruses multiply in the protoplasm. However, since 
in its physical and chemical properties blood differs considerably from 


VI. DEVELOPMENT FROM VIRUSES TO ORGANISMS 171 


the usual protoplosm, in order to multiply in blood the Rickettsiae 
may require a quite strong assimilase activity not present in viruses. 
As will be fully explained later, such a strong assimilase activity, in 
the main, is apparently based upon its large particle size. 

In addition to Rickettsiae, there are many other small bacteria-like 
bodies or organisms, found in higher animals on which they are 
parasitic. They are, like viruses, usually unable to multiply without 
cells of higher animals. The organism responsible for pleuropneu- 
monia of cattle may be cited as one of the specimens. This was once 
regarded as a kind of viruses, as it could pass coarse bacterial filters, 
but this undoubtedly cannot be a virus, for it can sometimes grow in 
nutrient media containing blood in the absence of living cells. It 
stains with proper bacterial stains and is microscopically visible. 

The organism of contagious agalactia is also filtrable, and likewise 
once thought to be a virus, but at present known to be an organism 
akin to the above one, and can be also cultivated in the absence of 
living cells. Furthermore, various bodies have been described by dif- 
ferent workers in close association with the red blood cells of man 
and animals suffering usually from certain types of anaemia. These 
bodies are believed to be definite organisms and called Bartonella. 
Non-pathogenic bodies similar to Bartonella are also found and named 
Grahamella. 

It seems that these bodies or organisms occupy a position between 
the Rickettsiae and the ordinary bacteria. Generally they can multiply 
only in living cells as do viruses, but sometimes some of them can 
manage to utilize blood proteins to produce their replicas. 

These organisms are distinct in the property to form specifically 
shaped bodies, so that it may be possible to distinguish them by their 
shapes in contrast to ordinary viruses. Primitive viruses may be able 
to exhibit their action only when the protein molecules are polymerized 
to a certain degree without being greatly concerned with their sizes 
and shapes. However, in highly differentiated viruses, such as those 
of lymphogranuloma inguinale and psittacosis, the property to form 
peculiar bodies is manifestly forthcoming, presumably as a result of 
the development of the structural pattern which may cause several 
elementary bodies to associate regularly into a larger body. A regu- 
larly associated product of the same molecules is a crystal, and its 
shape is determined by the structure of the component molecule. 
Since the assimilase is a liquid crystal of proteins, its shape should 
be likewise determined by the structural pattern of the proteins, again 
Since such a structural pattern is to be transmitted successively to 
daughter assimilases, the shape should be inherited. Thus, when some 
protoplasm proteins, the structure of which has been changed to be 


172 III. THE EVOLUTION OF VIRUSES 


identical to that of a Rickettsia, form a liquid crystal corresponding 
to the changed structure, the shape peculiar to the Rickettsiae will be 
developed in the protoplasm, with the multiplication of the peculiarly 
shaped body. 

In case of viruses, protoplasm proteins, changed in structure by a 
virus, occasionally form large ‘‘crystals’’ under some conditions; the 
crystals are termed inclusion bodies. Viruses are able to exhibit their 
function in the form of a single elementary body or sometimes even 
in much smaller particles. produced through the decomposition of the 
elementary body itself, so that such large crystals are never necessary 
for their function. However, if the function could not be set forth 
unless the proteins formed such a large ‘“‘crystal’’, the inclusion body 
would be regarded as the only feature of the virus. 

For the achievement of complicated functions, indispensable for 
usual organisms, large sizes are apparently necessary for the assimi- 
lase. Viruses therefore must have the property to form larger parti- 
cles in order to advance on a line of indisputable organisms. 

Of orgainisms called bacteria, there are many groups which are 
strictly parasitic, usually only capable of multiplying in the presence 
of certain cells, accodingly failing to grow in culture meida unless 
blood proteins are mixed. Neisseria Moraxella or the group of Morax- 
Axenfeld bacillus, and B. pneumosintes may be cited as such bacteria, 
which can be regarded as primitive bacteria evolved comparatively re- 
cently from viruses. 

The writer proposes the name, ‘‘secondary organisms’ to these 
organisms thus evolved from viruses, and accordingly the organisms 
in which the secondary organisms are advanced must be called “‘pri- 
mary organisms’’. 


CHAPTER VII 


CAUSES OF THE EVOLUTION OF 
VIRUSES 


1. Development of Instincts Indispensable for 
Organisms 


It has been described thus far that the viruses generated de novo 
can evolve into fixed viruses by variations, and that they can fur- 
ther advance into typical organisms through the stage of Rickettsiae. 
But, little discussion has been made on the problem why they have 
to evolve. In this chapter this problem will be fully dealt with. 

Darwin attributed the cause of the organic evolution to the sur- 
vival of the fittest set forth by natural selection; this likewise pos- 
sibly folds for the evolution of viruses, although there are some 
differences between usual organisms and viruses. 

One of the principal differences between them may lie in the fact 
that viruses have no instinct for self- or race-preservation in contrast 
to the indisputable organisms. Even in this respect alone it seems 
impossible to regard viruses as organisms. This is the instinct indis- 
pensable for organisms; organisms cannot exist without this instinct. 

Viruses are lacking in this important instinct, so that their exis- 
tence is of quite evanescence ; only some viruses which incidentally get 
possession of a property favourable for their existence, can continue 
to exist for some periods, while others are cancelled immediately 
after their generation. 

The character most essential for organisms is their continuity in 
existence. This is secured by the instinct, owing to which they have 
been, and will be able to accomplish their evolution. For higher 
organisms, however, Self-preservative instinct may be only of the 
secondary importance, merely answering the purpose of race-preserva- 
tion the important necessity for organisms, and an individual of higher 
organisms having already fulfilled this purpose may be entirely of no 
uSe in itself; the existence of individual may always be ready to be 
sacrificed for the race-preservation. 

On the other hand, in case of extremely primitive organisms 
existing at the stage of virus, there seems no distinct difference be- 
tween the self and race-preservation. In most cases the race-preserva- 


174 III. THE EVOLUTION OF VIRUSES 


tion may be accomplished by the self-preservation. 

Newly formed viruses would start on the long journey of the 
evolution as a group of particles of various individualities. Of them 
some particles with high resistances against environmental influences 
might be able to escape from the immediate inactivation to continue 
their existence, and again some particles with strong assimilase action 
might be capable of achieving extensive multiplication, so that par- 
ticles both with strong assimilase action and high resistance would 
be able to survive. Thus the survival of the fittest would be effec- 
tuated by a sort of natural selection. 

Viruses thus having survived would undergo variations, with 
the yieldance of various particles differing from one another in their 
individual properties, and again some individual having a property 
more favourable for their existence would be selected out of these 
particles and would continue their existence. Variation, selection, and 
the survival of the fittest would thus be recited over and over again. 
There seems no doubt that the characters resembling those of higher 
organisms are most desirable for the continued existence, so that 
viruses that happened to possess more organism-like characters would 
be able to survive much longer than do other viruses, and therefore 
the characters would become continuously more and more alike to 
those of typical organisms. 

The properties thus developed in selected viruses, resembling 
those of higher organisms, must be the properties favouring conti- 
nuous existence, and thus the instinct for the self- or race-preserva- 
tion would be developed gradually in the viruses until the stage of 
typical organisms was reached. The characters useful for the conti- 
nuous existence, if became unusually strong and firm, should be called 
instinct. Therefore, even if we assumed that the instinct belongs 
only to indisputable organisms, it would be impossible to draw a line 
between living and nonliving. 

The evolution of the shape or size of virus particles may be 
elucidated in like manner: In the case of tumour, such as cancers, 
newly formed structures can sometimes be transmitted to other normal 
cells by protoplasm particles, but commonly fail to be maintained in 
the particles, and accordingly as the structures have to share their 
fate with organisms in which they have generated they will be lost 
when the organisms are perished, and therefore their evolution will 
never take place; whereas since in the case of influenza or poliomye- 
litis the newly generated structures can be preserved in the particles, 
they are able to be liberated from the organisms, in which they have 
been generated, to be transmitted to other organisms. The continuity 
of their existence would thus be commenced, and the evolution would 


VII. CAUSES OF THE EVOLUTION OF VIRUSES 175 


thus become possible. 

Newly formed structure or a virus may be better maintained in 
larger particles, while readily lost in smaller ones as mentioned 
already (Part I, Chapter II), so that for the preservation of the struc- 
ture larger particles may be more favourable. In addition, assimilase 
action is considered to be directly proportional to the particle size, 
that is, larger particles possess greater faculty for their multiplica- 
bility as also pointed out already (Part II, Chapter II). Larger sizes 
are, therefore, advantageous to the viruses for these two reasons, 7. é., 
for the maintenance of the specific structure and for the enhancement 
of the assimilase action. Accordingly, viruses should have to secure 
the property to form larger particles in order to continue their ex- 
istence. 

This property like many other properties is determined by a 
structure of the protein; that is to say, the combination of many ele- 
mentary bodies to form a large body, may be achieved when the ele- 
mentary bodies have a peculiar structure favouring the large body 
formation. Hence, the virus which has incidentally secured such a 
structure by a variation may be in a better position to continue its 
existence, and the structure may be transmitted in succession. Thus 
the structure to form larger particles would be attained and streng- 
thened, with the result that assimilases which can be called organisms 
might always get possession of the property to form bodies larger 
than ordinary virus particles. 


2. Difficult Situation of Newly Generated Viruses to 
Continue Their Existence 


Viruses seem to be constantly generated from a variety of cells, 
so that strains or kinds of newly formed viruses must be innumerable 
and thus numberless kinds of viruses must be constantly being thrown 
out on the earth. However, the kinds of fixed, typical viruses never 
seem to be numberless, but limited to a rather small number. This 
may indicate that the probability of newly formed viruses of becoming 
fixed is extraordinarily small. Most of the viruses generated de novo 
possibly disappear immediately after their generation, because they 
have no instinct of self-preservation. 

If in some protoplasm a new structure is produced which is suffi- 
ciently stable to be retained in the protoplasm particles into which the 
protoplasm is decomposed, then such a particle may be able to transmit 
the structure to another organism as a virus. In order to evolve into 
a typical organism, however, the virus should be successively trans- 


176 III. THE EVOLUTION OF VIRUSES 


mitted to other organisms with vigorous multiplication, only thereby 
variations in a desirable direction become possible. Remaining in the 
same host must result in its extinction together with the host as in 
the case of tumours. 

On the other hand, In the case of common cold, for instance, the 
newly formed structure carried by particles may be able to spread 
easily through the coughing. The fact that common cold is always 
accompanied by coughing may enable the virus to exist for consider- 
ably prolonged periods and thus the virus may be able to become more 
and more virulent until it may come to appear even to be a kind of 
influenza virus. 

Like the virus of common cold, a large number of viruses, such 
as those of measles, mumps, poliomyelitis, and influenza, may be 
spread, though not so readily, by means of droplets of secretion from 
the upper respiratory tract. In the case of venereal diseases, such as 
lymphogranuloma inguinale, spread of viruses by direct contact can 
be afforded. In plant virus diseases, infections by direct contact is 
also conceivable. 

Many other ways may be possible for viruses to spread to other 
host individuals, but in this connection a strong reason for which the 
continued existence of viruses may become most difficult can be con- 
sidered. The reason is that, for the continued existence, viruses must 
be transmitted frequently to other organisms quite different from the 
customary host. 

As mentioned already, when a virus continues for a long time to 
multiply by successively affecting a series of the same kind of host, 
the virulence in the long run has to decrease until the virus itself is 
lost, because the virus will be gradaully assimilized by the host pro- 
toplasm to become quite identical to the latter. In order to avoid 
such a fate, viruses have to change occasionally their host. Insects 
appear to be universally utilized by viruses for this purpose as will 
be detailed in the next chapter. 


CHAPTER VIII 


VIRUSES AND INSECTS 


1. The Multiplication of Viruses in Insects 


Many virus diseases are transmitted by insect vectors, animal 
viruses usually by blood-sucking insects such as mosquitoes, lice, 
and sometimes by arthropods such as ticks, while plant viruses by 
aphides and leafhoppers. It is worthy of note that an insect which 
has been contaminated with a virus, as a rule, is endowed with no 
infectivity immediately after the contamination, but that only after a 
certain period of time becomes capable of transmitting the virus. In 
the case of yellow fever a kind of mosquitoes, for example, attains 
the infectivity 12 days after blood-sucking, and in dengue fever there 
is an incubation period of 7 to 10 or more days before the mosquitoes 
are infective to man. Also with a variety of plant viruses and their 
vectors the existence of similar incubation periods have been well 
established. 

The presence of these incubation periods can be readily elucidated 
by the assumption that viruses multiply in the insect bodies thereby 
the properties of the virus are changed and activated. In corroborat- 
ing this assumption, it has been confirmed by a number of workers 
that some viruses actually multiply in the insect vectors. For instance, 
Trager (89) has shown by cultivating the virus of equine encephalo- 
myelitis in sterile mosquito tissue that the virus can under certain 
circumstances undoubtedly multiply in the mosquito. Merrill and 
Ten-Broeck (90), working with the same virus, macerated the bodies 
of infected mosquitoes, and fed the juices to non-infective mosquitoes, 
and found that after ten transfers the mosquitoes still become infec- 
tive and since the dilution at each transfer was at least 1:100 it 
seems conclusive that multiplication must have taken place. Whitman 
(91) has presented evidences that a kind of mosquito is capable of 
multiplying yellow fever virus in its body. Following the injection 
of human blood, the content of virus falls for several days, reaching 
a minimum during the first week. It then increases rapidly until 
quantities of virus greater than those previously encountered can be 
demonstrated. 


178 III. THE EVOLUTION.OF VIRUSES 


Furthermore, working with the virus of aster yellows and its 
specific vector, Black (92) has confirmed that the virus could increase 
in the insect at least one hundredfold between the second and twelfth 
days of the inoculation. Maramorosch (93) has likewise ascertained 
the multiplication of corn stunt virus in its insect vector, a kind of 
leafhoppers. He has succeeded in transmitting the virus mechanically 
from an insect to another; the virus isolated from infected plant, 
when injected into insects, renders the latter infective and the juices 
of the insects can successively infect other healthy insects through 
mechanical’ injection. The minimum incubation period of the virus to 
become infective in the insects was found to be 6 weeks. A plant 
virus not mechanically transmissible from plant to plant can be thus 
mechanically transmitted between insect vectors. 

In spite of such numerous positive evidences, virus multiplication 
in insect vectors are not fully admitted, because there are some facts 
known which appear to be inconsistent with the concept. For instance, 
if viruses multiply in insect vectors it would be reasonable to expect 
that all the vectors, when once become infective, will remain so for 
long periods and probably for the whole of their lives, but this does 
not happen, for it has been found that different individuals remain 
infective for varying periods, some soon losing infectivity and others 
not. Moreover, it has been confirmed that the length of time for 
which insects remain infective depends on the length of time they 
have fed on the source of the viruses. Vectors may become infective 
after a few minutes feeding but do not remain infective for long, 
whilst if they have fed for hours or days they remain infective for 
long periods, often for the remainder of their lives. Sometimes it was 
even confirmed that both efficiency of the vectors and length of time 
for which they remain infective increases with increased feeding time 
on the infected plant (39). These evidences may appear at first sight 
to indicate that a virus remains in the insect as such without multi- 
plying, being contradictory to the above view, but in the theory of 
the writer these are by no means inconsistent with the view as will 
be argued in the following section. 


1. The Reversibility of Protein Structure 


The writer holds the opinion that the structure of protoplasm 
protein is springy or elastic, so that if the factor is removed which 
has changed to some extent the structure, the original structure may 
be recovered sooner or later. In short, protoplasm proteins can remem- 
ber the structure they once possessed and will resume it if possible. 


VIII. VIRUSES AND INSECTS 179 


A virus which has been inactivated by an agent can occasionally 
be reactivated when the agent is removed; this may result from the 
memory of the original structure. Numerous analogous phenomena are 
known and recognized in general as reversibility of protein denaturation. 

Usually yeast cells cannot ferment galactose, but if placed in 
contact with the sugar, they will subsequently acquire the enzyme 
necessary to ferment the sugar. Such an enzyme is generally called 
adaptive enzyme. The adaptive enzyme is extremely unstable in the 
absence of the specific substrate, and as a rule the enzyme will come 
to show a loss of the specific activity shortly after the substrate is 
removed from the medium. 

Spiegelman (94) held the opinion that adaptive enzyme is produced 
from the protein precursor by the action of the substrate and that 
since the change of the precursor to the enzyme is reversible, the 
enzyme will come back to the state of the precursor when the subst- 
rate is removed. This is almost in accord with writer’s opinion, 
according to which the precursor is the protoplasm protein itself whose 
structure is altered by the substrate to become complementary to the 
substrate structure, thereby the specific activity as the enzyme is 
revealed, but as the change is reversible, on the removal of the sub- 
strate which have caused the change, the original structure is reco- 
vered (12). 

In the case of allergic dermatitis, dealt with previously, it is 
supposed that the protoplasm protein of skin tissue cells is altered 
in its structure by some agent, the altered structure being able to 
spread successively in protoplasm and even from cell to cell, but 
that since the altered structure is unstable, having a high tendency 
to return to its original state, the skin tissue will soon recover from 
the disease when the agent is removed. The failure of the demon- 
stration of any virus in such dermatitis is probably dependent upon 
such a lability of the altered structure. 

Some workers claimed that the adaptation of yeast cells to a sugar 
could be transmitted by a virus-like agent, but some other workers are 
disinclined to accept this claim. Also in this case strong reversibili- 
ty of the structure may render the detection of the “virus’’ difficult. 

Protoplasm protein changed in its structure by a virus seems like- 
wise to be inclined to recover its original structure. The recovery 
from virus diseases may be accomplished mainly owing to this reversi- 
bility (12). The recovery of the original structure is nothing but the 
disappearance of the virus itself. 

Now we Shall return to the problem regarding the insect vectors. 
As above mentioned, insect vectors which have once become infective 
by feeding on diseased plant do not always remain infective for long 


180 III. THE EVOLUTION OF VIRUSES 


periods. This must be a most natural result since the normal struc- 
tural pattern should be recovered sooner or later. The action of virus 
as already discussed is generally directly proportional to its quantity ; 
the greater the quantity, the stronger the action. When the action 
is stronger, replicas produced will be more distinct and firmer. Thus, 
the above mentioned fact that the length of time for which insects 
remain infective depends on the length of time they have fed on the 
source of the viruses should naturally be expected, as the quantity 
of the virus taken by the insects must be greater when they have 
fed longer. 

The writer is of the opinion that antibodies are prcduced in entirely 
the same way as the adaptive enzymes. Namely, protoplasm proteins 
altered in their structure by antigens are the antibodies (12). Also in 
the case of this antibody formation, it is known that both the amount 
of antibody produced and the length of time for which the antibody 
production persists are inclined to be directly proportional to the 
antigen amount administered. The protoplasm structure changed by 
antigens is likewise reversible, so that sooner or later the original 
structure’ is recovered with the cessation of the antibody production. 

Kunkel (95) has found that a kind of the leafhopper carrying 
aster yellows virus is unable to transmit if exposed to high tempera- 
tures. Leafhopper exposed to 32°C. for a day lost the ability to infect 
healthy plants, but on lowering the temperature to 24°C. they quickly 
regained the ability without again feeding on a source of the virus. 
This may indicate that the structure of the virus is stable at lower 
temperatures, while at higher temperatures the normal protoplasm 
structure is more stable than the virus structure, so that at 32°C. 
the virus structure may disappear but at 24°C. it may reappear. 
Again, according to the above author the leafhopper carrying the 
virus, if heated for one day, regained the ability to transmit in a 
few hours at the lower temperature and those heated for a week 
required two days or longer, whereas those heated for more than 
12 days never regained the ability to transmit. This is analogous 
to the above mentioned phenomenon that the length of time for 
which insects remain infective is determined by the length of time 
for which they have been fed on the diseased leaves. The stronger 
and the firmer is the change, the more prolonged period of time will 
be required for the recovery of the original structure, no matter 
whether the latter structure be normal or pathological. Thus the 
reactivation of a denatured virus would occur the more readily, the 
slighter and the shorter was the inactivation process. It may be 
possible to consider that, in the case of reversible inactivation of a 
virus, the inactivated structure is stable in the presence of the inacti- 


VIII. VIRUSES AND INSECTS 181 


vating agent, whereas normal structure is stable in its absence, a 
view which will be dealt extensively in Part V. 


3. Heritable Changed Structure 


Although the reversible character of protoplasm-protein structure 
is generally revealed in such a remarkable way, profound distortions 
given rise to in the structure may render the recovery of the ori- 
ginal structure extremely difficult. According to the structural 
pattern of virus the degree of distortion which will be raised in the 
protoplasm may vary, and some viruses can induce most profound 
distortions, while others only slight changes. If the distortion 
is slight, the distortion or the changed structure may be unstable, 
and the original structure may be promptly recovered with the 
disappearance of the virus and thus the disease will be cured, 
whereas if the change is striking the changed structure may be stable, 
and the virus will persist in the host cells as long as the host 
exists. Thus when a host is affected by a strong virus it may be 
unable to become freed from the virus for the remainder of its life. 

In connection with this trend of thought an extremely important 
argument should be advanced. Strong viruses may thus be able to 
induce replicas in host firm enough to remain for the host’s life. In 
the opinion of the writer, if the template action of the viruses is 
still stronger, the change can be spread even into the germ cells 
through which the structure is transmitted to the offspring. Such a 
transmission of the virus structure to the offspring must be the 
transmission of the virus itself to the offspring through the germ 
cells. Now we shall see next the verity of this concept. 

It seems that the virus structures multiplying in insects commonly 
disappear sooner or later, yielding to the normal structures. It has 
been known, however, that sometimes viruses persist not only for the 
remainder of the host lives, but also can be transmitted to the pro- 
geny of the host. Fukushi (96) has found that the virus of dwarf 
disease of rice was passed from parent to offspring to the third 
generation without recourse to a fresh source of virus. Black (97) 
likewise has confirmed che transmission of the clover club-leaf virus 
from parent to offspring. He found that the virus can be inherited 
over a period of four years without the insect coming to contact 
with a fresh source of virus. Such hereditary transmission of viruses 
has been recorded also with animal viruses. The eggs of a kind of 
wood ticks apparently give rise to infective nymphs if the parent 
tick is infected with the virus of equine encephalomyelitis. Similarly, 


182 III. THE EVOLUTION OF VIRUSES 


e 


the virus of Nairobi sheep disease is said to be transmitted through 
the eggs of a brown tick, the vector (98). 

Usually seed from infected plant gives healthy progeny, but a 
few plant viruses are believed to invade and survive in the seeds set 
by infected plants, thus making possible the transmission of the 
virus through seed, a phenomenon analogous to the virus transmission 
through the eggs laid by infective arthropods. Seed transmission is 
said to be more characteristic. of viruses of leguminous plants than 
any others; a number of those have been described as seed-borne. 

However, not all the seeds set by diseased plants are infected, 
and the proportion of infected seeds to virus-free ones depends on 
the length of time the parent plant has been infected (98). This may’ 
support the customary idea that the virus is mechanically carried by 
the seed. But in the opinion of the writer this should be regarded as 
analogous to the above cited fact that the ability of insects to cause 
infection depends upon the initial charge of the plant viruses, and 
also to the fact that both the duration of antibody production and the 
antibody amount produced tend to be directly proportional to the anti- 
gen amount administered. 

Such transmissibility of virus to offspring through egg or seed 
involves, to the belief of the writer, the utmost important significance. 

As will be discussed in great detail in Part V, there are many 
good reasons to conclude that in germ cells the replicas given rise to 
by viruses are kept in an altered form. The virus in itself, accor- 
dingly, may unable to be demonstrated as such. The altered replica, 
however, may gradually recover its original form as the germ cell 
develops to an adult organism, so that the virus may be revealed at a 
stage of the development. 

Usually young organisms having emerged from the eggs of the 
infected insects are not infective immediately after hatching, but will 
become so only after variable waiting periods. For example, according 
to Black (97) no insect raised from eggs laid by an insect vector 
infected by clover-club leaf virus can be infectious until three weeks 
after hatching, and most infections are obtained when the insects are 
between 6 to 12 weeks old. Such a failure of newly hatched insects 
in inducing infection may not be impossible to be attributed to too 
small virus amounts contained in the egg to cause the infection; if 
so, the waiting period may be the time required for the multiplication 
of this virus. The writer, however, regards this fact as an example 
of the reversion of the replica in the developing insect. It should be 
noted that plants generated from the seed inheriting a virus like- 
wise never reveal the disease symptoms until the plants grow up to 
a certain extent. 


VIII. VIRUSES AND INSECTS 183 


Such a phenomenon may never be confined to insects or plants 
only. Human beings may not be exceptional. It may be possible that 
children inherit the structure of a virus from parent through the 
germ cell, but that as the structure is transmitted in an altered form 
the reversion of the complete virus form requires some periods after 
the birth of the children, so that the virus appears when the children 
grow up to some extent. The virus of measles may be one of the 
viruses which can raise, in human protoplasm structure, a change 
strong enough to be transmitted to progeny. Children who have 
inherited the structure of measles virus from the parent may suffer 
from measles, without the infection from outside, when the structure 
recovers its complete form. 

In this connection it should be mentioned that bacteria infected 
with phage may become lysogenic in which, however, the presence of 
the virus cannot be demonstrated, but the bacteria are bound to 
undergo lysis with the liberation of the virus if brought under pecu- 
liar environmental conditions (7). This fact must depend upon the 
presence of the virus structure in an altered, inactive form in the 
bacteria just as the structure of the measles virus may be in the germ 
cells or in the premature children. 

In Chapter III in this Part is shown the fact that the seasonal 
change in the morbility of measles strikingly resembles the change 
in human conceptibility. Now, the writer is in the position to eluci- 
date this phenomen clearly. Thus, the seasonal change in the human 
conceptibility may be represented in a smooth curve presumably be- 
cause of a regular seasonal change in the activity of hormonal glands, 
while, on the other hand, the complete structure of measles virus may 
be developed in human cells just as harmones are excreted under the 
regular influence of seasons. It may be said, therefore, that the re- 
version of the virus structure tends to occur in a definite season when 
the children grow up to a certain age. 

If the pattern of the structure of measles virus inherited from 
parent is developed in a child, the pattern will be engraved in the 
mother-germ cells of the child by the infection, and therefore the 
pattern will be transmitted from generation to generation. If, however, 
the normal structure of the protoplasm is sufficiently strong to reject 
the virus structure, or at least powerful enough to overcome the virus 
_ Structure before it can engrave its pattern into the germ cell, the virus 
will not be transmitted to the offspring. 

As is generally recognized, about 5 per cent of man do not suffer 
from measles throughout life, showing that some persons possess a 
strong predisposition to reject the virus. Even if the virus can afford 
to affect such a person of strong predisposition, the structure of the 


184 III. THE EVOLUTION OF VIRUSES 


virus may not only be unable to remain for a long period but may 
be defeated and assimilized soon by the normal structure. 

It is noted that not all larvae from eggs laid by insects carry- 
ing a plant virus become infective, suggesting those of a weak pre- 
disposition only admit the virus structure to develop. This appears 
to hold true also for viruses transmitted by seed. The proportions of 
infected seeds are reported to be highest with plants which are them- 
selves the progeny of infected seeds (39), a fact which is to be expected 
since the predisposition associated with a weak structure may be 
heritable. 

Not only the virus of measles but many other viruses of man 
may, more or less, be heritable. For instance, mumps, a virus disease 
of parotid gland, may be one of the heritable virus diseases. In this 
connection, it should be remembered that in the submaxillar gland of 
normal guinea-pigs can usually be demonstrated a virus, which, how- 
ever, inay become detectable only when the animals grow up to a 
certain age. 

Viruses such as those of measles and mumps may have extremely 
powerful structure, so that they may be able to pass though germ 
cells to progeny without being much concerned with the predisposition 
of man, but in the case of many other viruses, persons with a weak 
predisposition only may be affected and the structure may be trans- 
mitted to the next generation only when the virus affects most sus- 
ceptible persons. 


4. The Cause of the Fixation of Viruses 


Some viruses confer the animals they affected high degree of 
specific immunity, occasionally lasting for the remainder of their lives. 
The writer designates such viruses as fixed viruses, and holds the 
opinion that the immunity to a virus is caused by the engraving of 
the virus pattern into the protoplasm of the animal and that the 
immunity continues as long as the pattern is present in the animal 
(12). In other words, when some cells in an animal are endowed with 
virus structure, the animal is said to be immune to the virus. Since 
such cells are endowed with the structure of the virus itself, the 
virus which may invade afterward from outside will be preferably 
combined with the cells and assimilized without exerting any injuri- 
ous effect. 

If this concept is correct, a virus will be detected in organisms as 
long as the organisms are immune to the virus. In fact this is the 
case with plants. It is confirmed that the persistence of active virus 


VIII. VIRUSES AND INSECTS 185 


in plants recovered from virus diseases is an invariable and essential 
factor for the immunity. Thus, freedom from a second attack of an 
acute disease, or protection from the effects of virulent strains, persists 
only as long as tissues are infected. As for insects, after they become 
infectious on feeding on diseased plants, some remain capable for life 
of excreting the virus. Namely, the insects are ‘“‘immune’’ to the 
virus for the remainder of their lives. 

Likewise in the case of animals, it is known that, although they 
may apparently recover from: infection with some viruses, the animals 
can continue to harbor the agent in their tissue long after recovery. 
Accordingly, some workers have postulated that active immunity in 
virus diseases is dependent on ‘“‘infection-immunity’’, meaning that 
resistance to reinfection is due to the persistence of living virus in 
the body. The detection of viruses from recovered animals, however, 
is not always possible unlike with plants. It should be considered, 
therefore, that in the majority of animals, the structure of viruses in 
the host cells may be altered after the recovery from the diseases so 
as to be rendered unable to act as the virus itself but retaining the 
ability to combine preferably with the virus without being suffered 
from it. 

This concept is strongly supported by the fact ascertained with 
phage; bacteria may become immune to phage after the infection 
with the phage but usually no phage is detected in the bacteria. 
However, the phage will be released when the bacteria are brought 
under peculiar‘conditions (7). It must be emphasized that the acquired 
character to produce the virus is transmitted to progeny. In like 
manner, human beings also may be able to transmit the virus-produc- 
ing character acquired through the infection to the progeny. 

It may appear strange that bacteria that have become immune to 
the virus will be lysed when the active virus is produced. As is 
already suggested, this may be attributed to an unfolding of the 
protoplasm protein structure which is necessary for the acquisition of 
active virus structure. The virus structure in the bacteria may be 
inactive because of the folding of the active group, and the unfolding 
of the structure may result in the striking adsorption of water to the 
surface of the elementary bodies leading to the dissociation of the 
cell into the elementary bodies. This may be the case also with 
human cells which inherited virus pattern from parent. The virus 
particles thus released from the cells may be scattered about through- 
out the body to cause systemic infections. 

The persistent immunity must be based on the very powerful 
structure of the virus in question, because the host protoplasm will 
recover its normal structure by overcoming the virus structure unless 


186 III. THE EVOLUTION OF VIRUSES 


the latter is powerful. ‘Extremely powerful viruses can remain in 
the host not only for’ the host’s life, ‘but also can stick to its germ 
cells through which they can be transmitted to progeny. 

Since such firmness of the structure must be associated with the 
invariability of ‘the structure, fixed viruses’ with strong structures 
will hardly give rise to variants. The antigenic specificity of fixed 
viruses is, aS a rule, constant, and generally every fixed virus has its 
own antigenic specificity. Therefore, usually various types may, be 
demonstrated in a single fixed virus. For example, the virus of 
measles or of mumps has each distinct immunological specificity and 
hence various types of measles or of mumps are never found. Also in 
this respect fixed viruses may resemble a typical organism. 

On the other hand, newly generated viruses, as for example, those 
of influenza are provided with an extremely weak structure so that 
they may readily be overcome by the normal structure. Consequently 
such weak viruses will persist only for short periods in host cells. 
This must be the reason why newly 28 EES viruses can confer 
only transient immunities. 

The weakness of the structure should necessarily ‘be associated 
with the unusual variability. Indeed, influenza virus is noted for its 
marked variability. Newly generated viruses may have no constant 
immunological ‘specificity because of this marked variability- as well 
as of the suc¢essive generation of similar ‘viruses having’ varying 
specificities. The presence of such various immunological specifici- 
ties, therefore, must be one of: the characteristics of newly beaiitien os 
viruses. 


However; if a’ virus’ particle having a relatively rigid’structure 


is incidentally generated, it will remain longer ‘and multiply more 
abundantly than do other weaker individuals. Virus particles produced 
by such a ‘virus should inherit this relatively rigid structure. And 
again a particle having the strongest structure will naturally be sele- 
cted out of these ‘relatively strong particles, as the strongest can 
remain much longer and multiply much vigorously... Thus the struc- 
ture of a virus is to’ become naturally more’ and more rigid and 
powerful. 

The acquisition of the ‘strong structure appears to answer the 
purpose of race-preservation as well as of self-preservation of viruses. 
In short, viruses which can give rise to prolonged immunity are more 
fitted for existence than those which may confer only transient immu- 
nity and those which can spread to progeny through germ cells are 
still more fitted. As to plant viruses, the ones which can long persist 
in insect bodies are fitter, while the ones which can be transmitted 
through the eggs to the offspring must: be still more fitter. 


~ 
< 


VII. VIRUSES AND INSECTS 187 


Now we have to discuss the nature of the rigidity of virus struc- 
ture, the most important fector determing the virus evolution. As 
already pointed out, protoplasm structure must be springy. A spring 
may readily resile to its original state after a slight bend, but do so 
only with a considerable difficulty after a heavy distortion. The 
viruses capable of exerting on protoplasm such a heavy distortion 
may be said to be strong and rigid. The degree of the distortion 
may be determined by the structural pattern of the virus probably by 
the manner of spatial arrangement of polar groups, especially of amino 
acid residues. 

Since the structure to cause a heavy distortion can multiply in 
the protoplasm through the infection with the virus having the struc- 
ture, and since a heavy distortion must be the structural change 
which cannot easily spring back to the original structure, the virus 
which can cause a heavy distortion must be in itself stable and rigid. 
In short, the virus having a stable, rigid structure can cause a heavy 
distortion in the host protoplasm. The virus capable of producing a 
strong pattern, therefore, may accomplish vigorous multiplication, and 
at the same time the virus itself is stable and fitted for prolonged 
existence. 

In additicn to the chemical structure of the component protein, 
the amount and the kind of nucleic acids contained in the virus parti- 
cles may also play an important role in the determination of the virus 
strength. If a virus particle contains a nucleic acid in a richer amount 
than the others, it will act as a stronger virus owing to the hardening 
action of the acid already discussed. If, therefore, certain cells acuqire 
the property to synthesize nucleic acid more vigorously on the infec- 
tion with some virus, strong virus particles will be produced by such 
cells. Thus it may be concluded that the property of a virus to make 
the nucleic acid increase in the host cells is essential to its evolution. 

Particle size is considered to be another important factor for the 
virus evolution. As already pointed out assimilase action is, to some 
extent, directly proportional to the polymerization degree of the pro- 
teins, so that larger particles can reveal greater action. 

To sum up, in addition to the specific structural pattern of the 
component protein capable of causing marked distortion in the host 
protoplasm, the properties to agglutinate into large particle and to 
combine with large amount of nucleic acid appear to be essential for 
the evolution of viruses, that is, for the increase in their template 
strength, although these properties may be governed also by the specific 
structural pattern of the component protein. 

Even when the infected cells of the same kind are decomposed 
into virus particles, the particles produced may not be identical with 


188 III. THE EVOLUTION OF VIRUSES 


one another either in size or in nucleic acid content. In such a case, 
large particles rich in nucleic acid only may be able to act as a strong 
virus, whilst small particles with little or no nucleic acid may be 
extremely unstable, scarcely capable of acting as a virus, and may 
therefore disappear immediately after the production. 

Moreover, the replica corresponding to the virus pattern may not 
be uniformly produced in all the particles; in some particles complete 
replica may be formed but in others only incomplete replica. Particles 
endowed with such incomplete replica may be unable to behave as a 
virus. It can be said, therefore, that the demonstration of virus action 
can scarcely be achieved with all the particles produced by infected 
cells. However, the property to constantly produce similar large par- 
ticles, rich in nucleic acid, having complete replica should be required 
for a virus in order to evolve into a typical organism. 


CHAPTER IX 
THE REJUVENATION OF VIRUSES 


1. The Transmission of Viruses by Insects 


However strong the virus structure may be, it will gradually be 
assimilized by the host protoplasm if the virus continues to multiply 
in the same kind of the host. Since the protoplasm in which the 
virus multiplies is also assimilase similar to the virus, the protoplasm 
may always be striving to assimilize the virus though it is weaker 
than the virus in the assimilase action as to be assimilized, in the 
main, by the virus. In addition, as the protoplasm structure involves 
the reversibility, the protoplasm itself may be striving to recover its 
original structure even when it has been changed into a virus structure; 
that is to say, there must be a potential force in a virus to develop 
by itself the original structure of the protoplasm. It will be expected 
therefore that viruses themselves will be assimilized in the long run 
by the protoplasm of the host cells if they always adhere to the same 
kind of host. 

In order to avoid this fate, viruses have to change frequently 
their host. Insects appear to be commonly utilized by viruses for 
this purpose. Various animal and plant viruses are transmitted by 
insects in which they can multiply, thus host change becoming possi- 
ble. As for some plant viruses and their insect vectors, however, 
many cases are known in which virus multiplication in insects can 
never be considered, although the viruses are undoubtedly transmitted 
by insects. In such cases, insects can infect healthy plants immedi- 
ately after they have acquired viruses, and they soon cease to be able 
to do so, sometimes becoming non-infective within minutes and always 
within hours after leaving the infected plants. 

In view of these facts it may be said that the relationship between 
plant viruses and their insect vectors may be revealed in the following 
two ways: In the first, the insect acquires infectivity after a cer- 
tain incubation period and the infectivity continues for long periods, 
frequently for the remainder of its life, as already described; in the 
second, the insect becomes infective immediately after leaving a 
diseased plant and the infectivity is rapidly lost. 

Many workers may consider that in the latter case the insects 
act simply mechanically, transferring the virus as a contaminant on 


190 III. THE EVOLUTION OF VIRUSES 


the outsides of their stylets. Nevertheless. Bawden (39) claims that 
there is much evidences showing that the vectors do not behave 
merely mechanically. Thus, if insects acted solely by getting their 
mouthparts contaminated externally with virus, it is difficult to see 
why all those with similar feeding habits should not’ be equally effi- 
cient vectors, except that those with larger stylets might perhaps be 
expected to carry a little more virus. However, this is not so, and 
insects with similar feeding habits will transmit some viruses but not 
others, and, with viruses that have a number of aphids as vectors, 
one species is often much more efficient than the other. Bawden 
states further that if transmission by insect were merely mechanical, 
it might be expected that virus most readily transmitted by artificial 
inoculation could also be most readily transmitted by insects. And, if 
the viruses were merely acquired as a contaminant on the mouth 
parts, those occurring in infected plants in the greatest concentration 
might be expected to be most easily aquired and transmitted. But it 
is not so. . Tobacco mosaic virus and potato virus X are more easily 
transmitted by rubbing or by needle inoculation, and occur ‘in’ greater 
concentration in extracts of infected plants, than any of the viruses 
under: discussion. Neither of these normally appears to be aphid-. 
transmitted. é 

Moreover, Bawden emphasizes the fact: that insects can. cause 
infection with virus amounts much smaller than those needed - by 
ordinary inoculation method, :and he describes further the remarkable 
fact that the efficiency of insects as: vectors is much increased if they 
are prevented from feeding for some time before they are fed on the 
source of the virus. : 


2. The Mechanism of ‘Rejuvenation 


There seems no doubt that plant viruses above mentioned which 
are rapidly lost in the insect vectors fail to multiply in the insect 
bodies. It seems, however, reasonable to consider that transitory 
activation of the viruses may take place in the insect bodies. 

Prior to considering the mechanism of this virus activation, we 
have to discuss the nature of protein denaturation. Proteins, in general, 
when exposed to physical or chemical effects, may change their pro- 
perties so as to become ‘‘denatured’’ through the alteration of their 
structure. In this process the closely folded polypeptide chains in 
protein molecules may unfold transitorily and subsequently again may 
refold to give some other folding pattern different from the original 
internal structure, as shown in Fig. 21 (102). 


IX, THE REJUVENATION OF VIRUSES 191 


The peptide chains in the native protein molecules are folded as 
shown in: I of this Figure; in this state almost all the polar groups 
of the side chains may exist in mutual saturation, whereas, by the 


Fig. 21, Diagram of protein denaturation. After. Haurowitz; Chemi- 
stry and Biology of Proteins, 1950, (a little modified). 


action of denaturating agent, the chains are unfolded as II, thereby 
polar groups of the side chains are set free, but as this form is un- 
stable, refolding will follow soon to take the form III which is different 
from I. 

At first, mention should be made as regards the phase II in Fig. 21 
where peptide chains are unfolded and polar groups are set free. In 
this phase proteins may be in an activated state owing to this freed 
polar groups which enable them to combine with one another or to 
act upon other substances. For example, red blood corpuscles are 


192 III. THE EVOLUTION OF VIRUSES 


agglutinated by the antiserum, probably because of the denaturation 
of the surface protein of the corpuscle due to the antiserum, whereby 
free polar groups causing the corpuscles to combine with one another 
are set free. If an agent named complement is present in this process, 
the phenomenon called complement fixation may occur, in which com- 
plement is combined with blood corpuscles owing to the same polar 
groups set free. Such a state of corpuscles to achieve agglutination 
and complement fixation is actually known to be only transient, 
showing that unfolding of the peptide chains is soon followed by the 
refolding (12). 

Lesley e¢ al. (101) have suggested that infection of bacteria by 
phage stimulates some mechanism whereby phage adsorbed subse- 
quently to the cell is broken down extensively at the cell surface. 
This may be ascribed to the activation of the cell surface by the 
preliminary combination with phage; the activation resulting from the 
liberation of polar forces may enable the cell surface to cause a 
severe structural disturbance in the phage added at a later time. 

Some plant viruses can readily infect leaves by rubbing if the 
leaves injured by carborundum, indicating that the protein in the sur- 
face of the leaves is activated by the application of the agent to adsorb 
the virus. In this case the activated state is likewise temporary, as 
it is confirmed that the leaves regain their resistance to infection 
within three hours of the procedure, though they remain permeable 
to salts presumably because the cuticle is not repaired (102). 

Now we have to return to the problem concerning the transmis- 
sion of plant viruses by insects in which no virus multiplication takes 
place. The writer claims that also in this case viruses may acquire 
the activated state in insect bodies. The agent causing such an 
activation is presumably a protein, present in the alimentary tract of 
the insect, which can combine specifically with the virus, like an anti- 
body with the antigen. 

As already mentioned, some insects can be parasitic on certain 
plants because of the presence of common structure between their 
proteins, whereas the same relationship must be present between a 
plant and a virus which can affect the former. Therefore, also be- 
tween a virus and an insect living on the plant which the virus affects, 
there must likewise be common structure, through which the protein 
of an insect can combine specifically with virus. Thus, if the virus 
is taken into the alimentary tract of the insect, some protein present 
in the latter will combine with the virus. This combination should 
be analogous to that between antigen and antibody, and hence a kind 
of denaturation is to be raised in the virus, thereby the temporarily 
activated state will result in the latter. 


IX. THE REJUVENATION OF VIRUSES 193 


The presence of specific relation between insect and virus as well 
as the prompt disappearance of the infectivity can readily be explained 
by this concept. The efficiency of fasting of the insect upon the 
infection of the virus may be attributed to the increase by the fast- 
ing of the agent specifically acting upon the virus. It is fully ex- 
pected that for the occurrence of the activation proper proportion of 
the virus to the activating agent will be required as in the case of 
antigen-antibody reaction. It is said that the optimum conditions for 
transmission are to use insects that have fasted for at least one hour, 
feed them on the diseased plant for about 2 minutes and then transfer 
them immediately to healthy plants (39). Increasing the length of 
time the aphids feed on the infected plant greatly reduces the number 
of infections obtained, indicating the decreased activation due to too 
much amounts of the virus. 

Argument has been advanced thus far concerning the second phase 
of denaturation, whereas now we have to discuss the third phase 
shown in III in Fig. 21. If the environmental condition, under 
which protein molecules are present in the first state, is changed, 
thereby the structure of the protein being rendered unstable, then the 
protein will be changed in the structure to adapt itself to the new 
condition. It is considered that such a change is commonly accom- 
plished by the refolding of the peptide chain. 

Thus, if some protein is altered in its structure from form I to 
III in Fig. 21 by an environmental change, the structure represented 
by III must be stable under the new condition. Environmental change 
can be brought about by various physical or chemical agents, and 
hence, if some chemical agent causes the structural change from form 
I to III, it will be said that the form III is stable in the presence of 
the chemical agent. 

The shift of the form, however, may often fail to occur when the 
environmental change gradually arises, just as water may be super- 
cooled without being frozen, but when an adequate stimulus is applied 
to disturb the structure of the protein, the shift will immediately 
start to attain to the stable state just as super-cooled water is sud- 
denly frozen by stir. For example, we have frequently observed that 
phage samples having been inactivated by some unknown causes are 
activated by such manipulation as the addition of inorganic salts or 
heating to a proper temperature (103). Such a phenomenon can be 
explained by assuming that the phage remained in an inactivated state 
despite of the removal of the inactivating agent, and that the mani- 
pulation served only to initiate the recovery of the original activated 
State. 

In general, the mechanism of the reversible inactivation of physi- 


194 III. THE EVOLUTION OF VIRUSES 


ologically active agents such as viruses, toxins, and ferments can be 
illustrated in the same way. Thus the nature of the reversibility of 
protein structure discussed in the previous chapter is also made clear. 

As above discussed, some plant viruses may be activated in insect 
bodies, a phenomenon which has been explained by taking into account 
the second phase of the protein denaturation. This phase is considered 
to be brought about by some denaturating or inactivating agent present 
in the alimentary tract of insect, so that the third phase, reached by 
passing through the second phase, should involve the inactivated state. 
It is actually known that some agent capable of inactivating certain 
viruses are found in insect bodies. 

However, if the inactivating action of the agent is not so strong 
as to completely inactivate the virus, the virus will be disturbed in 
its structure without being inactivated, and the disturbance may 
enable the virus to recover its original structure which the virus 
possessed before having been damaged by the host protoplasm from 
which it is now liberated. 

Thus, if a virus which has been weakened by an unfavourable 
condition in the host cell, is removed from the host to an insect, a 
new host, in which no unfavourable condition is present and in which 
the virus can regain the original active state, then the virus can be 
said to have achieved ‘‘rejuvenation.”’ 

For the establishment of this rejuvenation in insect, some adequate 
stimulus to cause the shift towards the original active state may be 
necessary. Such a stimulus is possibly given to the virus through the 
contact of the virus with the insect protoplasm having a structure 
somewhat different from that of the virus. If such a stimulus is too 
strong, the virus will sooner or later be inactivated, although in the 
second phase of the inactivation or denaturation the transient activation 
may arise. In such a case plant viruses are transitorily activated in 
insect bodies without multiplication. 

On the other hand, if the stimulus is mild and adequate the virus 
will be able to regain its original active structure in the insect body 
without inactivation, and in addition the virus thus activated is pos- 
sibly capable of spreading its structure into the insect protoplasm, 
that is, the virus will be able to multiply in the insect body. Since 
different hosts must have different protoplasm consisting of proteins 
of different structures, although they may share some structure in 
common as they have the property to be affected by the same virus, 
the virus will possibly acquire some new structure through the multi- 
plication in a new host. 

The acquisition of a new structure on the multiplication in a new 
host means that the virus is assimilized to a certain extent by the 


IX. THE REJUVENATION OF VIRUSES 195 


new host, but by this process the virus will be able to get rid fully 
of the injurious assimilizing effect of the former host and thereby 
the rejuvenation will become quite complete. 

The concept that plant or animal viruses can multiply in an 
insect, an entirely different organism, may seem at first sight unrea- 
sonable, but as already pointed out, the fact that an insect is parasitic 
on a certain animal or a plant shows the presence of common struc- 
ture between the protein of the insect and that of the animal or of 
the plant; through this common structure the virus may be able to 
affect both the insect and the animal or the plant. In other words, 
the relationship analogous to that between an enzyme and its substate 
must be present between an insect and an animal or a plant on which 
the insect is parasitic, and a similar relationship must also exist 
between a virus and its host, and accordingly a virus can simulta- 
neously affect both an insect and an animal or a plant. 


3. Various Means for Rejuvenation 


Whereas the host change appears to be most essential to the evo- 
lution of viruses, it cannot be said that all fixed viruses are transmitted 
by insects. For example, viruses of such diseases as measles, mumps, 
or small pox seem to have no insect vectors, a fact which appears 
contradictory to the writer’s view. 

Rejuvenation is, however, indispensable not only for viruses but 
also for every organism without exception. Organisms are generally 
supplied with a splendid tool to perform the rejuvenation. This tool 
is sexual reproduction. By means of this tool a protoplasm having a 
certain structural pattern can combine with another protoplasm having 
different structure, thus being able to achieve the structural exchange, 
a process which may be called mutual assimilation, and by this pro- 
cess rejuvenation is accomplished. This appears quite analogous to 
the rejuvenation of virus by means of the host change. 

The writer holds the opinion that above cited viruses, such as 
those of measles and mumps which appear unable to perform any host 
change, can achieve rejuvenation by availing themselves of the sexual 
reproduction of the host. As fully discussed already, some fixed 
viruses can be transmitted to offspring through the germ cells of the 
host; in the case of insects the transmission is said to be usually 
established by eggs. Since egg cells are to be rejuvenated on the fer- 
tilization by the contact with sperm cells composed of proteins with 
somewhat different structure, the virus which has become a part of 
the egg cell should also be rejuvenated through this process. 


196 III. THE EVOLUTION OF VIRUSES 


When the structure of a virus becomes the stronger, the longer 
it may remain in the host, until it can even be transmitted to the 
progeny. It may be said, therefore, that a virus can attain of itself 
the property to perform the rejuvenation as it evolves to a certain 
extent. Thus, it follows that the strong structure becomes indispen- 
sable for a virus to continue its existence not only for the reason of 
the necessity of the stable nature but also of the need of the ability 
to make use of the rejuvenation of the host. 

Plant or animal viruses which can multiply in insect vectors may 
naturally avail themselves of the sexual reproduction of the customary 
hosts as well as of the insect vectors when the virus acquires struc- 
ture strong enough to spread their pattern to the germ cells. Thus 
it should be concluded that most evolved viruses are always to be 
transmitted to the offspring through the germ cells of the host. 

In order to acquire sucha strong structure the majority of viruses 
might have attained to the habit of host change; therefore, a virus, 
which have bocome transmissible by a vector, may be said to have 
come up to the easy course leading to a typical fixed virus. The 
writer has already expressed the opinion that viruses such as those 
of influenza and encephalitis may be newly generated viruses, but 
some of them might possibly have already arrived at such a course 
and hence it may be unreasonable to regard all of them as being new 
viruses. Indeed, it is generally accepted that some encephalitis viru- 
ses do posses insect vector and further that they can give rise to con- 
siderably long lasting immunity in their host. Likewise, some strains 
of influenza virus may be half-fixed through a host change unknown 
to us. It has been reported that swine influenza virus is actually 
disseminated by a lung-worm parasitic to the dog (104). The so-called 
pneumonitis virus, highly specialized virus-group causing pneumonia, 
may be an advanced form of influenza virus. 

It seems little doubt that insects or arthropods are generally 
utilized for the host change, but also other organisms may possibly 
be used for the purpose. For example, if a dog infected by rabies 
virus, it will become wild and maniacal and try to bite other animals, 
enabling the virus to be transmitted and at the same time to be reju- 
venated. Some plant viruses, such as tobacco mosaic virus, known 
to have no insect vector, may have some means of rejuvenation, if 
they cannot use of the sexual reproduction of the host plant. It 
is believed that various plant viruses can contaminate the soil and 
persist in it for long periods so that many of the outbreaks in tobacco 
and tomato crops originate from virus surviving in the soil; in some 
favourable soils some plant virus could be ascertained to survive for 
more than 9 years (105). Such a fact led a number of workers to be- 


IX. THE REJUVENATION OF VIRUSES 197 


lieve that some soil-inhabiting organism acts as a vector, presumably 
a nematode (39) 

Furthermore, it may be possible that some animal viruses multi- 
ply in certain bacterial cells to establish rejuvenation and subsequently 
to infect again the animals. If so, the bacteria would be thought 
pathogenic. The majority of workers believe that the causative 
agent of whooping cough is a kind of bacteria, whereas some others 
claim it to be a virus; the infection was reported actually possible 
through the filtrates of the patient excretes. 

Again, scarlet fever is usually believed to be caused by strepto- 
coccus*but repeatedly a virus has been suspected. Bringel (106) has 
claimed to have succeeded in isolating a filtrable agent which makes 
a Strain of haemolytic streptococcus capable of causing scarlet fever. 
This agent when present in the filtrate is so unstable as to be inacti- 
vated by heating to about 60°C. for 45 min. 

If a virus causes a venereal disease, it can be rejuvenated by 
being transmitted from one sex-to the other. Such a rejuvenation may 
be as complete as that brought about by sexual reproduction, since 
the virus is always to be transmitted in turn between both sexes. It 
is known that the virus of lymphogranuloma venereum, a _ highly 
advanced virus causing a venereal disease, fails to leave any immunity 
in the host in spite of its highly fixed character. For achieving the 
rejuvenation, the establishment of the immunity must be unfavourable 
for such a virus, so that the character not to leave the virus pattern 
which can contribute to the immunity might develop, the individuals 
having this character being selected as the fittest. Such a virus 
might evolve into bacteria, such as gonococcus, which have also the 
character to cause no immunity. 


CHAPTER X 
THE SECONDARY ORGANISMS 


1. Parasitism and Commensalism 


Since pathogenic filtrable agents are designated viruses, pathogeni- 
city must be an essential feature of viruses, whereas pathogenicity is 
by no means needed by protoplasm particles to exhibit the assimilase 
action. Only when the particles cause an injurious effect on the pro- 
toplasm of cells on which they exert their assimilase action they may 
be called viruses. Their existence therefore may not be realized if no 
injurious effect follows. Plant viruses may be called so because they 
can cause deleterious effectes in plants, but when transmitted to 
insect no effect is exhibited, so that despite the occurrence of their 
multiplication, their existence is only demonstrated when they are 
given back to the plants. In general, no difference can be found be- 
tween the insect carrying the virus and that carrying none. In such 
a case the virus may be called a, latent. 

It seems highly possible as already discussed that protoplasm par- 
ticles derived from normal, healthy cells act as a virus on some other 
cells, but it must be of most difficulty to distinguish a genuine latent 
virus from normal particles capable of acting asa virus. Presumably 
the term latent virus have been used up to the present without taking 
account of this problem. 

Assimilase action is usually involved in the protoplasm having its 
full form, only rarely the action being preserved in the decomposed 
particles. But, for the development of an assimilase into an indepen- 
dent organism, it must be quite necessary for the assimilase to retain 
its function in the form of decomposed particles. 

There is no reason to suppose, however, that pathogenicity is 
needed for the evolution of virus. On the contrary, it seems to be rather 
deleterious, because severe pathogenic action may have unfavourable 
influences on the multiplication of viruses. 

If a virus possessed a virulence so violent as to infallibly kill the 
host, it would soon be perished together with the host. It may, there- 
fore, be supposed that less virulent or non-virulent viruses can more 
easily pursue the course of the evolution than do more virulent. Thus 
the number of non-virulent viruses existing may be much greater than 


X. THE SECONDARY ORGANISMS 199 


expected. However, we are unable to demonstrate their existence 
because a virus is detected always on the basis of its pathogenicity. 

Nevertheless, when viruses have highly evolved as to arrive at 
the state of Rickettsiae, their existence may become demonstrable on 
account of the peculiar shape and size of their particles even if they 
have no pathogenicity. Thus, although only six or seven species of 
Rickettsiae have been found in association with disease of mammals, 
over forty non-pathogenic species have been described in various in- 
sects and other arthropods (88). In addition, isolation of various non- 
pathogenic pleuropneumonia-like organisms from the genitourinary 
tract has frequently been reported (107). Such organisms are con- 
sidered to be intermediate between Rickettsiae and bacteria. It must be 
emphasized that the majority of the bacteria thus evolved from viruses 
are likewise non-pathogenic. 

Some bacteria are not only non-pathogenic but sometimes appear 
to have favourable effect on the host. This may be the case also with 
some viruses, because in doing so the viruses may produce in the host 
more beneficial conditions for their multiplication and accordingly for 
the continuance of their existence, though it is also conceivable that 
some proper injurious‘effect upon the host may sometimes be desirable 
for their multiplication, for it is occasionally recognized that emaciated 
organisms are more readily affected by a virus, a phenomenon which 
is also well confirmed in some Rickettsiae. 

Most workers have failed to find any significant differences be- 
tween infective and non-infective individuals of insect vectors. Com- 
monly infective individuals live as long as and breed as non-infective. 
However, a beneficial effect from feeding on celory and aster plants 
infected with aster yellow virus has been described by Severin (108), 
who found that several leafhopper species multiply more freely, pass 
through nymphal stages sooner, and live longer than those feeding 
on healthy plants. In most cases the difference was quite clear-cut, 
the insects dying off in a few days when put on healthy plants, but 
building up flourishing colonies when put on diseased ones. There 
are numerous leafhopper species which live on diseased celory and 
asters, but when transferred to healthy plant they die. Likewise 
with aphid and leaf-roll mosaic, Arenz (109) has found that the vector 
multiplies substantially faster on diseased potatoes than on healthy. 
These may show the favourable effect of the virus upon the host, but 
at the same time they may be regarded as being only the examples 
where injuries given rise to by virus upon the plant benefit the 
proliferation of the insect (110). However, in discussing numerous 
cases of an association between insects and microorganisms for mutual 
benefit, Leach (111) has claimed that there is no reason why association 


200 III. THE EVOLUTION OF VIRUSES 


of insects and viruses may not have arisen in a similar manner. 

Sonneborn (112) has found in a kind of paramecia a virus-like 
agent designated kappa; the individuals of this protozoan containing 
this agent produce a substance called paramecin which is able to kill 
another kind of individuals having no kappa, but when a large 
amount of this agent is present, the protozoan acquires the property 
not to be killed by paramecin. Protozoa not containing kappa or with 
only a few are readily killed by paramecin. The presence of kappa, 
therefore, confers a Survival advantage to the protozoan. This agent 
is believed by some workers to be a kind of Rickettsia-like micro- 
organisms parasitic on the protozoan. Likewise in various arthropods 
there are found Rickettsia-like bodies, known as bacteroids; they have 
rarely, if ever, been grown outside the living host. It is possible, 
however, to eliminate them in a certain insect by treatment with 
penicillin or with certain sulfa drugs, and in this way to show that 
bacteroid-free individuals invariably die (113). This is no doubt an ex- 
ample of commensality. It is a noticiable fact that the bacteroids are 
said to be transmissible to progeny through the eggs like viruses. 

Anyhow, it may safely be mentioned that numberless seeds of 
secondary organisms are still continuously being poured upon the 
earth, but that only exceedingly small portions of them are to evolve 
into organisms. The seeds are, of course, protoplasm particles with 
assimilase activity having become independent of the protoplasm. They 
are recognized as the virus when able to confer some injurious 
effects upon certain cells, but the majority of them may silently be 
pursuing the course of the evolution leading to the secondary organism 
without being recognized. 


2. Important Significance of Parasitism 


The primary organisms were presumably generated and developed 
in water. They were unable to leave the water until evolved into 
highly developed creatures, whereas the secondary organisms were 
produced in cells of other organisms already created, and developed 
in the cells, so that their parasitic nature would be of most obstinacy. 
For the second organisms the liberation from the parasitism, there- 
fore, would be as serious and as difficult as would be the release from 
the water for the primary organisms. 

In this respect any parasitic organisms, living outside the water 
despite their comparatively primitive nature should be regarded as the 
secondary. Accordingly, at least the majority of bacteria and protozoa 
should be considered to be the secondary organisms. To be sure, 


X. THE SECONDARY ORGANISMS 201 


bacteria are mostly parasitic, and all the major groups of protozoa, 
except the sporozoa and the sustoria, involve both free-living and 
parasitic forms. Sporoza are entirely parasitic and sustoria are 
sedantry, although they are often found attached to the surface of 
fresh-water organisms. Many parasitic protozoa, especiallly sporozoa, 
undergo extremely complicated life cycles, involving not only several 
successive hosts but also distinct evolutionary states that are morpho- 
logically different. 

It is noticeable fact that exceedingly vast number of species are 
known in protozoa, over 15,000 species having been described, and that 
most modern authors are inclined to consider many groups of them 
as so many phyla (114). This would follow naturally if protozoa were 
evolved from viruses continuously being generated de novo. Parasi- 
tism must be the most distinct characteristic feature of the secondary 
organism, but such an existence of a vast number of groups, between 
which no phylogenetic correlation appears to be present, must likewise 
be one of their chief characteristics. 

The so-called mesozoa, which was once recognized to be intermediate 
between protozoa and metazoa, are totally parasitic, and there seems 
no doubt in their secondary nature. Notwithstanding their highly 
evolved state, animals belonging to platyhelminthes such as flukes and 
tapeworms are still sticking to their parasitic nature. These animals 
are not only parasitic but also still clinging to the habit of virus of 
changing host and unable to proliferate without the troublesome host 
change. Thus host change must also be one of the characteristics of 
the secondary organisms. Malaria-producing protozoa and a kind of 
mosquito, trypanosomes and a fly, spirochaetes of reccurent fever and 
a tic or louce,—these are all the examples of the attachment to the 
habit of host change. 4 

In the case of viruses the host change may be indispensable for 
the purpose of rejuvenescence, but it is rather strange that the 
secondary organisms are frequently clinging to the habit still at present 
when they have acquired much more suitable means, namely sexual re- 
production, for the purpose. The habit may be beneficial for the trans- 
mission as in viruses, but this cannot be supposed to be the only reason 
for the persistence. If eggs of a parasite such as a tapeworm or a flute 
acquired the faculty to develop in the host bodies without trouble- 
some host change, it would be said that a remarkable progress was 
established for their proliferation, but the host should be perished 
because of the dreadful proliferation of the parasite, resulting in the 
extinction of the parasite itself. During the long course of evolution, 
parasite capable of developing without host change might appear, but it 
seems no doubt that the host of such dreadful parasites would soon 


202 III. THE EVOLUTION OF VIRUSES 


be perished, followed by the extinction of the parasites themselves. 
In this way the habit of the host change might be left as an indis- 
pensable character. 

This may hold also for the plant kingdom. Smuts parasitic on 
higher plants are known to perform most complicated host change or 
life cycle; not only smuts but generally pathogenic fungi are mostly 
confirmed to be transmitted by insects. Thus insect-fungi relationship 
is highly organized and has broad biological and evolutional signifi- 
cance. The insects known to be vectors of plant disease-producing 
fungi are grasshoppers, crickets, aphids, scale insects, beetles, true 
bugs, flies, wasps, and bees (115). Plant diseases caused by bacteria 
are also numerous, and in the dissemination of pathogens from field 
to field, insects have been recognized as vectors since the days of 
very early investigations (116). 

Likewise nematodes such as ascaris and filaria belong most pro- 
bably to the secondary organisms. Ascaris has no insect vector, but 
its eggs cannot develop unless they are exposed to low temperatures 
outside the host body, probably contributing to the prevention from 
its too vigorous proliferation as mentioned above, but also serving to 
the achievement of the rejuvenation as will be mentioned later. It 
has been established that certain mosquitoes are vectors of filaria, 
causing elephantiasis in man. Several other species of nematodes 
associated with insects possess likewise a heterogenic generation in 
their life cycle. In addition, each of the three fundamental groups of 
annelids also gives rise to parasitic species that show, in almost 
every case, definitely specialized characters of parasitism. 

The arthropods, constituting by far the largest phylum of the 
animal kingdom, may be the most advanced secondary animals, although 
the majority of them have already succeeded in getting rid of parasi- 
tism. Of crustaceans of the arthropods, all stages of parasitism occur 
especially in copepods. Further, mites and ticks are the parasitic 
acarina. Nearly all the groups of insects contain species that fail as 
yet to become free from parasitism either in the larval or in the adult 
stages. However, only very few insects are totally parasitic in all 
stages of their life history and these are practically restricted to the 
true lice and sucking lice. 

It is customarily believed that parasitic organisms have been 
evolved from free living ones on adapting themselves to parasitism. 
If this were true, parasitic organisms should have been more advanced 
than the free living. However, the truth is that the most primitive 
organisms such as bacteria and protozoa are almost parasitic, whilst 
the most advanced organisms such as vertebrates including man are 
entirely free-living without a single exception, presenting a fair con- 


X. THE SECONDARY ORGANISMS 203 


trast to the fact that Rickettsiae as well as viruses are all parasitic. 
Is it really conceivable that man and bacteria share common ances- 
try and that the latter have waited and waited throughout a dread- 
ful long span of time in the most primitive unicellular form for their 
brethren to be evolved into man, on which they were bound to be 
parasitic? Is there any doubt in assuming the parasitism as being 
the most primitive mode of life of organisms ? 


3. The Limit of the Secondary Organisms 


The writer holds the opinion that free-living organisms can never 
enter parasitic life unless they have a phylogenetical experience of 
parasitism. Parasitic life involves too specialized and too limited en- 
vironment to be newly adapted. 

Certain animals especially some insects are known to become 
parasitic at their-adult stage although free-living at the larval. As 
will be fully discussed later, this must be a kind of atavism, never 
the new acquisition of parasitism. This occurs generally much more 
frequently in females than in males; it can be supposed that parasi- 
tism is more profitable for females because of some physiological 
function particular to females such as egg production or egg laying, 
and hence females that could accomplish the reversible parasitism or 
atavism might have been selected as the fittest. 

Parasitic botflies are known to show a complicated life cycle. 
Botfly eggs can hatch only under the combined influence of the 
horse’s tongue and saliva with which they are carried into the mouth, 
whence they burrow into the mucosa of the tongue or the palate and 
tunnel their way through until they reach the pharyx. The second 
larval stage is now reached. The maggots emerge from the mucosa 
and attach themselves on the surface, close to the epiglottidean region. 
They finally pass down into the intestine and each species takes up 
its abode in a particular region, some in the stomach, the others in 
the rectum or the duodenum. They generally abandon their host 
before pupating and are evacuated with feces (117). The life history 
of ascaris is somewhat similar to this, indicating a similarity in their 
parasitic nature existing between nematodes and insects. Insects, in 
general, possess at their larval stage a form strikingly resembling 
that of nematodes, a fact in which a relic of parasitism is seen. 

Metamorphosis, especially distinct in insects, may suggest the rapid 
phylogenetic occurrence of the liberation of themselves from parasitism 
just as the sudden release from water of a tadpole with the establish- 
ment of metamorphosis. 


204 III. THE EVOLUTION OF VIRUSES 


It was said that the difference between a carnivore and a para- 
site is simply the difference between living upon capital and income, 
between the burglar and the blackmailer, but this view may not be 
legitimate, for there seems an essential difference between them. A 
carnivore may eat flesh of every kind of animals, whereas between a 
parasite and its host there is a specific relationship which should be 
found between a virus and its host or even between an enzyme and 
its substrate. Caterpillars feed on the leaves of plants, but there is 
a definite relation between a caterpillar and the sort of the plant. 
Again, there is also a specific relationship between a mushroom and 
the kind of trees on which it grows. 

As to the plant kingdom, not only mushrooms but generally fungi 
can be regarded as the secondary organisms. Schizomycetes or bacteria 
may be regarded as the secondary as above stated. Likewise phy- 
comycetes or slime fungi, at least plasmodiophorales may be so, since 
these are especially distinct in their parasitic nature. The groups of 
fungi proper, including phycomycetes, ascomycetes and bastdiomycezes, 
that constitute the overwhelming large phyla of the plant kingdom 
are all parasitic to more or less extent. They may possibly be the 
organisms having evolved chiefly from various plant viruses. 

It should be noted that most of the fungi, though prefering moist 
locations, do not thrive if submerged in water; but their parasitic 
nature is marked, indicating clearly that they are never the primary 
organisms generated and advanced in water. Among them the rust 
fungi, protobasidiomycetes, are striking examples of obligate parasite, 
that is, organisms unable to live except in living tissues. They are 
incapable of living in culture media, even when the media are made 
from the host plant upon which they thrive. 

Almost all the fungi are thus parasitic, but algae are free-living 
although many of them are extremely primitive both in form and func- 
tion. For example, the cyanophyta, or blue-green algae, are so primitive 
that there is no morphological separation of nucleus and cytoplasm, 
chromatin being distributed throughout the cell. Non-parasitic nature, 
however, would be no gainsaying their possibility of being secondary 
organisms. It may be highly possible that the secondary organisms 
having been generated from higher plants, which can utilize the en- 
ergy of sunlight by virtue of the catalytic properties of chlorophyll, 
may also be able to do so in their extremely primitive state because 
they may inherit the faculty from the plant. 

The fact that phyla or classes in the plant kingdom are extraor- 
dinarily numerous and complicated, no phylogenetical correlation 
appearing to be present among them, suggests that at least the ma- 
jority of them may be the secondary organisms. It may rather be 


X. THE SECONDARY ORGANISMS 205 


possible to suppose that all the plants may be the secondary. The 
thallophyta, to which algae and fungi belong, include plants of great 
diversity, but many of which have little in common save the small 
size and simplicity. 

On the other hand, it is a noteworthy fact that there is entirely 
no parasitic species in chordates, the most advanced animals, among 
the classes of which the most orderly phylogenetic relation can be 
found. This suggests that chordates are the primary organisms which 
might provide the first scaffolding to the secondary organisms for 
their evolution as well as for their generation. Moreover, it should 
be noted that no species of parasitic echinoderms is known, although 
certain kinds of echinoderms provide the host to parasitic snail, but 
they themselves are never parasitic, whereas it is generally accepted 
that there seems a closely related phylogenetical connection between 
echinoderms and chordates. This fact also suggsts that echinoderms 
as well as chordates are the primary organisms. 

Now we must consider in this connection of mollusks, one of the 
most highly organized phyla of animals. It is remarkable that differ- 
ent groups of mollusks are parasitic. In unionidae parasitism is re- 
sorted to by larval forms only, known as glochidia, parasitic on fishes, 
whereas in parasitic snails the adults are parasitic and the larval 
forms free-living, the hosts being always provided by echinoderms 
(117). In contrast to arthropods, however; the parasitic nature of 
mollusks is rather exceptional, only being observed with some 
peculiar kinds. Nevertheless, since parasitism does exist in mollusks, 
if as relics, it may be not unreasonable to regard mollusks as the 
biggest senior of the secondary animals that were evolved from 
viruses in the oldest age. 

Of course, if a secondary organism was developed to a certain 
extent, viruses would naturally be generated in it, and the viruses in 
turn would be evolved into organisms in this senior secondary orga- 
nism, and hence it cannot be said that the secondary organisms are 
always generated and evolved in the primary organisms. 

The writer thus has come to possess the strong view that viruses 
represent a transitional stage from non-living to living whereas many 
authors consider that viruses have evolved by a process of retrograde 
evolution from higher organisms, that is, present-day viruses are 
direct descendants of peculiar forms that were once free-living. This 
idea appears to be based upon the fact that saprophytic organisms 
sometimes lose the ability to synthesize certain essential growth re- 
quirements, involving the loss of structure and functions that are no 
longer needed, thus being degenerated both in their function and forms 
to become apparently very primitive organisms. They believe that in 


206 Ill. THE EVOLUTION OF VIRUSES 


a similar way viruses have evolved from higher organisms as a 
result of degeneration due to the parasitism. It seems, however, that 
this idea might be based on a grave misunderstanding as regards 
parasitism and the basic principles of life phenomena as well. 

It is true that parasites seem to be often very highly modified in 
their structure to meet the demands of their particular enviroment. 
Fixed parasites generally have neither sense organs nor well-developed 
locomotive functions, and intestinal parasites do not need highly. 
organized digestive tracts. However, parasites must be specialized, 
often to a very high degree, to adhere to or to make their way in 
their particular host, or the particular part of the host in which 
they find suitable conditions for their existence. Still more remar- 
kable are the specialization of parasites in their reproduction and 
life history to insure, as far as possible, a safe transfer to new hosts 
for succeeding generations. .Every structure, every function, every 
instinct of many of these parasites appears to be modified, to a certain 
extent, for the sole purpose of reproduction. 

The apparent degenerancy associated with parasitism is, there- 
fore, nothing but the highly developed specialization wonderfully 
adapted to the peculiar, extremely limited, environment. As above 
cited, viruses are distinct from typical organisms in their lack of 
self- or race-preservation instinct, whereas in ordinary parasites this 
instinct is found to be most manifest as just stated. 


4. Inclusion Bodies and Metamorphosis 


Many virus infections are characterized by the presence of peculiar — 
bodies, usually referred to as inclusion bodies, in the nucleus or cyto- 
plasm of parasitized cells. The bodies are rounded, oval, or irregular 
in shape. It has proved possible to photograph with the electron mic- 
roscope inclusion bodies such as those of mouse ectromelia and human 
variola, and they are seen to consist of aggregations of virus particles 
clumped together. Thus it is difficult to regard inclusion bodies as 
other than intracellular aggregations of virus particles. 

It has already been stated that plant-protoplasm particies are 
liable to aggregate into larger bodies sometimes resembling protozoa. 
This may be regarded as the production of inclusion bodies 7” vitro. 
The inclusion bodies may be produced in the host cells when virus 
structure is enveloped by the normal structure of the protoplasm; 
since both structures can not melt into each other because of their 
different structures, the virus structure may have to exist in forming 
a mass distinctly separated from the host protoplasm. 


X. THE SECONDARY ORGANISMS 207 


If a virus structure was readily assimilized and destroyed by the 
normal structure when existed in separate particles, whereas in a 
large aggregated state it was stable and could resist the assimilase 
action of the protoplasm, then the large mass only would remain as 
the virus. In view of the virus nature already discussed such an 
occurrence seems highly probable. 

The shape of inclusion bodies are said occasionally to tend to be 
peculiar to the kind of viruses and of the host cells. Since the bodies 
formed by the aggregation of virus particles may be regarded as a 
kind of liquid crystals like the protoplasm, the shape may naturally 
be exerted some influences by the kind of protoplasm as well as the 
sort of the virus. The shape accordingly may change with the host cell 
as well as with the virus. It should be remembered that the shape of 
crystals is, as a rule, determined by the solvent or the environment, 
in which they are formed, as well as by the component substance 
of the crystal. 

If some viruses or some microorganisms could exist only in a form 
like that of inclusion bodies, they would vary their shape with the host 
or with the surrounding condition. This may be the reason why para- 
sitic microorganism, existing in the form of virus-aggregate, alter 
their shape whenever they change the host. As already stated, the 
protoplasm can be regarded as a virus-aggregate or a mass composed 
of elementary bodies. The mechanism of metamorphosis may thus 
be developed. 

The earlier workers regarded inclusion bodies of some viruses as 
protozoa, and pictured them as varying stages of an elaborate life-cycle. 
From the above point of view, it may be said that this supposition is, 
in a sense, correct. In fact, at present, the majority of workers con- 
sidered that highly advanced viruses such as those of psittacosis and 
lymphogranuloma undergo a developmental life cycle, in which virus 
particles or elementary bodies enter a cell, and then increase in size 
to become initial bodies; a larger form of inclusion known as a plaque 
or morula is then formed from the initial bodies, and comes to con- 
tain large numbers of elementary bodies, which eventually rupture 
from the cell (118). This phenomenon can be interpreted as ‘“‘crystal- 
lization’’ of virus particles or elementary bodies in the host proto- 
plasm into larger bodies, which are decomposed when the environ- 
mental condition is changed. 

It follows from the theory of the writer as regards the nature of 
protoplasm and virus that inclusion bodies should be produced by 
proper chemical or physical agents other than viruses, if structural 
changes similar to those induced by virus are given rise to in the 
protoplasm. In fact, it is known that a part of protoplasm having 


208 III. THE EVOLUTION OF VIRUSES 


undergone a change by some injurious agent is separated from the 
normal protoplasm in forming an inclusion-like body which sometimes 
is expelled from the cell. 

Yamafuji ef al. (119) by applying peroxides have succeeded in 
producing in silk-worms inclusion bodies similar to polyhedral bodies 
peculiar to polyhedrosis. The generally confirmed mutagenic activity 
of peroxides in microorganisms shows that protoplasm is liable to be 
changed in its structure by these chemical agents. Of course, the 
formation of an inclusion body must be entirely different from the 
production of a virus. 


CHAPTER XI 
THE SUMMARY OF PART III 


Organisms may sometimes be changed in their properties when 
acted upon by proper chemical or physical influences. The change 
is termed mutation or variation and is probably caused by an altera- 
tion of the protoplasm structure including genes. If an altered 
structure thus produced is strong and retained in the protoplasm par- 
ticles, the particles may be able to behave as a virus towards some 
other cells having the protoplasm of a weaker structure. Special 
case of such change involves adaptation, and the production of virus- 
like agents in the adaptive change of microorganisms is occasionally 
reported. 

Even protoplasm particles of normal structure, provided the struc- 
ture is preserved in the particles, may act as a virus upon certain 
cells having weaker protoplasm. Phage produced by some lysogenic 
strains may be regarded as such normal particles. Similarly, phage 
present commonly in chicken feces may be normal protoplasm particles 
of chick cells. Many other examples can be cited showing that normal 
protoplasm particles can behave as a virus. 

Cells affected by a virus may acquire the faculty to produce the 
virus, even if they have not fallen into a pathological state, as a 
result of mutation or variation due to the virus. Since the protoplasm 
structure of a cell is to be changed into virus structure when affected 
by a virus, the cells affected by a virus naturally produce the virus. 


2 


If a new protoplasm structure, exhibiting assimilase action stronger 
than that of the original structure, is produced in a cell or cells of 
some tissues or organs the newly formed structure will spread into 
the surrounding cells by assimilizing the protoplasm. Allergic der- 
matitis or various tumours including cancer may occur in such a 
way. The general failure of virus detection in these cases is presum- 
ably due to the lability of the structure unable to be retained in 


210 III. THE EVOLUTION: OF VIRUSES 


the protoplasm particles. 

If the protoplasm structure in question is preserved in decom- 
posed particles of protoplasm, the structure will be transmitted to 
other cells or organs through such particles, and if the cells or the 
organs fall into a pathological state on the transmission of the struc- 
ture, the latter particles will be regarded as a virus, and the patho- 
logical state will be called an infectious disease. Tumour diseases 
are commonly not infectious, as the structures cannot, as a rule, 
remain in the protoplasm particles. Although viruses are occasionally 
demonstrated in certain tumours, it cannot be said that the structures 
are Stable enough to make tumours regarded as infectious diseases 
always caused by a virus. 


3 


The structure of virus can be altered by various causes. If a 
virus retains the faculty to act as such after a change is induced in 
its structure, the changed structure can multiply, so that a virus 
strain having the changed structure may be produced, because the 
only function of viruses is to confer their own structure to the proto- 
plasm of cells which they affect, thus the altered structure being 
inherited to ‘‘progeny.’’ Since the property of viruses is determined 
by their structure, the alteration in the structure is to be accompanied 
by the change in the property, and therefore the changed property is 
to be inherited with the altered structure. In such a case the virus 
is said to have undergone a mutation or variation. 

Viruses may easily be generated and each particle of them may 
have individuality even immediately after their generation. In addi- 
tion, they can variate in various possible directions. Among the par- 
ticles with various properties thus having arisen, some ones with 
suitable property for the continuity of their existence may continue 
to exist in escaping the extinction. However, the probability of inci- 
dentally acquiring the property to become a virus able to continue to 
‘exist for prolonged periods is apparently extremely small, presumably 
far smaller than that of spermatozoa of higher organisms to meet an 
egg-cell to develop into a full individual. This may be attributed to 
the lack of the property in the newly generated viruses compatible 
with the self- or race-preservating instinct of higher organisms. 

In order to escape the extinction, high stability of the structure 
is essential for the virus. If the structure is too unstable to be pre- 
served in the protoplasm particles, virus may scarcely be found as in 
the case of eczema. 


XI. THE SUMMARY OF PART III AU 


Even when the structure can be retained in the particles, it will 
soon be destroyed if it is labile, whereas stable and firm structure 
may continue to exist, imparting the stable pattern to the host proto- 
plasm, thus the virus with the strong and firm structure can multiply 
far from becoming extinct. The more stable and the stronger is the 
structure of a virus, the longer periods it will exist and the more 
abundantly it will multiply. 


4 


Viruses may affect certain cells because they are stronger in the 
assimilase action than the protoplasm of the cells; in other words, 
in order to multiply in the prototoplasm viruses must beat the proto- 
plasm in the competition for assimilase action. The protoplasm on its 
part, however, may be making a continual effort to assimilize the 
virus. Accordingly, if a virus sticks to the same kind of cells for 
prolonged periods, it will gradually be assimilized until at last becomes 
identical with protoplasm, resulting in the complete disappearance of 
its action. Viruses with unstable structures will be soon assimilized 
and cancelled, whereas the ones with strong structures may resist 
the action of the protoplasm to continue their existence. 

Organisms once affected by a virus may acquire the resistance 
to the virus, and become immune to it. Since the protoplasm struc- 
ture of the organisms is to become identical in its structural pattern 
with the virus following the infection, it may only be a natural 
result that the organisms will become indifferent to the virus which 
shares the structural pattern in common with the organism. Such an 
immunity should last as long as the virus structure remains in the or- 
ganisms. Strong viruses can remain long periods, so that immunity 
induced by a strong virus is of a long duration, whereas weak viruses 
may cause only transient immunities. 

In many virus diseases, such as measles, mumps, and yellow fever, 
the immunity persists for prolonged periods, usually for life, while 
in some other diseases, such as poliomyelitis and influenza, it is 
only transient. It may be supposed therefore that the viruses of the 
former diseases are highly advanced and are endowed with strong 
structures, whereas the viruses of the latter diseases are probably 
newly developed and unstable. 


212 III. THE EVOLUTION OF VIRUSES 


5 


The structural change of protein is liable to be reversible. This 
reversibility is especially distinct in the protoplasm. Thus distorted 
protoplasm can spring back like a spring to its original state when 
an agent causing its distortion is removed; that is, the protoplasm, 
in remembering its former structure, is inclined to recover the struc- 
ture it once possessed. 

The recovery from virus diseases may chiefly be achieved owing 
to this reversibility of protoplasm. The reversibility tends to persist 
even when the protein is freed from the protoplasm, resulting in the 
fact that protein denaturation is generally reversible to some extent. 
Thus inactivated viruses may sometimes be reactivated when the 
inactivating agents are removed. A virus once subjected to a varia- 
tion will tend to recover its original property if released from the 
causative agent leading to the variation. 

The failure of virus demonstration in some inflammatory diseases, 
such as allergic dermatitis, may be attributed to a high reversibility® 
of the distortion. On the other hand, the distortion induced by strong | 
viruses may be so intensive that the recovery of the original structure 
is rendered impossible. Since such a distortion means a structural 
change of the protoplasm, a distortion induced by a virus must involve 
the structural patttern of the virus itself, and accordingly the irrever- 
sible distortion is nothing but the multiplication of the strong irre- 
versible virus. 


6 


However strong and firm the structure of a virus may be, it will 
gradually be assimilized and annihilated by the assimilase action of 
the host protoplasm if the virus continues to multiply in the same 
kind of host, thereby the virus structure becomes identical with the 
protoplasm structure. In order to escape this fate virus has to change 
the host. 

Host change enables a virus, which has already assimilized to 
some extent by the host protoplasm, to come into contact with the 
protoplasm of a new host having a structure different from the former 
host.. Through this contact the virus will recover the original, non- 
assimilized structure, and at the same time structure of the new host 
will be introduced into the virus; thus the rejuvenescence of the 
virus is established. 


XI. THE SUMMARY OF PART III 213 


A virus can affect different individuals if these belong to a simi- 
lar kind of organisms, since similar organisms share some protoplasm 
structure in common, through which the virus can affect them. A 
virus affecting customarily a certain animal, however, is occasionally 
able to infect a blood-sucking arthropod, parastitic on the animal, and 
multiply in it. This is probably due to the existence of common 
structure between the animal and the arthropod because it is con- 
sidered that the relationship analogous to that between viruses and 
their hosts or between enzymes and their substrates is present be- 
tween the host animal and the arthropods. This appears likewise to 
hold true between plants and their parasitic insects. Viruses make 
use of such arthropods for their host change not only to be saved 
from the extinction due to the assimilase action of the host but also 
to be rejuvenated with the enhancement in their activity. 

Viruses having insect vectors are, as a rule, not mechanically 
transmitted by insects, but rejuvenated in them. To evade extinction 
newly generated viruses presumably have to acquire such means of 
rejuvenescence; those incapable of succeeding in the acquisition of 
such means would soon become extinct. 


7 


Whereas numerous viruses are transmitted by insect vectors, 
some viruses, which are never thought to be newly generated appear 
to possess no insect vectors. Such viruses are supposed to be capable 
of establishing rejuvenescence without insect vectors, presumably by 
making use of the sexual reproduction of the host. 

If a virus has evolved highly by acquiring a strong structural 
pattern, it may become able to impart to the host protoplasm life- 
long lasting distortion, giving rise to the immunity lasting as long. 
It is considered that the distortion caused by an extremely strong 
virus persists in the host for life but also may be transmitted to the 
progeny through the germ cells of the host. Many evidences are 
actually known indicating that some viruses are transmitted to offspring 
through eggs or seeds. Certain plant viruses can also confer a pro- 
found distortion of this kind on their insect vectors; as a result the 
viruses can be demonstrated in the offspring of the vectors. 

The rejuvenescence of organisms is generally accomplished by 
sexual reproduction, in which a germ cell is rejuvenated by fertiliza- 
tion through the contact with a germ cell of the other sex, just as 
a virus is rejuvenated by a host change through the contact with 
protoplasm of a new host. If the structural pattern of a virus is in- 


214 III. THE EVOLUTION OF VIRUSES 


volved in a germ cell, the pattern will be rejuvenated together with 
germ cell by the fertilization. The viruses, such as measles and 
mumps, can probably be rejuvenated in this way without insect vec- 
tors. Structural pattern of virus present in the germ cell, if ever 
present, possibly exists in an altered state, so that it may fail to act 
as the virus itself, but when the germ cell develops into an adult 
form after the fertilization, the pattern may also be developed into 
its original form, thereby becoming capable of acting as the virus; 
thus the virus may appear in the progeny only when germ cells have 
developed to a certain extent. As will fully be discussed in Part V, 
such is not a phenomenon restricted to virus pattern but may occur 
generally in various, normal patterns which may develop to produce 
adult forms. 

Virus demonstration is commonly impossible in larvae hatched 
from the eggs deposited by an infected insect, but as they develop 
into adult forms the demonstration may become possible. In like 
manner, measles virus may not be demonstrated in a newly born 
baby, but the full pattern of the virus will develop as the baby grows, 
and thus the baby or the child will suffer from measles by the virus 
developed in its body. 

The structural pattern of a virus will naturally be spread to the 
germ cells, when the structure of the virus becomes strong, and 
thereby the virus can acquire by itself the means of rejuvenescence. 
This must be the general course of the virus evolution. Thus, the 
virus possessing insect vector can be rejuvenated by both the host 
change and sexual reproduction of the host. Such an intimate corre- 
lation of a virus with the host results in the excretion of the virus 
from apparently normal organisms. In such a case the virus may be 
called ‘“‘latent,’’ but to distinguish latent viruses from newly generated 
ones may be difficult. 


8 


The increase in the strength of virus structure answers not only 
the purpose of self-preservation of the virus, but also that of race- 
preservation as just pointed out. Therefore, as the virus structure 
becomes the stronger the virus may become the fitter to continue to 
exist, and thus the property of self- or race-preservation will become 
more marked until it can be called instinct. 

Both the strength of the virus action and the structural stability 
of the virus seem to be directly proportional to some extent to the 
polymerization degree of protein molecules, 7. é@., the size of virus par- 


XI. THE SUMMARY OF PART III 215 


ticles, so that the increase in the strength of virus structure will 
follow the increase in the particle size. Since virus particles as well 
as the protoplasm, the decomposed particles of which are virus parti- 
cles, are presumably a kind of liquid crystals, the “‘crystal form’’ will 
be determined by the property of the protein component, which, in 
turn, will govern the structural pattern of the virus or of the proto- 
plasm, and therefore the pattern of a virus producing large “‘crystal’’ 
will be transmitted to the host protoplasm; as a result a large sized 
virus will reproduce a similarly large sized virus. 

For the continuity of the existence various properties similar to 
those of organisms, including size and shape, are essential for viruses. 
Therefore viruses mostly resembling organisms will become fitter and 
develop up to the stage of Rickettsiae until they come to get possession 
of the faculty to assimilize even proteins present in blood which can 
be regarded as a streaming protoplasm. Thus the faculty of multipli- 
cation without living cells will be obtained together with the stage 
of undoubted organisms. 


9 


When some protoplasm particles affect certain cells which are 
weaker than the particles in their assimilase action, and can make 
cells fall into pathological state, then the particles will be called 
viruses. However, particulate assimilases may not always exhibit the 
pathogenic action. 

The presence of virus is acknowledged only by the pathogenicity ; 
therefore the demonstration of the non-pathogenic virus must be most 
difficult, if not impossible. Latent viruses are usually non-pathogenic, 
but when environmental conditions are altered the pathogenic property 
may sometimes be revealed to prove their existence. 

When a virus is developed up to the stage of Rickettsiae, its ex- 
istence will be readily recognized, in contrast to undeveloped viruses, 
by its peculiar form even without pathogenicity. Thus, nonpathogenic 
groups of Rickettsiae known are much more numerous than the patho- 
genic ones. 

For the evolution of particulate assimilase into organisms, any 
intensive pathogenicity may not be needed. On the contrary, in most 
cases it may prevent the evolution, because virus may be unable to 
find any favourable condition for their multiplication in the severely 
injured host, and, therefore, less pathogenic viruses may become fitter. 
From this point of view, non-pathogenic viruses existing are expected 
to be much more numerous than pathogenic ones as in Rickettsiae, 


216 III. THE EVOLUTION OF VIRUSES 


although the existence of non-pathogenic viruses cannot be demon- 
strated so readily as in Rickettsiae. Moreover, the existence of viruses 
exerting favourable effects upon the host is conceivable, that is, the 
symbiosis between viruses and their hosts may be possible. 


10 


The writer designates the organisms having evolved from viruses, 
pathogenic or non-pathogenic, as the secondary organisms. The orga- 
nisms having served as the first scaffolding for the secondary organisms 
are naturally to be called the primary organisms. 

Whereas the primary organisms were generated and evolved in 
water, the secondary organisms depended on the protoplasm of senior 
organisms both for their generation and for evolution. Therefore, 
for the secondaty organisms, the liberation from the dependence upon 
the senior organisms might be a matter as difficult as the release of 
the primary organisms from the water. Accordingly parasitic, pri- 
mitive organisms living outside the water should be regarded as the 
secondary organisms. From this point of view, at least the majority 
of bacteria, protozoa, and fungi, and also presumably more advanced 
parasitic animals such as platyhelminthes and nematodes may be 
secondary organisms. Furthermore, arthropods are considered to be 
likewise so, most of which have already succeeded in liberating from 
the parasitism. 

If arthropods are the secondary organisms, it should naturally 
follow that the relationship between insects and plants or animals 
on which they are parasitic is analogous to that between viruses and 
their hosts; and this latter relationship may, in turn, be regarded as 
similar to that between enzymes and their substrates. 

Some adult arthropods enter into parasitism after free-living larval 
stages, a fact which is to be interpreted as an atavism. It is consi- 
dered to be impossible that free-living organisms can incidentally 
enter into parasitism, a most specialized, and accordingly extremely 
limited environment. Therefore, it may be proper to regard the orga- 
nisms as the secondary ones if they reveal parasitic nature at any 
stage in their life. In this respect, mollusks may be supposed to be 
the secondary animals generated in the oldest age. _It should be noted 
that there is no parasitic groups in chordates and also in echinoder- 
mates which the latter are generally believed to be intimately related 
phylogenetically to the former, suggesting that they are the primary 
organisms in the animal kingdom. 

It is a remarkable fact that various secondary organisms are still 


XI. THE SUMMARY OF PART III 217 


clinging to the habit of cumbrous host change which was indispensable 
for the rejuvenescence at their viral stage but which has lost its 
essential significance now that the organisms have acquired the ability 
to perform the sexual reproduction, the better method for the rejuve- 
nation. This shows how difficult is for the organisms the abandon- 
ment of their long abiding habitat. 


218 Ill THE EVOLUTION OF VIRUSES 


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S- 
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Ne eas CUED ES ES) 


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PART IV 


THE GENERATION OF 
THE PRIMARY ORGANISMS 
AND THE FUNDAMENTAL PRINCIPLES 
OF LIFE PHENOMENA 


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CHAPTER I 


THE MATRIX’ FOR THE GENERATION OF 
THE PRIMARY ORGANISMS 


1. The Sedimentation of Globulin and Lipids in 
Primeval Oceans 


As discussed in the previous Part, for the generation of the 
secondary organisms the living substances already produced are indis- 
pensable. It may be said, therefore, that the discussion on the 
generation of the secondary organisms has nothing to do with the 
problem as to the true origin of life, but as will be fully discussed 
later it should be considered that the primary organisms were gene- 
rated principally in the same way as the secondary organisms. 

The writer has advanced so far the argument into the problem of 
the possible manner in which organisms were newly generated in the 
living protoplasm. In like manner the primary life might have been 
created on the primeval, lifeless earth, if there were present any 
matrix acting as the protoplasm. On the other hand, there are actually 
good many reasons to consider that such a matrix was present on the 
lifeless earth. 

As already stated in Part II, Chapter IV, protoplasm particles 
isolated from leaves of certain plants formed protoplasm-like masses 
when left in a weakly acid solution at the laboratory temperature. 
Usually the mass appeared homogeneous and tended to take a shape 
peculiar to the plant from which the particls were prepared. In most 
cases these particles fused by hundreds into a mass of a bacterial 
size, some forming a bacillar form and other a coccoid, and sometimes 
the mass grew further until it became a protozoa-like body. 

The writer has succeeded by using protein particles isolated from 
castor beans in preparing beautiful protozoa-like bodies in which 
usually various granules were contained, some of which occasionally 
appeared to be nucleus. Such a protozoa-like body would coagulate 
or disintegrate into minute particles as does the ordinary protoplasm 
when pressed mechanically under cover-glass or added with chemical 
agents such as alkali. 

This fact suggests that protoplasm particles produced by the 
coagulation of protoplasm.can solve the coagulation when stand in 


224 IV. THE PRINCIPLES OF LIFE PHENOMENA 


an aggregated state at the isoelectric point, thereby presumably folded 
polar groups of the protein are set free enabling the particles to com- 
bine intimately with one another. Thus a homogeneous mass is formed 
in which protein molecules are arranged regularly just as in the pro- 
toplasm proper. At the isoelectric point both NHz and COOH of the 
protein molecules, constituting the surface of the particle, may become 
free enabling the particles to fuse and to arrange orderly as in the 
protoplasm. Accordingly, this may be regarded:ias the reversion of 
protoplasm 7” vitro. 

In the primeval oceans various organic substances might be pre- 
sent abundantly as there were no microorganisms which would devour 
them up, and if proteins having the globulin-like property were syn- 
thesized by certain means which we shall have an océasion to discuss 
later, the protein would be precipitated since the water had possibly a 
weakly acid pH, the isoelectric point of the protein, on dissolving car- 
bon dioxide which is considered to have been present in abundance in 
the air of the ancient lifeless world. Globulin in ‘its nature may 
preferably involve lipids in its precipitate, and therefore, if lipids were 
present together with the protein, the globulin-like protein in the 
ancient ocean would precipitate with the lipids forming minute coagu- 
lated particles; as will be mentioned later, globulin molecules possess 
the character to form virus-like particles in combining with lipids at 
the isoelectric point. 

Particles composed of the globulin-like protein and lipids would 
be thus formed and precipitated on the bottom of the ocean, where 
when they were standing in forming aggregates, the protein molecules 
would be unfolded to be fused into homogeneous masses, and thus 
protoplasm-like masses just referred to above would be produced. 

If the globulin-like protein and lipids were synthesized successi- 
vely in the primitive ocean, they might continuously be sedimented 
in such a way in forming protoplasm-like masses, which might be 
accumulated on the bottom, possibly presenting a picture as if the 
bottom were composed of protoplasm-like masses. The writer believes 
that the matrix for the generation of the primary organisms was 
thus established. All the organisms existing at present are, without 
exception, provided with the protoplasm composed of globulin-like 
proteins and lipids, suggesting that the primeval organisms were also 
created from such chemical components. in addition many evidences 
can be presented to show that life phenomena are revealed solely on 
the stage composed of such materials. 

If only a part of carbon which constitutes all the organisms exis- 
ting today was present in forming carbon dioxide, it might be suffi- 
cient to reduce the pH of the primitive sea water to about 5.5 at which 


I. MATRIX FOR THE GENERATION OF THE PRIMARY ORGANISMS = 225 


globulin was most readily sedimented. 

For the isolation of protoplasm particles, including viruses, by 
means of the isoelectric point precipitation method we used acetic acid, 
whereas carbon dioxide has been generally used by many workers for 
the isolation of substances related to globulin such as serum-euglobulin 
and the stroma of red-blood cells. The fact that both serum-euglobu- 
lin and stroma are precipitated at pH about 5.5 in the form of virus- 
like particles and that both can be regarded as a kind of protoplasm 
particles was already stated. The application of carbon dioxide is 
very convenient to adjust a globulin solution to its isoelectric point 
for the precipitation of the globulin. It may .be a relic of this pre- 
cipitating property of the primeval protoplasm in the ancient oceans 
that still now the substances related to protoplasm proteins are readily 
precipitable by carbon dioxide. 


2. The Properties of Artificial Cells 


As above stated, the writer has been able to prepare protoplasm- 
like masses from certain plant materials; especially from castor beans 
most, demonstable, beautiful masses were obtained. The preparing 
method was as follows: 

The emulsion of castor beans ground up ina mortar is centrifuged 
to eliminate precipitable coarse materials together with lipids, which 
are raised to the surface. The pH of the emulsion from which the 
lipids and the coarse materials having thus been eliminated, is adju- 
sted to 5.5 with acetic acid, whereby a precipitate is formed, which is 
subsequently separated by centrifugation, and washed with distilled 
water for several times, each time being separated from the water 
by centrifugation. The substance thus obtained proves to be composed 
of virus-like particles, which will fuse into protoplasm-like masses if 
stand in a solution of the weakly acid pH for several days at the 
laboratory temperature. It appears that peculiar conditions are neces- 
sary for the production of such masses, for frequently the experiment 
would fail. Presumably temperature at least is an important factor, 
and a good result was commonly obtained if the solution was lelt at 
a temperature near 10°C. 

At any rate, the masses may be called artificial cells or proto- 
plasm, the properties of which are described in the following. 

As shown in Fig. 22 the masses are globular with sizes of 10 or 
more in diameter. Small sized particles similar to red-blood cells are 
sometimes abundantly produced, and if their size is thus small they 
are quite homogeneous, but in larger sized particles various granules 


226 IV. THE PRINCIPLES OF LIFE PHENOMENA 


are seen, some of which occasionally are of considerable sizes and 
have an appearance of nucleus. Consequently they strikingly resemble 
in appearance some primitive single-celled organisms. They are also 


t——1 l0z 


Fig. 22. Protozoa-like masses prepared from castor beans. 


similar to the organisms in their chemical composition, and composed 
of globulin-like proteins and lipids, which the latter are contained in 
an amount about 1/4 of the former. 

Proper chemical or physical effects administered to the mass or 
the artificial cell cause to produce in the body minute particles which 
will again disappear gradually if the effects are removed. Thus, the 
reversible coagulation can take place as in the usual protoplasm, 
although the reversibility is very tedious. This fact indicates that 
the artificial cell can exhibit an irritability which is generally believed 
to be an essential character of living. If the stimuli are too severe 
the total body will be coagulated and then disintegrated into minute 
particles just as in ordinary cells. 

If minute particles prepared from castor beans are given to the 
artificial cells, the former will be gradually fused into the latter, that 
is to say, the artificial cells eat the particles to grow, and when they 
grow to a certain extent, they will undergo fission into smaller bodies. 


I. MATRIX FOR THE GENERATION OF THE PRIMARY ORGANISMS = 227 


Thus, they can accomplish both growth and multiplication, though 
the fission appears not to occur spontaneously, proper environmental 
changes, such as those of temperatyre, or some mechanical effects 
appearing to be necessary for it. 

From what has been described above, it will be evident that the 
mass is capable of exhibiting all the properties believed to be essential 
for living organisms, and therefore it may be said that a living orga- 
nism is produced 7” vitro. 

It should be a remarkable fact that such artificial cells can also 
be prepared from Merck’s ricin preparation, in which no virus-like 
particles are contained. Therefore, it seems improper to regard the 
formation of the artificial cell as merely an example of reversible 
decomposition of protoplasm. The Merck’s ricin preparation with 
which the writer carried out the experiment was composed of water 
soluble protein containing entirely no virus-like particles, but when 
its water solution was added with ethanol of 1/3 of its volume and 
left in an ice box for several days after the pH had been adjusted to 
5.5 by adding acetic acid, the protein would precipitate together with 
lipids contained in the preparation in forming virus-like particles. 
These particles after isolated by centrifugation were suspended in 
water of pH 5.5 and left at the laboratory temperature, thereby the 
particles were fused into protoplasm-like masses. 

This fact indicates that the globulin-like protein exhibits the pro- 
perty to polymerize with lipids to form virus-like particles, which 
in turn can fuse into larger protoplasm-like masses. As stated in 
Part II, elementary bodies of protoplasm are considered to be a parallel 
array of thread-like protein molecules among which lipids are inserted, 
and the protoplasm is assumed to be composed of such elementary 
bodies associating mutually also in parallel alignment. The virus-like 
particles prepared from ricin are comparable to the elementary bodies ; 
the particles can combine with one another in a regular alignment to 
form larger masses which may in turn be comparable to protoplasm. 
The association among the particles in the mass, however, is not 
sufficiently strong to make the mass remain always in the same homo- 
geneous state, so that upon the addition of a certain stimulus, the mass, 
like the protoplasm, will be decomposed into the particles, stable poly- 
merization units of globulin-like proteins. 

It should be considered, therefore, that globulin-like proteins possess 
in themselves the property to form protoplasm-like masses. Namely, 
globulin-like proteins possess the property to polymerize to virus-like 
particles by arranging themselves in parallel alignment in their stre- 
‘tched polypeptide chains in inserting lipids among them; these particles 
tend to combine further with each other in parallel arrangement to 


228 IV. THE PRINCIPLES OF LIFE PHENOMENA 


form larger masses. If proteins synthesized in the primeval oceans 
had a similar property, the formation of protoplasm-like masses would 
be only a natural result. 

The property of proteins of globulin nature to form virus-like 
particles is also shown in an experimental result obtained by Gross 
(1), who has found that virus-like threads or filaments sedimentable 
by ultracentrifugation are produced in water solutions of crystalline 
trypsinogen added with a few crystals of trypsin preparation after 
incubation at 5°C. for 3to 7 days. Takahashi and Ishii (2) have isolated 
from plant tissues infected with tobacco mosaic virus a protein which 
formed at a weakly acid pH high molecular weight aggregates readily 
sedimentable. in the ultracentrifuge. This plymerization was rever- 
sible. 

In short, it seems little doubt that proteins with globulin nature 
are able in a weakly acid solution, probably under an assistance of 
lipids, to form protoplasm-like masses. The primary organisms were 
most probably generated and evolved in the matrix composed of such 
masses, which on the other hand, may be regarded as the most primi- 
tive primary organisms. Oparin (3) stated that the primitive life might 
be originated from coacervates, while it is possible to regard the pro- 
toplasm-like mass above mentioned as a kind of coacervates composed 
of protein and lipids. 


3. The Evolution of the Primitive Organisms 


The Protoplasm-like masses produced in the primitive ocean might 
be similar both in their appearance and chemical composition to the 
protoplasm of the organisms existing at present. It is highly pro- 
bable, however, that the component proteins and lipids of the primeval 
protoplasm, being not fitted for exhibiting the complicated life pheno- 
mena, were never similar to those of present organisms, and hence the 
ability to change the structure in accordance with the environmental 
effects might be insignificant as compared with the present organisms, 
and moreover if they could exhibit the property to rearrange the 
structure of weaker masses, 7. é@., the property to act as assimilase, it 
would also be insignificant. 

Nevertheless, however trivial these properties might be, the 
masses might change their structure when certain stimuli were given, 
and the change thus raised would spread in the masses. Since the 
change produced in the protein structure may generally be reversible, 
the changed structure in the masses might also recover their original 
structure on the removal! of the stimulus, but when some irreversible 


1. MATRIX FOR THE GENERATION OF THE PRIMARY ORGANISMS 229 


or difficult-reversible change was brought about and the changed 
structure was able to exert a physicochemical effect stronger than the 
original one, then the new structure would ‘‘assimilize’’ the sur- 
rounding masses. By the repetition of such a production of stronger 
structure the masses would become stronger and stronger in their 
structure, and more and more fitted to act as strong assimilase. 

In short, the evolution of the primary organism at the primeval 
stage might be quite similar to that of the secondary organisms or 
viruses. In the case of viruses, however, the matrix in which they 
have been evolved is the preexisted, perfect protoplasm, which itself 
is the strong assimilase and in which various differentiated mechanism, 
as for example, that of the spreading of changed structure, are already 
provided, so that the stronger structure acting as the viruses would 
easily be produced, whereas in the case of the primeval organisms, 
as the matrix itself was quite incomplete, the enhancement of the 
strength of the structural pattern would be much more difficult; 
accordingly far longer span of time would be required for their 
evolution than for the virus. 

As fully discussed in the previous Part, when a virus continues 
to multiply in the same kind of host cells, the virus structure is to 
be gradually assimilized by the host protoplasm until it becomes 
extinct. In order to avoid such a fate the virus must frequently 
change the host. This may account for the difficult situation in the 
continuance of their existence. The same might hold true for the 
primeval primary organisms. 

The state in which the protolasm-like masses accumulated on the 
bottom of the ocean of the primitive age, exerting mutually structural 
influences upon one another, might be similar to the state of the 
viruses multiplying always in the same kind of cells. Therefore, even 
strong structures which might appear and spread in the accumulated 
massses would likely be influenced in the long run by the weaker 
masses existing in the overwhelming majority until it became iden- 
tical to the weaker masses, or at least it became weaker in the 
structure. 

In order to avoid such a decrease in the structural strength, the 
mass which might acquire a strong structure would find it necessary 
to move to another accumulation of the masses of a different type 
corresponding to a different kind of host cells in the case of viruses. 
In the movement of masses, the water in motion might play an im- 
portant role. Thus, if the masses on the bottom of the ocean was 
carried near the shore, say, by a tidal current, they would always 
have good chance to contact with other masses of different structures 
by the constant motion of waves. If there was a structure in common 


230 IV. THE PRINCIPLES OF LIFE PHENOMENA 


between two masses thus brought into contact, they would combine 
with each other and the one with the stronger structure would over- 
come and assimilize the other. This must have been a struggle for 
existence, a struggle to eat or to be eaten. Thus the individuals 
with the stronger assimilase action could survive and prosper the 
more extensively. 

The strength of the structure might be determined chiefly by 
both the arrangement and type of polar groups in the protein which 
constituted the mass, but of the masses of the similar proteins, the 
nucleic acid content might play an important part as in the case of 
viruses. The masses therefore capable of getting possession of great 
amounts af nucleic acids or nucleic acid-like substances would become 
powerful in their assimilase action, and accordingly would become 
more fitted for the continued existence. 

In this problem, however, there seems to be involved a great 
dilemma. As stated in Part II, for the response to stimuli and for 
the spread of the structural change produced by the stimuli, lipids 
must be inserted among protein threads of the protoplasm, but in 
such a state the assimilase action of the protoplasm is extremely 
weak, and will be easily assimilized by other assimilases. On the 
other hand, if nucleic acids are contained in rich amount, the struc- 
ture will become rigid and exhibit a strong assimilase action, but in 
this state, as a natural result, structural changes essential for life 
phenomena will scarcely occur. Primeval organisms had to find their 
way out of this dilemma in order to continue their existence, and did 
actually find a splended way as discussed in the next chapter. 


CHAPTER II 
THE PATTERN OF PROTOPLASM 


1. Protoplasm as a Mixed Crystal 


A crystal is usually composed of similar molecules, occasionally 
different molecules in mutual connection can form a crystal, known 
as a mixed crystal. Molecules capable of forming a mixed crystal 
are different in their chemical composition, but similar in their arange- 
ment of polar groups and in their molecular sizes. 

The protoplasm can be regarded as a crystal, but since it is by 
no means composed of the same molecules, it must be a mixed crystal. 
If the protoplasm is a mixed crystal, the proteins constituting it 
must be immunolégically identical, if not uniform in chemical compo- 
sition, since the component proteins should be identical in their arran- 
ment of polar groups. 

It is generally recognized, however, that there exist several types 
of immunological different proteins, such as serum-albumin and serum- 
globulin, in the blood plasm which can be regarded as a pool of 
a liquid protoplasm. At first sight, this fact may appear to disprove 
the above concept. Nevertheless, the writer interprets this as being 
attributable to an artificial separation of protein fractions which 
are present in the plasm in a State of equilibrium exerting mutual 
influences to form a definite pattern specific to the plasm; the arti- 
ficial separation may destroy the equilibrium to make each fraction 
reveal its own pattern. 

Sérensen (4) has stated that proteins in biological fluids are never 
single chemical entities but systems of similar molecules in an equili- 
brium with each other and with other constituents of the solution, 
and that proteins isolated by the customary method are not identi- 
cal with those originally present. Kleczkowski (5) observed that when 
pairs of proteins are heated, they combine in the initial stages of 
denaturation to form complexes. If one protein is present in larger 
amount the complex will not precipitate with antiserum to the minor 
component. The failure to precipitate with antiserum to the minor 
component could be reversed by peptic digestion of the major compo- 
nent. Addition of aniserum to the protein present in larger amount 
precipitates the entire complex. This fact may indicate that the in- 


232 IV. THE PRINCIPLES OF LIFE PHENOMENA 


fluence of the pattern of protein present in larger amount is greater 
in the establishment of the equilibrium. It has already been stated 
that a weak virus cannot exert its structural influence when present 
in a small amount, but can do so if present abundantly. This is the 
“‘dosis effect’? recognizable in many other cases as detailed in Part II. 

Again, according to Zoet (6), the antiserum against a heated 
raixture of horse and pig serum can react markedly with this mixture, 
but scarcely with a mixture of the two sera heated separately. 
McFarlane (7) stated that one protein in a mixture even can effect the 
molecular size of another. _ 

It is well acknowledged that apparently similar serum-albumins 
from different animals exhibit markedly different antigenicities, 
whereas there is not so distinct immunological difference between two 
distinct proteins, 7. e., serum-globulin and serum-albumin, if they are 
separated from the same animal, showing that proteins isolated from 
one and the same biological system cannot reveal immunological pat- 
terns so different as do those derived from different systems, although 
each protein may be able to recover its own pattern when separated 
from the system. It should be remembered in this connection that 
only substances with structural patterns almost similar can make a 
mixed crystal. 

Various substances in protoplasm must be present in such a 
system of equilibrium. The main component element of the system 
may be the elementary bodies composed of globulin and lipids. These 
bodies may be fused into mixed crystal, exerting mutually structural 
influences to form a definite pattern specific to the protoplasm. When 
exist in the protoplasm they may be compelled to take this definite 
pattern, but if liberated from it, some bodies.may change the pattern 
according to their own peculiar structure, for all the bodies constitu- 
ting a cell cannot be identical in their chemical compositions. Bodies 
with high contents of nucleic acid may retain the pattern specific 
to the protoplasm, even when the particles with little or no nucleic 
acid undergo a change, since the former bodies cannot be altered in 
their structures on account of the presene of nucleic acid. Moreover, 
such bodies can act as strong assimilases for the same reason, and 
hence behave as viruses. 

The same may occur when the bodies are present in protoplasm. 
Thus, when a stimulus is given to cell, most elementary bodies in it 
would change the structure in accordance with the stimulus, but not 
the bodies with highcontents of nucleic acid, which, therefore, when 
the stimulus is removed, can bring back the other changed bodies 
to the original state by their strong assimilase action. The bodies 
thus capable of maintaing the original pattern of the protoplasm may 


II. THE PATTERN OF PROTOPLASM 233 


be called genes when exist in the cells of higher organisms. Genes, 
therefore, are probably elementary bodies governing the structural 
pattern of the protoplasm. 

If a primeval organism had a few number of such bodies, while 
it mostly being composed of bodies rich in lipids, having no or little 
nucleic acid, then the organism would be able to.respond readily to 
various stimuli without losing its original strong pattern. This must 
be the splendid means by which the organisms could escape the dilem- 
ma pointed out in the previous chapter. The most advanced means 
along this line must be the gene system of the higher organisms of 
the present day. Chromosomes which may be regarded as aggregates 
of genes are actually confirmed to contain uucleic acid in rich amount 
but without lipid (8). 


2. The Evolution of Protein Molecules 


Before discussing the problem of genes, it may be proper to make 
some argument regarding the above mentioned phenomenon that 
different proteins in a solution may exert mutual influences in the 
establishment of an equilibrium system. That each component protein 
in such a system can recover its own structural pattern on the isola- 
tion, may be a natural result of the reversibility of protein structure. 
However, it is noteworthy that adequate heating appears to render 
the change irreversible as indicated in the above cited both findings 
by Kleczkowski and by Zoet. If one component protein in a system 
exerts a particular strong influence upon the other the pattern of the 
system will be chiefly determined by the strong component. The 
effect of some agent, like heating, on such a system may render the 
change of the weaker components irreversible so that the change 
may become durable, that is, the weaker component proteins may be 
assimilized perpetually by the strong. 

The writer assumed that assimilase action is given rise to only 
when protein molecules of the same type are orderly polymerized. 
However, from what has been mentioned above, it may be said that 
the assimilation is a general phenomenon occurring between different 
types of proteins, although the assimilase action may only become 
distinct on a,regular polymerization; in other words, it is considered 
that protein molecules can act in themselves as assimilase upon other 
molecules having weaker patterns. 

The synthesis in the primeval oceans of high molecular proteins 
with a globulin nature might be possible on the basis of this pro- 
perty of proteins as assimilase. Thus it seems most probable that 


234 IV. "THE PRINCIPLES OF LIFE PHENOMENA 


high molecular proteins were produced in the same way as primeval 
organisms; primeval organisms might be different from usual pro- 
teins only in their orderly polymerization. Possibly even at the stage 
of primitive proteins there might also be a struggle for existence, 
thereby weaker molecules were assimilized by the stronger, and thus 
the evolution of proteins were established just as in the case of pri- 
meval organisms. 

There seems no doubt, however, that such a “‘struggle for exis- 
tence’’ among the protein molecules, when the proteins had not yet 
been endowed with any highly evolved structure to produce a strong 
structural influences, was only trivial, and accordingly their evolution 
would be extremely tedious. The speed of evolution of assimilase or 
of protein would be directly proportional to the differentiation degree 
of the protein structure, and even when the proteins were able to 
form orderly polymerized masses, the speed would increase with the 
degree of differentiation of the polymerized mass, or assimilase. It 
should be said, therefore, that the highly evolved creatures of the 
present day may have the greatest speed of evolution and still now 
be evolving most rapidly. This reasoning may naturally lead to the 
view that the evolution of viruses which can make use of highly 
evolved protoplasm for their matrix for evolution might be much more 
rapid than that of the primary organisms. 

The very important fact discussed above that proteins are able to 
exert their structural influences upon other proteins should be, on the 
other hand, interpreted as indicating that proteins themselves are 
enzymes, and therefore the evolution of proteins can be regarded as 
the evolution of enzymes. As regards this view further discussions 
will be made later. 


3. The Reason for the Presence of Optically Active 
Amino Acids in Protoplasm 


Organic substances synthesized by organisms are always optically 
active, as for example, amino acids constituting the proteins of 
protoplasm are as a rule laevorotary, whereas optically inactive 
racemic compounds are always yielded by artificial synthesis. 

This fact has long been regarded as one of the biological enigmas, 
but this may naturally follow the crystal nature of the protoplasm. 
The fact that optical isomers are distinguishable by their different 
crystal shape has well been known since the brilliant finding of 
Pasteur. Substances of dissimilar optical activities can by no means 
exist in one and the same protoplasm. 


II. THE PATTERN OF PROTOPLASM 235 


The writer is unable, however, to find any firm reason for which 
all the amino acids synthesized by organisms must be laevorotary. 
Presumably some individuals of primeval primary organisms were 
decomposed of L-amino acids, while at the same time the others were 
of D-amino acids, but there could be any synergetic connection between 
these two groups, as the one could not make use of the other because 
of the principal difference in their crystal shape, so that there might 
be a distinct line of demarkation, and perhaps an iron curtain hanged 
between them as in the case of the two groups of man in the present 
day. 

There are actually ample evidences showing that D-amino acids 
are not only useless but sometimes injurious to the present-day L- 
organisms. The inhibition of growth of both gram-positive and gram- 
negative bacteria by D-amino acids has been well known (9) (10). 
Especially D-serine appears to be injurious, as for example, the growth 
of FE. coli is injured even when pD-serine is present in so small an 
amount as 57 per ml. (11). Furthermore, D-serine exhibits a nephro- 
toxic action on rats (12). 

It has become evident that D-amino acids occur occasionally in 
nature, particularly in products derived from certain molds and bac- 
teria. Thus, D-proline occurs in a number of ergot alkaloids, ;whilst 
several of antibiotics, particularly penicillin and gramicidin are notable 
for their content of D-amino acid residues. In the case of penicillin, 
the D-amino acid residue is one of critical structural features, since the 
L-analog is without activity. The high molecular polypeptides in the 
capusular substance of bacteria of the mesentericus group contains 
also D-amino acids. : 

It should be noted that all these substances are injurious to some 
organisms, indicating evidently that optically different amion acids 
are incompatible with each other. The supposed p-group of the or- 
ganisms might have been extinguished by the injurious effect exerted 
by some products of L-group, leaving the latter alone on the primeval 
earth. If the L-group thus gained a victory over the D-group and left 
alone, all the secondary organisms generated from them should also be 
L-group and accordingly all the organisms have come to be L-group 
without exception. It is supposed, however, that some L-organisms 
existing at the present time have acquired the ability to synthesize 
D-amino acids to exert injurious effects upon the rivals in order to 
gain the victory in the struggle for existence. 

It has been shown that lactobacilli, which have lost the ability to 
synthesize piridoxin, can grow in a medium which contains pD-alanine 
even in the absence of piridoxin. Organisms grown in this medium 
are devoid of piridoxin and its derivatives, whereas organisms grown 


236 IV. THE PRINCIPLES OF LIFE PHENOMENA 


in media containing piridoxin but no D-alanine are found to synthe- 
size D-alanine. This finding has led to the discovery of a new enzyme 
called ‘‘racemase’’ which produces DL-alanine from L-alanine (13). Kégl 
and his collaborators (14) have repeatedly claimed that cancer cells 
contain D-amino acids in considerably high amounts, while Christensen 
et al. (15) have observed 7m vitro that a wide variety of amino acids, 
natural and unnatural, of the D- as well as L-configuration was accu- 
mulated in the free cells of mouse ascites carcinoma. The reason 
why cancers are injurious may be partly accounted for by this fact. 


CHAPTER III 


THE NATURE OF GENES 


1. The Generation of Genes 


As referred to above, primeval organisms presumably could 
succeed in maintaining their structural pattern despite their easily 
changeable character which they had secured for the purpose to 
respond to stimuli, by making use of elementary bodies having high 
content of nucleic acid. 

Such bodies or particles would decide the fate of a primeval orga- 
nism when the organism combined with another type of organisms 
having a different structure; the one containing the nucleic acid-rich 
particles of stronger structure would overcome and assimilize the 
other, since the particles could govern the whole structure of the 
organisms. Owing to their strong structure due to the high content 
of nucleic acid, the particles even after freed from the organism 
would be able to transmit their pattern to the other organisms having 
weaker particles. In short, the same would result either when the 
particle combined in a free state with weaker organisms or when com- 
bined without being freed from the organism. 

At least strongly active phage particles produced from lysogenic 
bacteria can be regarded as such representative bodies of the bacterial 
cell. Only one type of a certain virus can multiply when two 
different types simultaneously affect a host cell, a fact which is known 
as interference phenomenon, and which has been studied with phage 
especially in detail. 

The multiplication of a phage in a bacterial cell is the transmission 
of the phage structure to the bacteria, whereby the bacteria are assi- 
milized by the phage. Accordingly, it should necessarily follow that 
in a bacterial cell affected simultaneously by two different phages 
solely a strong phage can multiply, the weaker one being unable to 
exert any influence. If, however, the strength of two phages is alike 
both may multiply in a cell at the same time without interference; 
this is found to be actually the case. 

In this connection should be mentioned a very remarkable fact. 
When a single bacterium is infected with a pair of phages like Tory 
and Tur, one finds among the emerging viral progeny types which 


238 IV. THE PRINCIPLES OF LIFE PHENOMENA 


are different from either of the infection viruses, having peculiarities 
of both strains. In the example found by Delbriick and Bailey (16), 
the new types are such as Teor and Tary. 

Similar phenomenon has been likewise observed by Burnet and 
Edney (17) with influenza viruses. From mixed infections in mouse 
brain of the neurotropic influenza strain NWS and the non-neurotropic 
strain MEL, virus strains which differ sharply from either of the 
originals have been obtained. 

This phenomenon can be readily explained by the assumption 
that the strong structure is restricted to a portion of the protein 
molecules. If nucleic acid was inserted in a portion of the polymeri- 
zation product of the thread-like protein molecules, only the portion 
would have the strong structure, although it is well conceivable that 
some portion of the protein threads themselves can possess the strong 
structure. At any rate, if two particles, one of which has the strong 
structure at portion A and the other at portion B, affect simultane- 
ously a single protoplasm, portion A of the protoplasm protein will be 
changed by the former and portion B by the latter independently 
from each other. If the portions in which the structural patterns of 
the two viruses are to be impressed are either identical or present 
very near, the interference phenomenon may occur, whereds if the 
portions are separated enough both patterns will be replicated with- 
out interference. 

The formation of a replica of a strong structural pattern at a 
certain portion of the protoplasm protein may lead in the organisms 
to the appearance of property, which is determined by the structural 
pattern of the portion regardless of the kind of replica of the other 
portions. Namely, the particle having a strong structure at portion 
A will give rise to the property determined by the structure at portion 
A, and the particle having a strong structure at portion B will reveal 
the property determined by the structure at portion B, and thus the 
new progeny having the peculiarities of both particles will be gene- 
rated. In such a case the particles will be regarded as genes capable 
of determining the character peculiar to each particle. The mecha- 
nism for causing localized changes in limited portions of the protoplasm 
protein is apparently most advanced in the structure of genes of the 
present-day higher organisms. 


2. The Structure of Genes 


As the organisms advanced the higher, their character would 
become the more complicated, and at the same time the various loca- 


II. THE NATURE OF GENES 239 


lized structures governing each of their differentiated and complicated 
characters would be arranged the more orderly in the protein mole- 
cules constituting genes. 

The protein components in the nucleoprotein in the nucleus were 
believed to be basic proteins,-such as histone and protamine, but at 
present a series of workers claim that the chief protein in the chro- 
mosomes is acidic one named chromosomin as already stated in Part 
II. In general, protoplasm consists of protein of globulin nature, which 
is also of acidic nature, and hence chromosomin may be a protein of 
globulin nature, forming genes by combining with nucleic acid, whose 
threads are presumably oriented parallel to the protein threads (18). 
Stedman (19) who was the first to find that the main protein compo- 
nent of chromosomes is acidic protein, objects to the opinion concerning 
the importance of nucleoprotamines and nucleohistones in heredity, 
because the structure of protamines is so simple that it could hardly 
account for their alleged importance as carriers of heredity. 

In short, all the characters of organisms are subjected to the 
structures of protoplasm protein, which in turn are governed by the 
structure of genes. In the gene of higher organisms of most compli- 
cated characters, there must be present a great number of localized 
strong structures each of which may direct each component factor of 
the complicated characters; and a single gene may have only a single 
localized structure so that the number of genes may be equal to that 
of localized structures. If there are two genes which govern the 
structure of the same portion, the structure of the portion should be 
determined by the stronger gene, thus the reason why there are do- 
minant and recessive genes may be readily explained; this must of 
course be an interference phenomenon between genes. 

This concept is elucidated in Fig. 23, in which structures of the 
restricted portions are shown by various patterns; those given by 
dotted lines are weak structures, which are subjected to the strong 
patterns of non-dotted lines. Each gene in a pair is inherited from 
each of the parent and the structure of the restricted portion is 
determined by the stronger one, the dominant gene, which accordingly 
directs the character subjected to the structure. 

Genes are, therefore, virus-like polymerization products of proteins, 
a portion of which is endowed with a particularly strong structure. 
Each of a certain number of the particles which are comparatively 
similar to one another may aggregate into each larger body at a 
stage of the cell division; such aggregates may be termed chro- 
mosames. Since different genes sharing the site of determination 
in common may repel each other, each gene in a pair may be distri- 
buted to different chromosomes so that every gene in a chromosome 


240 IV. THE PRINCIPLES OF LIFE PHENOMENA 


may have each site of determination. Mendel’s laws of inheritance 
can be well explained by this concept. 

However strong the reversibility of the protoplasm structure may 
be, the pattern will more or less be damaged when the protoplasm is 
repeatedly altered in its structure by various stimuli. The pattern 
thus damaged will be repaired by genes which are always able to 
retain the original pattern. 


Cytoplasm 
Doninant === Se eee oe ocaoee 

Gene I : Cg aay Sum “ies = 
Recessive / Ne IY eee igs go 
‘ ' ‘ . ’ . 1 
1 us 7 Sass \ ' 

EE ES EES) 
= z FA \ 

(ne fae a ae 

I ni 1 ‘ N ‘ Ne 1 

1 7 se su 1 i} 

SSS 

. 1 ni oa oe 1 sat 

: Dominant ee 
ne I { —— 
Gere Recessive bere EA pote 
Need RENCE Ki 4 

| SS 

: fi eer ALA ie 1 

{ 1 " iy ‘\ fe 8 ! 
1 ‘ nh as Shoe g * ; 

‘ SS 
maeadan Aa ae | 
brent hed ‘ Ae Sok Rit 
| , 1 28 Siete \ 1 
_——— 


et 


Dominant 
Gene Ill Recessive 


Fig. 23. Diagram of genes. 


Chromosomes as above suggested may be coagulated aggregates of 
genes of relatively similar structures, so that in the chromosomes the 
active groups of genes may be present ina folded state. Accordingly, 
in this state genes may be unable to accomplish their function, which 
may develop only when the coagulation is solved and gene particles 
are set free. This may occur at a certain stage of the cell division, 
at which nuclear masses are mixed with the cytoplasm. 

If genes always exerted their strong structural influence upon 
the cytoplasm, the latter would be unable to change freely its struc- 
ture, and accordingly the exhibition of various vital functions would 
become impossible which must only be accomplished by movement of 
the protein molecules constituting cytoplasm. 

It is said that chromosomes are present as such only at a certain 
stage of cell division, in the living interphase nucleus being optically 
homogeneous, and that chromosomal structures appear only after 
injury or treatment with histological fixations; in the living nucleus 
the chromosomes are in a greatly extended state, filling the nucleus 
homogeneously and upon injury the chromosome condenses and becomes 


III. THE NATURE OF GENES 241 


visible (20). If isolated from the cytoplasm, the genes would be 
unable to exert their influences upon the latter even when their active 
groups were not folded. Anyhow, there should be in a cell a mecha- 
nism in action by which continuous effect of genes is prevented. 

The mutation of genes may result from their irreversible struc- 
tural change, and since chromosomes can be regarded as a kind of 
aggregates or even crystals of genes, the shape of the chromosomes 
composed of genes undergoing mutation may sometimes be altered. As 
is generally recognized, the shape of chromosomes is occasionally 
changed following a mutation of the organism. 


3. The Size of Genes 


The majority of workers consider that the dimensions of genes 
are not very different from those of viruses or of fibrous molecules. 
For instance, from genetic data it has been inferred that the shape of 
the genes is rod-like and their length is approximately 125 y, their 
width being 5-20, (21). An electron microscopic study of salivary 
chromosomes has shown that giant chromosomes were composed of 
series of granules of 210-330 my in diameter (22). 

On the other hand, a number of workers claim that genes must 
be much smaller than viruses, because small as they are virus parti- 
cles seem to be endowed with many component characters each of 
which is inheritable independently, a fact already discussed in the 
beginning of this chapter. 

If genes are regarded as entities as shown in Fig. 23, they must 
have a dimension of viruses, but if only unit structures directing 
each component character are taken into consideration, the dimension 
will naturally appear much smaller. However, since unit structures 
themselves cannot exist independently of the whole protein molecules, 
it should be proper to regard the genes as virus-like polymerization 
product composed of protein and nucleic acid. 

In Fig. 23 only three unit structures are shown, but in reality 
the structures must, of course, be present in a much larger number. 
Since a single molecule of globulin may be composed of more than 
1,000 amino acids, if a single amino acid residue can behave as a strong 
unit structure directing a character of an organism, more than 1,000 
determinant structures may be able to exist and accordingly more 
than 1,000 kinds of genes may appear to be present in a cell, because 
in a single gene particle only one structure of them may act as a 
directive site. 

Viruses can be regarded as genes, though not so differentiated, 


242 IV. THE PRINCIPLES OF L4FE PHENOMENA 


so that each unit structure in a particle can behave occasionally, like 
in genes, as each determinant site. For example, if a virus with 
three unit structures, A, B, and C, affects a cell together with another 
virus having unit structures, A’, B’, and C’, and if the degree of 
strength of each pair of the structures is as follows: A>A’, B<B’, 
and C<C’, then newly formed virus will be A, B’ and C’, whereas if 
the former virus combines with a virus, A’’, B’’, and C’’, and the 
degree of strength of the structures, being A>A’’, B>B” and C<C”, 
then the new virus will be A, B, and C’’.. In such a manner, each 
unit structure of the virus can be revealed separately appearing as if 
it were composed of many genes. 

It may, however, be unreasonable to assume that in a gene particle 
only a single unit structure can always have the supremacy, directing 
a property without being influenced by other structures. There is a 
good reason to suppose, as will be mentioned later, that it is difficult 
to change a restricted portion in a protein molecule without altering 
other portions. Moreover, it is possible that the manner of arrange- 
ment or of combination of different structures may sometimes deter- 
mine a character. A normal organism is said to be the resultant of 
action of some thousand of genes; for instance, in Drosophila about 
eight thousand of genes are believed to be in action (23). Presence of 
such a great number of genes cannot be explained by the assumption 
that a component character is always directed by only a single unit 
structure. 

Antigens, like viruses, are capable of producing a sort of replica 
of their own in the protoplasm of certain cells, though the replicas 
produced by them are not so perfect as those produced by viruses, 
and moreover though the cells in which the replicas are to be formed 
are the cells which are generated for the purpose of antibody produc- 
tion. Anyhow, there seems no doubt that antigens are a replica- 
producer just as viruses or genes, and in this respect genes may be 
regarded as the strongest antigens in the cell protoplasm. 

By the famous researches of Landsteiner and his coworkers (24) 
it has become evident that the specificity of antigenic character of 
proteins resides in the chemical structure of their molecules. The 
average size of a specific group is said to be of the order of 600 to 
1,000 in molecular weight, but occasionally very simple artificial che- 
mical groupings may act as specific determinant (25). It seems, how- 
ever, that the specificity of natural proteins is due not to a single 
type of group but to a specific arrangement of different polar groups 
in the surface of the protein molecule (26). The same may hold true 
for genes. 

In general, the substances able to behave as antigens are proteins. 


II. THE NATURE-OF GENES 243 


This fact indicates that the property for replica formation appears to 
be a characteristic of proteins, at least proteins appearing to have the 
property most suitable for the replica formation. This is one of the 
main reasons why the writer has taken into considerations only pro- 
teins in the argument into the nature of virus or of protoplasm. 
Since substances such as sugars and lipids in protoplasm may merely 
be subjected to the pattern of the protoplasm directed by genes, the 
argument can be advanced without regard to their existence so far as 
the problem is concerned with the replica formation. 


CHAPTER IV 
THE SUPREMACY OF GENES 


1. The Type of Nucleic Acids and Its Biological 
Significance 


In cells there are two kinds of nucleic acids, one of which is 
desoxyribonucleic acid (DNA) and the other ribonucleic acid (RNA). 
DNA is usually present in nuclei, and the nucleic acid in genes is 
believed to be DNA, whereas RNA is found as a rule in cytoplasm, 
mostly being contained in particles known as microsomes which there- 
fore can be regarded as elementary bodies or their aggregates contai- 
ning RNA in rich amount. In addition, particles called mitochondria 
are said also to contain this nucleic acid, but the particle present are 
much fewer in number than microsomes which the latter are reported 
to occupy even 15 to 20 per cent of the cytoplasm. 

These particles must have directing influences upon the whole 
cytoplasm when genes are separated from the latter, since they 
contain nucleic acid in rich amount and form large sized particles. 
Most likely they, if liberated from the cell, may first and foremost 
behave as viruses, no matter whether the cell has the normal struc- 
ture or the structure changed by a virus; in the latter case particles 
will be regarded as the virus having multiplied in the cytoplasm. 

The nucleic acid in phage particles separated by ultracentrifuga- 
tion are reported to be mostly DNA, while their lipid content is poor. 
On the other hand, chromosomes which may be regarded as aggre- 
gates of genes similarly contain DNA but no lipid, a fact which may 
enable one to consider the bacterial particles isolated as phage to be 
bacterial genes. Cytoplasmic particles such as microsomes and mito 
chondria contain nucleic acid in rich amount as mentioned above, but 
in contrast to genes the nucleic acid is RNA and in addition, 30 to 40 
per cent of the particles are composed of lipids (27) (28). 

Such large lipid contents of these cytoplasm particles may partially 
account for their submission, in spite of their high nucleic acid 
contents, to the genes. The difference in the kind of nucleic acids, 
however, may have on this point a much more important significance. 

According to the writer’s concept, when inserted among protein 


IV. THE SUPREMACY OF GENES 245 


molecules, nucleic acids may cause the polymerized product of the 
protein molecules to become rigid and strong, thereby the polymerized 
product is endowed with the ability to behave as strong assimilase. 
In this hardening action DNA may be more effective than RNA, since 
it is known that RNA is split by the action of solium hydroxide, 
whereas DNA is more stable and resists this treatment. Application 
is made of this difference for the quantitative determination of each 
nucleic acid in the presence of both of them; after the destruction of 
RNA by sodium hydrooxide, DNA is precipitated by the addition of 
proteins in acid solution (28). Presumably organisms make use of this 
difference and allocate DNA to genes which require the strongest 
pattern, and RNA to cytoplasmic particles to be subjected to the genes. 

In addition, owing to its rather weaker structure RNA may 
readily split off the energy-rich phosphate bond which may provide 
the energy necessary for the protein synthesis as will be discussed in 
greater detail in Chapter IX in this Part. This may be the more im- 
portant reason why RNA is allocated to cytoplasmic particles where 
the protein synthesis may chiefly take place. 

There appear to exist substances other than nucleic acids which 
may be able to exert hardening effect upon protein complexes. Di- 
valent cations, such as Ca, and polysaccharides, for instance, may be 
regarded as such substances, but it should merit attention that only 
nucleic acids seem to contribute the template action of viruses or 
virus-like agents including genes. This may be ascribed to the 
ability of nucleic acids, unlike other hardening agents, to stabilize 
the fine, specific pattern needed for the template action. It is claimed 
that there is as great a variety of nucleic acids as proteins (29); and 
again it is emphasized that the fundamental differences between 
groups of virus are correlated with their nucleic acid composition 
(30), and moreover that nucleotide composition of a viral nucleic acid 
is characteristic of the species (31). If nucleiotides are arranged ac- 
cording to the specific pattern of the protoplasm, nucleic acids with 
the specific composition will be produced. According to Hershey ef al. 
(32), phage DNA can be distinguished by its hydroxymethylcytosine 
content from normal bacterial DNA, which contains cytosine, indicating 
that bacterial pattern changed by the virus is revealed in the pattern 
of DNA. 

The analogies between genes and viruses have been discussed by 
numerous workers. Distinct difference between them seems to lie in 
that viruses can proliferate indefinitely in the cell, whereas genes 
appear to multiply only in an orderly and restricted way. However, 
the patterns of genes like those of viruses do multiply indefinitely in 
the cell, that is, the directing influences of the genes can spread 


246 THE PRINCPLES OF LIFE PHENOMENA 


throughout the cell to make the protoplasm identical in the structural 
pattern with the genes, although the multiplication of papticles itself 
capable of acting as a gene is restricted. Thus the multiplication or 
spreading of genic pattern must be equal in the nature to the prolife- 
ration of viruses. 

One cannot regard the multiplication of the genic pattern in the 
same light with the proliferation of particle itself capable of acting 
as a gene. The indefinite multiplication of genic pattern is desirable 
for organism but the indefinite proliferation of gene particles rich in 
nucleic acid must be fatal to them, because, as was already discussed 
in detail, for the development of various vital phenomena protoplasm 
must be composed mostly of elementary bodies containing large 
amount of lipids without nucleic acid. Accordingly, if genes rich in 
DNA multiplied unrestrictedly, the cell would naturally be soon 
destroyed. This may hold true likewise for cytoplasmic particles 
such as mitochondria and microsome whose multiplication is also 
restricted like genes. In short, it is evident that the organisms 
have been able to continue their existence to be left on this earth as 
the fitter, only through the development of the mechanism by which 
the numbers of the particles, such as genes and microsomes, are held 
constant. 

As stated above protoplasm is regarded as a mixed crystal. On 
the other hand, it is known that the crystal of a certain substance can 
take into it another substance to make a mixed crystal, but that the 
amount of another substance to be taken into it without effecting the 
original crystal pattern is limited. For example, MgSO,7H.O forms 
rhombic and FeSO,7H.O monoclinic crystal, and these two salts can 
form a mixed crystal with each other, but the quantity of each salt 
to be mixed with the other in order to form a mixed crystal without 
exerting any influence on the original shape of the other is limited. 
Thus the Mg-salt can dissolve at most the Fe-salt at 18.8 per cent to 
form a rhombic crystal. It may be not utterly impossible that simi- 
larly in the protoplasm the quantity of substances other than proteins, 
such as nucleic acid and lipid, which can be taken into it without 
effecting its original crystal shape may be limited. 

At any rate, it may be reasonable to consider that the mixed pro- 
portion of various substances in the protoplasm may be determined 
by some physicochemical laws. The unbalance induced in the pro- 
portion of nucleic acid to protein by the nucleic acid increase may be 
the cause of the reduction division or meiosis, by which the proper 
proportion would be restored. Thus, the physicochemical force acting 
to maintain the mixed proportion constant might be best availed by 
organisms for the proper restriction in the multiplication of genes or 


IV. THE SUPREMACY OF GENES 247 


other granules. 

‘ It is generally accepted that vigorous propliferation of cells is 
usually accompanied by a marked increase in nucleic acids. This 
may mostly be attributed to the unbalance in the mixed proportion 
caused by the increase of nucleic acid. It seems that some structural 
change in protoplasm may lead to the increased capacity of the pro- 
toplasm to combine with nucleic acids, as for example, mechanical 
injuries or chemical stimuli cause the increase in nucleic-acid content 
of the cell. Such nucleic acid increase would naturally bring about 
the vigorous synthesis of protein in order to alleviate the unbalance in 
the mixed proportion; this may account for the well known fact that 
various stimuli generally lead to the marked proliferation of cells. 

The virus infection, in general, may bring about the nucleic acid 
increase in the cell. For instance, when bacteria are affected by phage 
nucleic acid, especially DNA, markedly increases (33). During the 
course of normal growth the cells of E. coli synthesize almost three 
times as much RNA, but susceptible cells suspended in a synthetic 
medium and infected with phage will show a rate of DNA synthesis 
approximately four times as great as that observed in normal 
cultures. This may suggest that the structural disturbance in the 
bacterial protoplasm caused by phage may alter the mixed proportion 
of DNA to the protein. _ 

If a structural disturbance in the protoplasm caused by some 
stimulus resulted in the increase in nucleic acid, especially in DNA, 
the structural change, if occurred after the disturbance, would be well 
retained in the protoplasm particles, because these particles would be 
endowed with strong structure and accordingly with powerful assimi- 
lase action owing to the high content of the nucleic acid so that they 
might be able to act as a newly produced virus. When this virus 
affects some cells to produce the replica, in the protoplasm of the 
cells will be raised the structural disturbance which may cause the 
same nucleic acid increase, since the virus structure may have the 
property to cause the increase in nucleic acid. ‘This may be the reason 
why nucleic acid is increased in the cells affected by a virus. A 
virus which failed to cause a change leading toa marked nucleic acid 
increase in the protoplasm would not be able to continue to exist. ; 

Lysozyme can cause lysis of bacteria like phage, but fails to 
multiply. The characteristic chromatic granules of the bacteria are 
seen on the phage infection indicating the occurrence of striking 
change in the nucleic acid, whereas lysozyme fails to induce any 
similar change in the granules (34). The inability of lysozyme to 
become a virus would be accounted for partly by this failure to in- 
fluence the nucleic acid content. In the presence of proflavine bacteria 


248 IV. THE PRINCIPLES OF LIFE PHENOMENA 


infected with phage undergo lysis with the liberation of phage-like 
particles which have no phage activity. The ultraviolet absorption 
Coefficient of these particles revealed that their nucleic acid content, 
if any, is much lower than that of usual phage particles (35). 

Bacterial strains having acquired increased resistance to the lethal 
effects of X-rays or ultraviolet rays have been found to synthesize 
several times as much DNA as do the sensitive strains (36). Such 
bacterial strains might have increased the resistance, because they 
have undergone a structural change to cause the increase in DNA. 

In the presence of cobalt ions the growth of some bacteria is 
prevented but RNA content may be raised (37), and certain other 
bacteria, when grown under unfavourable conditions, may accumulate 
large amount of RNA (38). It is considered that also in these cases 
there might occur structural changes which caused the alteration in 
the mixed proportion. 


2. Plasmagenes 


A number of workers claim that the characters of organisms are 
determined not only by genes but also by cytoplasmic particles 
designated as plasmagenes. In the theory of the writer, it may be 
. said that protoplasm is composed of a sort of genes, since all the 
elementary bodies of protoplasm are assimilases capable of exhi- 
biting a function like a gene. However, as the bodies present in the 
nucleus possess the strongest assimilase action in the cell, the pattern 
of the whole protoplasm is usually directed by such bodies, which are 
accordingly called genes. It may be possible, therefore, that some 
cytoplasmic particles attain to the supremacy if the genes proper ex- 
hibit only a weak effect. 

A factor called kappa found by Sonneborn (39) in Paramecium 
was regarded as a kind of plasmagenes. This factor can exhibit a 
strong influence on protoplasm structure of this protozoan and provides 
the protozoan with the faculty to produce a substance named paramecin. 
As this substance can kill a sensitive strain of the protozoan, the indi- 
viduals producing paramecin are named killer. In short, kappa seems 
to be able to overcome the genes proper to direct the structure of the 
protoplasm. As a result the property to produce paramecin arises, but 
in the presence of some strong genes kappa fails to exhibit its influ- 
ence and is perished. 

This factor, however, now appears to possess many characteristics 
of microorganisms such as a Rickettsia (40). The size of the factor is 
similar to that of Rickettsiae, being 0.2 to 0.8. In addition, it should 


IV. THE SUPREMACY OF GENES 249 


be noted that kappa particle contains a large amount of DNA. Its 
strong effect may be attributed to this nucleic acid in addition to its 
large size. 

It is probably true to say that the gene system has been evolved 
amazingly in higher organisms because it is the most ideal system for 
their development. Therefore, presence of particles in cytoplasm such 
as kappa, which can strongly interfere with the gene, may not be 
regarded as a normal feature. Also from this point of view the con- 
cept of the parasitic nature of kappa appears to be reasonable. 

The results obtained by L’Heritier and his collaborators (41) in 
their studies on the physical basis of sensitivity to carbon dioxide in 
the fruit fly, Drosophila, are in many respects strikingly similar to 
the results on kappa in Paramecium. Reciprocal crosses showed that 
sensitives to carbon dioxide differed from resistants, not in any nuclear 
gene, but in some cytoplasmic factor. This factor was named sigma 
and at first regarded as a kind of plasmagenes, but there are also 
some evidences for its parasitic nature, although the factor has not 
been seen or counted. Presumably it is much smaller than kappa, 
more alike in size to a small virus or a nuclear gene. 

Likewise, in higher plants a similar cytoplasmic factor causing 
male sterility is known; the individuals having the factor fail to form 
functional pollen (42). This may also be of a parasitic nature. It has 
been demonstrated that this factor is inherited through the cytoplasm. 
Sigma is likewise inheritable; the flies that developed from the in- 
fected eggs are sensitive to carbon dioxide and transmit sigma to 
subsequent generations through the egg cytoplasm. Discussions have 
already been made on the fact that viruses are transmissible to 
offspring through germ cells (Part III, Chapter X). It should be borne 
in mind that not only viruses but also these parasitic Rickettsia-like 
organisms can be inherited to progeny through host germ cells. 

Mitochondria are usually regarded as normal cytoplasmic particles, 
but some workers consider them to be parasitic microorganism. This 
is not an inconceivable concept, though nucleic acid in mitochondria 
is RNA, so that they are always subjected to the genes, not disturbing 
the beautiful control of the protoplasm by the latter. 

To sum up, since the character of organisms having evolved to a 
certain degree is determined by genes, and since this mechanism of 
character determination is’ considered to be most suitable for the orga- 
nisms, cytoplasmic particles which can strikingly interfere with the 
function of genes may be, at least in the majority of cases, an abnor- 
mal existence. 

However, it should be mentioned here that there is an interesting 
evidence showing that the normal cytoplasm can exert a considerably 


250 IV. THE PRINCIPLES OF LIFE PHENOMENA 


great influence upon the character of amoeba. Danielli (43), using 
two large species of amoeba, 7. e., Amoeba proteus and A discoides, 
has carried out transplantation of the nuclei of individuals of one 
species to those of the other species from which their own nucleus had 
been previously eliminated and found that the form of the amoeba and 
the diameter of the nucleus are mainly determined by the cytoplasm, 
although the nucleus, of course, has some influence and in the long 
run it comes to exert a more pronounced influence, most of the amoe- 
bae becoming to have an intermediate form between discoid and 


proteus. 


CHAPTER V 


RELATIONSHIP OF GENES TO 
ENZYMES 


1. The Nature of Enzymes 


Two protein molecules of different types do not combine with one 
another because of the presence of the repulsive polar forces resulting 
from the different structures, whereas if they have structures, comple- 
mentary to each other, having strong attractive forces, the combi- 
nation will be established. However, repulsive polar forces, if present, 
will exert mutually disturbing influences when the two molecules 
having the complementarily shaped structures are drawn near by the 
attractive force. For the establishment of the combination of two such 
protein molecules the attractive force must be stronger than the re- 
pulsive force, but the mutual repulsive action will become very mani- 
fest when the combination is established. 

Now, if the repulsive force of protein A is much greater than 
that of protein B, the structure of this latter protein will be changed 
on being defeated by the repulsive force of the former, thereby all the 
structures in B will be changed to become complementary to those in 
A. The structural change of the protein B to become complementary 
to A in its structural pattern is nothing but to become identical with 
A itself; accordingly through this change B is assimilized by A. In 
this case A is assimilase. When polymerized orderly, the structure of 
protein molecules will become stable with the enhancement of their 
structural influence. Assimilase is therefore usually polymerization 
product of protein molecules of the same structure, and can adsorb 
protein molecules through the complementary structure to assimilize 
and to eat the latter molecules, thus being able to grow. Accordinly 
assimilase is the primitive form of life itself, and at the same time it 
is a kind of enzymes. 

When the influence of A is not so great as to make the structure 
of protein B identical with it protein B will only be disturbed in its 
structure. If the protein is ‘‘denatured’’ as a result of such a distur- 
bance and deprived of its faculty to combine with the protein A, the 
protein B will be liberated form A. A can thus combine again with 
another protein which will in turn be denatured and again liberated. 


252 IV. THE PRINCIPLES OF LIFE PHENOMENA 


In such a case, A can denature protein B successively and hence may 
be termed denaturase. 

When B has a structure to be easily decomposed into smaller 
molecules by the structural disturbance induced by A, or when Acan 
exert a disturbing effect which is specifically fitted for the decomposi- 
tion of B, the latter will be decomposed and liberated from A on 
account of the disappearance of the combining faculty due to the 
decomposition. Thus A can act as proteolytic enzyme upon B. 

If A and B are similar in their disturbing action, both will be 
denatured by the mutual effect on the combination. This will occur in 
antigen-antibody combination. In such a case, as A is likewise to be 
denatured, it will lose its function as a denaturase, and consequently 
will be consumed in this reaction. A cannot be called on such an 
occasion an enzyme in a Strict sense. 

This is the general outline of the writer’s idea as regards the 
nature of enzymes. From this point of view all the proteins may be 
regarded as enzymes. 

All the protein molecules produced in the oceans of the primitive 
age might likewise be in this respect some kinds of enzymes. As 
above stated, the evolution or the production of protein molecules in 
the primitive age is considered to be established by the ‘“‘struggle for 
existence’’ of the protein molecules, whereby weaker patterns were 
devoured by stronger patterns. Enzymatic nature can be said, there- 
fore, to be the essential characteristic of proteins. It cannot be sup- 
posed, however, that the protein molecules in the primitive age were 
endowed with enzyme activities as elavorated as those of the enzymatic 
proteins existing in the present day. The present-day enzymes must 
be the proteins highly evolved for the purpose of acting as enzymes. 
The evolution of organisms might be involved in the evolution of pro- 
teins, which in turn might involve the evolution of enzymes. Pro- 
teins and enzymes, therefore, may be inseparable from each other. 


2. The Relationship between Genes and Enzymes 


According to the writer’s concept, the protoplasm is a system 
composed of various substances especially proteins. In this system all 
the proteins must have a similar arrangement in polar forces, and 
the pattern of the arrangement is to be directed by genes. On the 
other hand, the action of a protein as an enzyme should be determined 
by the manner of arrangement of its polar forces, so that the func- 
tions of enzymes in a certain cell must be subjected to the genes of 
the cell. 


V. RELATIONSHIP OF GENES TO ENZYMES 253 


In fact, vast number of evidences are known to show the existence 
of intimate relation between genes and enzymes. In some lactobacilli, 
for example, loss of ability to ferment sorbitol and manitol is correlated 
with the loss of ability to produce the polysaccharides responsible 
for specific agglutinability (44). Such a loss of ability to produce the 
specific” substance is ascribed to a change in the protoplasm pattern 
brought about by a change in the gene system which the latter is 
responsible for the change in the agglutinability. Furthermore, in 
the case of different mutable strains of colon bacilli, colonial dissocia- 
tion, which shows a genic change, invariably occurs concomitant with 
the metabolic variation resulting in rapid fermentation of lactose (45). 
Again, passage of lactobacillus plantarum from the S to the R form 
is associated with the loss of ability to produce acid from certain 
sugars (46). Whereas the R form can grow in sugar-free peptone, the 
S form does not grow in the absence of carbohydrates. It is also 
reported that the: growth of staphylococcus in the presence of strepto- 
mycin is associated with the emergence of resistant mutants; at the 
same time, the emergence is linked with the ability of the cells to 
synthesize aspartic acid, phenylalanine, efc., in the presence of stre- 
ptomycin (49). 

It may be possible that each partial pattern of the protoplasm is 
connected with the action of each enzyme in a cell, so that it may 
be possible that a certain enzyme is directed chiefly by a certain 
gene. It cannot be considered, however, that one gene-one-enzyme 
hypothesis (48) holds always true; it may rather be reasonable to 
suppose that, though a certain gene tends to direct chiefly a certain 
enzyme, an enzyme is generally connected with several genes, or a 
gene has some connection with several enzymes, for it may rather 
be exceptional that the property of an enzyme is determined by a 
restricted partial structure only. 

It is claimed that almost all the proteins present in a bacterial 
cell are provided with enzymatic action of various kinds (49), a claim 
which is also raised from the writer’s view. The pattern of the pro- 
- teins present in the protoplasm is subjected to the genes, and since 
an enzyme with a specific active group must be governed by a gene 
directing chiefly the specific group, the enzyme will be changed with 
the gene. 

As stated in the previous section, the usual enzymes may not 
necessarily require for their action the regular polymerization of 
proteins which is indispensable for the assimilase action, but the 
polymerization may possibly favour the action of the usual enzymes, 
for the structural effect of proteins may, in general, be strengthened 
by the polymerization. Cytoplasmic particles such as mitochondria 


254 IV. THE PRINCIPLES OF LIFE PHENOMENA 


and microsomes appear to behave as assimilase and accordingly as 
viruses owing to their rigid polymerization resulting from the high 
content of nucleic acid, while at the same time they are known to 
have various enzymatic actions. For example, it has been noted that 
almost all the lactose activity in rat kidney homogenates can be sedi- 
mented with microsome fraction. Mitochondrial fraction, on the other 
hand, has been reported to have the major portion of the activity 
of a number of tissue enzymes (50) (51). It has already been stated 
that the structural pattern of these cytoplasmic particles is directed 
by genes, and hence their enzymatic actions must also be governed 
by the genes. 

Since viruses can be regarded as pathogenic genes, which determine 
the pattern of the host cells after the infection, the enzymatic action 
of the cells may likewise be changed according to the kind of viruses. 
It has been found by Bauer (52) that chick eggs inoculated with 
yellow fever virus showed a marked rise in xanthine oxidase acti- 
vity of embryo and of chorio-allantoic tissues. Both virus titer and 
enzyme activity reached a peak at about the 13th day and then de- 
creased. This may be a result of the change in the protoplasm 
pattern raised by the abnormal gene, namely, yellow fever virus, 
which may produce in the protoplasm protein the structure capable of 
acting as xanthine oxidase. Fredericq and Gratia (53) have described 
the phenomenon of stimulation of lactose fermentation by lactose- 
negative bacteria submitted to the action of a certain type of phage, 
a phenomenon indicating the production of the enzyme on the struc- 
tural change by phage. 

Further, Kun and Smith (54) investigating the respiration of the 
allantoic membrane infected with myxoma virus, found that zymo- 
hexase activity was increased and lactic acid production was cor- 
respondingly increased. On the contrary, if a virus pattern is incom- 
patible with the pattern of some normally existing enzyme, the enzyme 
will be destroyed by the virus infection. According to Bauer (55) 
cholinesterase activity in mouse brain infected. with yellow fever virus 
falls gradually during the incubation period and reaches a minimum 
value at the time when symptoms of encephalitis appear. 

Paramecia affected by kappa, as already stated, produce paramecin, 
which can kill other paramecia having no kappa. This killing sub- 
stance, paramecin, can likewise be considered as an enzyme directed 
by a gene or a virus named kappa. Since paramecin may be the pro- 
tein of the protozoa endowed with the pattern of kappa, it will strive 
to give the pattern to normal paramecia, but paramecin itself cannot 
multiply probably because of its failure of producing its exact replica, 
although it can cause disturbance great enough to kill the protozoa. 


V. RELATIONSHIP OF GENES TO ENZYMES 255 


Even the kappa bodies themselves can neither cause the production of 
paramecin nor protect the protozoa from the killing action of parame- 
cin, if they are contained only in small amount within the cell (56). 
If the pattern of the protozoa is changed into that of kappa, paramecin 
may be produced and, at the same time, the protozoa may become not 
to be killed by paramecin. 

. It has actually been confirmed that both kappa and paramecin 
contain desoxyribonuleic acid, both being present in the bodies of 
killer organisms, and both are unstable and inactivated at rates varying 
with temperature. Paramecin is inactivated by pepsin, chymotrypsin, 
and desoxyribonuclease, indicating that both protein and desoxyribonu- 
cleic acid are compounds essential for its killing action, and more- 
over paramecin particles are liberated from killer cells mechanically 
disrupted and they are easily centrifuged down (57). 

Since most proteins present in a cell are synthesized under the 
influence of the pattern of the genes, their pattern may be originally 
identical with that of genes. But some proteins may reluctantly be 
submitted to the genic pattern because of their peculiar configuration 
which makes it difficult to form the pattern directed by the gene. 
Such proteins may take their own natural patterns, if freed from the 
control of the gene. 

Various kinds of proteins which are somewhat different in their 
immunological specificity can exist in one and the same blood plasm. 
Similarly, proteins with different original patterns may be present in 
a single cell. But their original, peculiar patterns may be revealed 
only when they are liberated from the cell, in which they have been 
compelled to submit to the pattern of the gene. Recessive genes 
possess extremely peculiar pattern of their own, although in the 
presence of the dominant genes they are forced to change their 
patterns to be submitted to the direction of the dominant genes. 
However, when once freed from the dominant genes, they recover their 
original patterns and behave as independent genes in some cells where 
there are no dominant genes. ss 

The cells especially those of bacteria are believed to consist of 
various antigenic substances varying in chemical nature and biological 
properties, and termed antigenic mosaic. However, according to the 
theory of the writer as above stated, the cells are never the antigenic 
mosaic; the substances contained in the same cell should be equal in 
the antigenic pattern at least as long as they are present in the cell. 
The isolation in a “‘pure form’’ may lead either to a damage of the 
pattern or to the recovery of the original pattern as in recessive genes. 
The protein components constituting bacterial cell bodies such as 
those of pneumococci are called the somatic antigen, the specificity 


256 IV. THE PRINCIPLES OF LIFE PHENOMENA 


of which is not so stiking as that of the capsular antigen which is 
believed to be type-specific and to be polysaccharide in chemical nature. 
The type-specific structure may be fine and unstable, so that the 
pattern may be lost in the somatic protein during the purification 
process, only the solid, stable pattern being left untouched, whilst 
the fine, type-specific pattern may be retained in the polysaccharide 
even after the isolation owing to the rigid nature of its structure. 

The writer considers that the pattern of lipids is not only extremely 
labile but their existence results in the decrease in the action of 
assimilase as the rigidity of the pattern is lost by their presence. 
However, the fact that the pattern is not always labile in the sub- 
stances called lipids is fairly demonstrated by the work of Yamakawa 
and Suzuki (58) who have shown that a glucolipid isolated from red 
blood cells is evidently different in chemical structure between man 
and the horse. On account of its peculiar chemical composition, pre- 
sumably owing to the presence of sugars, the glucolipid may be able 
to retain its species specific pattern even after the isolation though it 
is called lipid. 


CHAPER VI 


FACTORS INTERFERING 
WITH GENES 


1. Hormones 


The pattern of proteins in protoplasm is generally determined by 
genes, but there are many evidences showing that factors other than 
genes can also play an important role in the determination of the 
pattern. Hormones appear to be substances which are produced by 
organisms for the purpose to interfere with the gene action. 

Insects will generally change in a remarkable way their shapes 
and functions during their developmental process, that is, they undergo 
metomorphosis. Such metamorphosis may be caused by the rapid 
development of the structural change in the protoplam protein. In 
other words, metamorphosis may be a result of a rapid change in 
“crystal shape’’ of the protoplasm; and the ‘“‘crystal shape’’ may be 
altered by the change in the structure of the component substance. 
On the other hand, it is generally accepted that hormones are involved 
in metamorphosis. The removal from insects of endocrine glands, 
such as prothracic gland and corpora allata, can induce profound 
effects in the feature of matamorphosis. This fact strongly suggests 
that hormones may be able to change the structure of protoplasm 
proteins. 

Such effects of hormones upon the ‘‘crystal shape’’ of protoplasm 
can generally be observed in animals. As is well known when fed 
thyroid gland, the metamorphosis of tadpoles is accelerated and pigmy 
frogs will result, whilst when fed thymus, the metamorphosis is 
delayed to make giant tadpoles. Likewise in man disorders in endo- 
crine secretion lead to abnormal shapes and functions. It may be 
concluded, therefore, that hormones are factors capable of strikingly 
interfering with the action of genes. The effect of hormones on the 
“crystal shape’’ of protoplasm seems to be of extremely high physio- 
logical significance; organisms can establish their normal development 
and achieve various functions owing to this effect of hormones. 

As we have seen above, enzyme systems are directed by genes, 
whereas it is believed that hormones constitute an important set of 


258 IV. THE PRINCIPLES OF LIFE PHENOMENA 


enzyme-regulating factors. It should only be natural that enzymes 
are regulated by hormones if hormones can interfere with genes. 
Definite patterns of changes in enzyme concentration occur in tissues 
and organs under the influence of hormones; this is well illustrated 
by the changes in the alkaline phosphatase, acid phosphatase, adeno- 
sine-triphosphatase, succinic dehydrogenase, malic dehydrogenase, and 
total glycolysis of corpora lutea of the rat during pregnancy and 
lactation (59). The action of hormones on enzymes has also been 
demonstrated with the effect of insulin and some adrenal and pituitary 
hormones on hexakinase. Insulin can increase glycogen formation 
and glucose utilization in rat diaphragm incubated 7m vitro, and fur- 
ther it also increases the incorporation of alanine into diaphragm 
protein (60). Again, phosphorylase in the rabbit is inhibited by cor- 
tisone and activated by adrenalin administration (61). These facts 
may be attributed likewise to the influence of the hormone upon the 
enzyme systems. 

An interesting finding throwing light on the nature of hormones 
has been reported by Scatchard ef al. (62). They have found that 
albumin is stabilized remarkably by the addition of small quantities of 
substances such as sodium caprylate and sodium salt of acetyltrypt- 
ophan. By the addition of these substances the protein becomes so 
stable as to stand heating at 60°C. for 10 hours. It should be noted 
that these substances, which exhibit such a remarkable effect upon 
albumin, have no influence on 7-globulin, whereas this globulin is 
stabilized by glycine and by certain simple sugars which are in 
effective to albumin. This fact may indicate that the structure of a 
protein is stabilized by the presence of a certain substance. It is 
worthy of note that substances which can stabilize albumin fail to 
effect any change in globulin, while grobulin is stabilized by other 
substances which exhibit no effect on albumin. 

Hormones may be regarded as an agent which can exert such 
specific effects on the structure of protoplasm protein. ‘The deficiency 
of a certain hormone may result in the destruction of certain struc- 
ture in the protoplasm on which the hormone exerts its specifically 
directive influence. Presumably hormones like genes can change the 
structure of the protoplasm proteins in various ways according to 
their specific properties and can maintain the structure. Further dis- 
cussions on this subject will be made in detail in Part V. 

Some organisms appear to produce hormone-like substances for the 
purpose of destroying some other organisms by taking advantage of 
the striking effect of the substances to change the ‘‘crystal shape’’ of 
protoplasm. The so-called antibiotics produced by microoganisms can 
be regarded as such substances. The phenomenon of synergism and 


VI. FACTORS INTERFERING WITH GENES 259 


antagonism which is generally recognized with hormones has been 
observed also with these substances (63). 

Penicillin, like phage, can cause lysis in bacteria (64). It has 
already been stated that lysis of bacteria by phage may be attributed 
to the structural disturbance caused by phage in the protoplasm of 
bacteria. Penicillin may likewise be able to change the protoplasm 
structure of bacterial cell, wherein severe disturbance may be raised 
which may cause the dissociation of elementary bodies leading to the 
decomposition of bacterial cell into the elementary bodies. The lysis, 
however, may not account for the effectiveness of penicillin, for the 
lysis may thus only be a result of the disturbance of protoplasm 
structure due to the drug. The disturbance in the structure may lead 
to the universal derangement in enzyme systems which must be 
the main cause of the effect. 

Antibiotics can produce specific resistance in bacteria, indicating 
the occurrence of specific change in protoplasm structure in response 
to the structure of the drug. Entirely a similar phenomenon will 
take place when phage affects bacteria. Thus, the bacteria become 
resistant specifically to the phage, and bacterial protoplasm particles 
can act as the phage following the infection. As pointed out in 
the previous Part, bacteria having become immune to a certain drug 
produce virus-like agent able to transmit the resistance to other 
organisms. 


2. Inorganic Salts 


Inorganic salts are considered likewise to have hormone-like action 
and can interfere with the genes. 

Lehmann (65) has succeeded in controlling chorda formation. by 
treating the gastrula of Triton or Rana with lithium chloride. More- 
over, it was found by Stockard that when, at a certain critical period, 
the developing eggs of Fundulus, a common sea minow, were subjected 
to the action of various magnesium salts dissolved in sea water, a 
large percentage of them developed a single median eye instead of 
ordinary pair (66). A similar effect of salts has been also demonstrated 
with the cephalopod Loligo vulgaris and other species. The modifica- 
tions were produced by lithium chloride and magnesium chloride. 
The most conspicuous feature of the modifications involves the differ- 
ential inhibition of the head region, resulting in all degrees of 
approximation of eyes to complete cyclops, reduction in sizes, anoph- 
thalimia, and acephalic (67). Such a striking action of inorganic 
solutions on animal forms may only be explained by assuming the 


260 IV THE PRINCIPLES OF LIFE PHENOMENA 


hormone-like function of the ions. We have found that the properties 
of virus-like polymerization product of proteins are remarkably in- 
fluenced by inorganic salts: Protoplasm particles, including viruses, 
are agglutinated at weakly acid pH values and exhibit high turbidi- 
ties, which are markedly changed by the presence of inorganic salts 
(68) (69). The isoelectric point of the particulate proteins, at which 
the turbidity is most manifest, is generally shifted to more acid sides 
with the increase in the salt concentration, whereby the turbidity at 
the pH values more acid than the isoelectric point becoming higher. 
These effects vary, in a most remarkable way, with the kind of ions. 


Grade of turbidity 


14 
12 
1 ee 
ggeaaiatan 
2 3 4 5 6 7 
pH 
Fig. 24 


The influence of inorganic salts upon the turbidity of vaccinia 
virus-protein solution. 

Concentration of the virus protein: 0.196; concentration of 
salts : 0.2m. 

The degree of the turbidity at pH 7.0 when no salt was present 
was taken as 10. 


An experimental result obtained with vaccinia virus is shown in 
Fig. 24. Although the influence of ions on the turbidity degrees is. 


VI. FACTORS INTERFERING WITH GENES 261 


somewhat different with the kind of particles, the gradient of the 
effect of various ions is almost similar regardless of the kind of 
particles, and so the result similar to that shown in this figure can 
be attained likewise with phage, rennin particles, and also with 
protoplasm particles having no virus action (70). 

The inorganic salts which have particularly great effects upon the 
turbidity also exert remarkable deleterious influences upon the virus 
activity. Thus vaccinia virus particles are particularly injured by 
salts, such as K,SO,, KI, KNO, KBr, efc. while these salts have 
striking effects upon the turbidity. The virus remains active for the 
longest if preserved in water containing no salt. 

Since a single active particle is regarded as producing a single 
plaque on agar, the deleterious action of inorganic salts on phage can 
be easily indicated in numerical values as seen in Fig. 25. In this 


Decrease in phage titre 
(Log.) 


Fig. Zo. 


Influences of various potassium salts and also of NaF upon phage. 


figure the degrees of the phage inactivation following the heating to 
50°C. in the presence of various salts are shown. 
The rennin sample prepared by the writer consisted of virus-like 


262 IV. THE PRINCIPLES OF LIFE PHENOMENA 


particles, the activity of which was also easily shown with numerical 
values as in the case of phage, as the action was readily estimated 
by the length of time required by the enzyme for the coagulation of 
milk. As indicated in Tables 7 & 8, likewise with this enzyme it 
was clearly shown that inorganic ions exhibited striking effect, the 
gradient of ions in the effect being almost similar to that observed 
with the viruses. 

Such remarkable influences of inorganic salts upon virus and rennin 
would be readily explained if we assumed that they could distort the 
structure of the particulate protein in various manners according to 
the kind of ions. This assumption seems reasonable, for the ions, like 
hormones, apparently interfere with the genes in changing the function 
of animals as above pointed out. 

The production of phage by E. coli is remarkably decreased if the 
media, in which the bacteria are cultivated, contain neither calcium 
nor magnesium, or if a citrate or an oxalate is added to the media. 
According to our finding, the phage, yielded with difficulty under such 
conditions, would produce extremely small plaques only, its virulence 
to the bacteria being remarkably reduced. It is a noteworthy fact 
that this changed property was occasionally heritable; that is to say, 
the phage yielded from bacteria affected by such a changed phage 
tended occasionally to be of the same changed property, forming only 
small plaques. This property was inherited even when the bacteria 
were cultured in usual media, a fact which may show the occurrence 
of a “‘mutation’’ in phage (71). 

Lack of the two-valent cations may cause a distortion in the pat- 
tern of the bacterial virus, giving rise to the ‘‘mutation’’ which is 
possibly induced by the alteration of protein structure in the proto- 
plasm. As stated in Part II, Chapter VI, minute quantities of cal- 
cium can maintain the virus particles or protoplasm particles ina 
rigid form, so that the particles are photographed under the electron 
microscope in a complete shape, indicating that the lack of calcium 
results in a structural distortion of the particles. 

Many evidences are known that calcium ions are involed in the 
transmission of structural change of proteins as in the case of blood 
coagulation. For example, it has been demostrated that calcium and 
magnesium ions in trace amounts are essential for haemolysis by 
antibdoy and complement, and possibly for bacteriolysis, as well as 
for enhancement of phagocytosis by antibody and complement (72). A 
number of the parthenogenetic agents lose their effectiveness when 
they are applied to eggs in calcium free sea water (73). 

When one breaks the membrane of an amoeba or sea-urchin egg, 
the outflowing protoplasm soon forms a new membrane, but if the 


‘VI. FACTORS INTERFERING WITH GENES 263 


Table 7. 


Effect of Inorganic Salts upon the Inactivation of Rennin due 
to the Neutralization. 


The elapsed time (min.) after 


’ the neutralization; pH 4,6 > pH 8,4 
Medium 


25 | 5,0 | 75 | 10,0 | 12,5 | is 
Water::-:++++++-- 69 140 220 700 
BaClp--++-+++++++- 300 290 330 560 
GAG lyn sink ce 290 250 270 310 


mfolclsiesisiscces'= 180 


eee eee eneeneee 


see eeeeeeeee 


stew eeeeenee 


eee eee eee rere ery 


ete e ee eeeeee 


stew ene eeeeee 


eee eee rr 


The numerals indicate the time (sec.) necessary for the occurrence 
of the coagulation of milk. The concentration of the salts was m/10. 


Table 8. 
The Influence of Inorganic Salts upon Rennet Action. 


Medium in which the rennin 
was dissolved 


Exp. I | Exp. Il Exp. III | Average 


WATE en ad a ee u i 12 ll 
Tae(GIN S Scesuadecocasastbanacceeccos cosesee 43 41 42 42 
124 fa gagece ten coagsoa cane aco ssaCBeSache ace 29 31 28 29 
DoS Onppoae ee eb iat Ae 24 24 %6 25 
122) 2. i aes GE en oe Eetioe hak 292 23 | 23 23 
iE oe ce pees per See eee 23 20 24 22 
Reales es corset sh cick vow taka eee 18 19 18 18 


———EEEEEE————— EEE ee ee eee 
Numerals indicate the time (sec.) necessary for the coagulation of 
milk, when lc.cm. of the rennin solution (0,01 9) was mixed with 
lc.cm. of milk. The concentration of the salts was m/10, and the 
pH of the solution being 5,4~5,6. The experiment was carried out 
at laboratory temp. 


264 IV. THE PRINCIPLES OF LIFE PHENOMENA 


cell is kept in a solution completely lacking in calcium after its 
membrane is broken, all the protoplasm flows out of the cell and 
scatters through the surrounding solution, no new membrane being 
formed. Again, phosphatases require the presence of magnesium as 
coenzyme, and pyrophosphatase is optimally active only in the presence 
of definite ratio of magnesium and calcium ions (13). The action of 
such coenzym may be based on the ability of the ions to retain the 
structure of the enzymatic protein in a proper form. 

A great number of evidences have been presented to show that 
the majority of vitamins behave as various coenzymes, suggesting that 
vitamins are also substances which have influences upon the structural 
pattern of proteins, especially that of enzymes. Anderson (74) has 
found that phage needs amino acids, tryptophan in particular, as co- 
factors for virus adsorption. These adsorption co-factors appear to 
combine reversibly with the virus, without them the virus being 
unable to combine with the bacteria. Presumably a distortion may 
occur in the virus structure when the amino acids are absent. It is 
said that D-tryptophan ‘is inactive despite the marked effectiveness of 
L-tryptophan, indicating that this amino acid is involved in the deter- 
mination of the ‘‘crystal shape.”’ 

The ‘‘crystal shape’’ of protoplasm may be influenced by the 
presence of various substances especially inorgenic ions, although the 
shape is, in the main, directed by the genes; the change in the mixed 
proportion of the ions will be followed by the alteration in the shape. 
Since the primary organisms were generated and evolved in the sea 
water, their protoplasm will retain its normal form only when their 
body fluid is similar, in the proportion of ions, to the sea water of 
the primitive age. An alteration of the proportion may, therefore, 


Table 9. 
The Stabilizing Effect of NaOH upon Red Blood Cells. 


pH of the (No‘od- 5.5 5.6 5.8 | Hel 
cell-suspension* dition) (n/80 3 1) | (n/80; 2) | (n/80; 3) | (n/ 1) infi0s 1)| ito 2) 
Degree of hemo- 
lysis by 0.7 9% NaCl 72 45 22 
solution (9%). 
Degree of hemo- 
lysis by 0.4196 NaCl) 100 100 100 100 38 29 
solution (9%). 


* Figures enclosed by parenthesis indicate the concentrations of NaOH 
and number of its drops added to 0.5cc of the blood-cell suspension. 


VI. FACTORS INTERFERING WITH GENES 265 


lead to the destruction of the normal structure, causing severe injury 
in the organisms. 

From what has been discussed above it should naturally be ex- 
pected that the hydrogen ion has an effect upon organisms similar to, 
or rather more than that of the usual inorganic ions. It is of no 
use to emphasize the striking effect of pH in the body fluid. 

We have found that the addition of minute quantities of alkali to 
blood-cell suspensions im vitro provides the cells with the faculty to 
resist the haemolytic action of hypotonic saline solution, probably 
because the structure of the stroma becomes stronger through this 
procedure (75). An experimental result showing this fact is cited in 
Table 9; rabbit’s red-blood cells are enabled to resist markedly to 
hypotonic saline solutions by the addition of minute quantities of 
NaOH. It may appear strange in this Table that pH of the cell 
suspension has the value of 5.5. Blood corpuscles washed and su- 
spended in the physiological saline solution, always shows this pH 
value. This suggests that the cell stroma itself isof this pH. Alkali 
salts other than NaOH exhibit the same effect, indicating that the 
effect is due to OH ion. Several minutes are required for stabilizing 
the cells. But the additions of hydrogen ion, on the contrary, instabi- 
lize the cells and render them liable to undergo lysis. The stabili- 
zation due to OH ion and the instabilization due to H ion are com- 
pletely reversible. 

It may be a natural result that the protoplasm, a mixed crystal, is 
influenced in its character, to a more or less extent, by the substances 
present in it. Inorganic salts are substances normally present in 
large quantities, and accordingly they have great influences upon the 
character. Hydrogen ion is also a nermal component and its effect is 
compatible with that of heavy metals, having much greater effect than 
the usual inorganic ions. 

As above stated, the embryonic development of some animals is 
much influenced by inorgnic ions; and similar effects are said to be 
induced also by the application of simple organic subatances, such as 
ether and alcohol, indicating that non-physiological substances, even 
if they have no peculiar, complicated structures, can cause a distortion 
in the protoplasm pattern when they enter the cell. It has been 
reported that trypan blue can induce in mouse embryos malformations, 
which bear a striking resemblance to those genically determined (76). 
This may indicate that the dye like a gene has the property to exert 
a peculiarly distinct distortion on the pattern. 

To sum up, the pattern of protoplasm is in the main determined 
by the genes, but substances, such as hormones and inorganic salts, 
which are contained physiologically in the protoplasm may interfere 


266 IV. THE PRINCIPLES OF LIFE PHENOMENA 


with the genes and modify the pattern directed by the latter. Pre- 
sumably higher organisms, by making use of these substances, are 
able to reveal different forms and functions that may vary with the 
varying parts of the body, even if the genes of different parts be 
equal. 


CHAPTER VII 


SEXUAL REPRODUCTION AND 
REJUVENESCENCE 


1. The Origin of Sexual Reproduction 


The primary organisms in their extremely primitive stages were 
presumably able to come into contact with other individuals by virtue 
of the motion of the water in which they were generated. If two 
different individuals shared some structures in common they would 
combine with each other and the one having the stronger structure 
would overcome and assimilize the other. Such a combination might 
be a struggle for survival, but on the other hand the stronger 
individual might be able to establish rejuvenescence by this combi- 
nation. 

When the organisms were evolved to such a degree that some 
elementary bodies in them were provided with nucleic acid in rich 
amount and that the structure of such bodies was endowed with a 
strong reversibility, these bodies would be able to recover their ori- 
ginal structure when they were liberated from the organisms which 
had been assimilized or devoured by some stronger individuals. Ac- 
cordingly, such bodies, if liberated from the defeated, weaker indivi- 
duals, would be able to assimilize the structure of still weaker 
individuals if they could enter the latter. The combination of two 
individuals at such a stage, therefore, may be regarded as a very 
primitive type of sexual conjugation. It may be said that the primary 
organisms at this stage had only a pair of genes, and the combination 
may be comparable to a phenomenon in which weaker bacteria are 
changed into stronger ones when these two strains are incubated 
together. 

Thus a strain of bacteria producing phage, 7. e., a lysogenic strain 
can change another strain, which is susceptible to the phage, into 
the lysogenic strain if these two strains are brought into contact, as 
was discussed already. Again, R-type of pneumococcus is changed to 
S-type when comes into contact with S-type. In these instances, 
nucleic acid-rich particles freed from the stronger strain can substitute 
the bacterial cell. These particles may be called free genes or viruses, 


268 IV. THE PRINCIPLES OF LIFE PHENOMENA 


but at the same time it may equally be possible to regard them as 
spores or primitive gametes, and so the combination may be said to 
be a primitive form of the sexual conjugation. 

However, since in such a conjugation the weaker individual is 
completely defeated, the stronger one will be unable to adopt any 
specific character of the former, although it can anyhow accomplish 
rejuvenation through the contact with different structure. If a part 
of the structure of the weaker individual was intermixed with that 
of the stronger by this conjugation, an individual having a new 
character would be produced, with a much contribution to the advance- 
ment of the evolution. 

This state of affairs would possibly result when protein structures 
of nucleic acid-rich particles of primeval organisms were differentiated 
to some extent. With certain bacteria the presence of such a differ- 
entiated structure is actually confirmed. As was already described, 
if two different viruses, A and B, affect simultaneously a strain of 
bacteria, sometimes a new virus is produced which has peculiarities 
of both A and B. Instead of two kinds of viruses, two types of 
bacteria yield the same result; the conjugation of two strains of 
bacteria occasionally produces a new strain of bacteria which have 
the characteristics of both strains. 

Thus Lederberg and Tatum (77) (78) have actually demonstrated 
such a sexual reproduction of bacteria. They have made use of 
biochemical mutant strain which are unable to synthesize certain 
essential substances and which consequently can grow only on media 
to which those substances have been added as nutrients. For example, 
one strain used was unable to synthesize biotin and methionine, ano- 
ther prolin and threonine. When two such double mutants are grown 
in mixtures in minimal medium, lacking the nutrients above mention- 
ed, there appear appreciable numbers of structural recombinations 
which can synthesize all the enumerated substances and consequently 
survive. Lederberg has also made use of strains which differ in 
the ability or inability to ferment lactose, and in resistance or suscepti- 
bility to a specific bacterial viruses. 

In this case also protoplasm particles can substitute the bacterial 
cells as has been demonstrated by Hayes (79) who has found that 
sexual conjugation of bacteria can be achieved by a virus-like agent. 
These particles, therefore, should be regarded as primitive gametes. 
However, both individuals may be unable to make use of such particles 
for their sexual reproduction, since the multiplication of the structure 
fails to be accomplished by the combination of two particles only, and 
so for the multiplication, one of them should be present in a cell or 
at least should be present in combining with large amount of 


VII. SEXUAL REPRODUCTION AND REJUVENESCENCE 269 


cytoplasm into which the new structure is to be spread. For this 
reason sex had to be differentiated; the particles, active gametes, 
which are liberated from the one are called male gametes, or sperms, 
and produced in much greater numbers than larger female gametes, 
or egg cells, which are provided with great quantities of cytoplasm. 
In the case of the structural recombination of two viruses above 
cited, there are host cells in which the new structure can multiply; 
without host cells new structure, of course, cannot multiply. 

If a weaker individual is devoured completely, its structure will 
be of no use, but if a part of the structure can be intermixed with 
the structure of the stronger individual, some character of the weaker 
one can remain in the combined structure to make the newly formed 
individual more fitted for existence; the part of the structure which 
can remain in the newly formed individual must be strong and so 
may give rise to a distinct character. Therefore, the individuals of 
assimilase or primeval primary organisms, which had succeeded in 
obtaining the faculty for the sexual reproduction would accomplish 
much more rapid progress in their evolution than those lacking the 
faculty, and consequently would become the fittest with the result 
that at present higher organisms without exception perform sexual 
reproduction. In short, the first aim of the conjugation may be the 
rejuvenation, but the yield of new structures may be another import- 
ant aim. 

It is true, however, that sexual reproduction is very troublesome, 
and the easiest and simplest way must be the reproduction by fission 
without sexual conjugation. This must be the reason why certain 
organisms choose the alternation of generation, or metagenesis, chara- 
cteristic of plants, also occurring among a few animals. It involves 
an alternation of a sexually-reproducing with an asexually-reproducing 
generation; in this latter generation vigorous multiplication occurs 
because of its easiness, while in the former generation rejuvenation 
may take place with best chances to form new individuals more fitted 
for existence, although the multiplication in this generation is not so 
easily done as in the asexual generation. 

Such a mode of multiplication must have been highly developed 
presumably because it was the best way to become the fittest for 
some plants and animals, whereas it must have been inconvenient for 
some other organisms exhibiting certain other forms and functions 
and therefore these organisms achieve Sexual reproduction only. 

Microorganisms, such aS paramecia or other certain protozoa, 
which multiply usually by binary fission and have not yet acquired 
the undoubted, distinct mode of metagenesis, appear to accomplish 
rejuvenation by performing now and then an irregular conjugation. 


70 IV. THE PRINCIPLES OF LIFE PHENOMENA 


It is said that paramecium and other protozoa after a few hundred 
divisions apparently die out from senescence, unless conjugation takes 
place. Ciliate, uvoleptus, after some two hundred or more divisions, 
in the absence of conjugation, the division rate slows down and the 
individuals pass into a decline followed by structural degeneration 
and death. . 

Since the organisms that cannot accomplish rejuvenation by means 
of conjugation must undergo senescence and be perished, the indivi- 
duals having the property to perform conjugation can solely continue 
their existence. Individuals having the property to dislike the con- 
jugation would certainly fail to produce their offspring. Therefore, 
the sexual desire has developed amazingly in the present day or- 
ganisms, and has become the instinct for the race preservation. 

The somatic cells of higher organisms continuously perform cell 
division throughout the developmental process of the individual, and 
accordingly the division is comparable to asexual reproduction of 
microorganisms, so that it may be expected that the cells may under- 
go a senescence. This seems, however, not to be the case, for there 
is a mechanism by which the cell is rejuvenated at every cell 
division, in which is involved the disappearance of the nucleus as a 
definite body, whereby the genes get into contact with the cytoplasm 
whose pattern has been deformed by the life activity during the 
inter-division phase, and the deformed pattern is to be adjusted to the 
original, normal pattern by the genes. This adjustment to the normal 
pattern must be the rejuvenation itself. Although somatic cells can 
be rejuvenated in such a way at each cell division, the individual 
itself, as a whole, cannot avoid a decade, the cause of which, how- 
ever, should. be explain-d from another point of view as will be 
considered later. 

It should be noted here that in some strains of paramecium con- 
jugation seems to be unnecessary. In such strains, a process of nu- 
clear reorganization, termed endomixis, occurs every forty or fifty 
generations, in which the old macronucleus disintegrates and the 
micronucleus divides and redivides as if in preparation for conjugation. 
Then, there is the same disappearance of micronuclear material, and 
from the single micronucleus remaining new macro- and micronuclei 
are formed. This may be regarded as an extremely advanced feature 
of rejuvenation mechanisms which is seen in the mitosis of somatic 
cells. 

The cyanophyta or blue-green algae are undoubtedly the most 
primitive of all green plants, and no evidence of sexual process has 
been observed in these algae, reproduction apparently occurring ex- 
clusively by means of simple fission. If so, since they have no para- 


VII. SEXUAL REPRODUCTION AND REJUVENESCENCE Zr 


sitic nature, they may have proper rejuvenation means like this para- 
mecium. 

In addition, certain organisms are apparently able to accomplish 
rejuvenation to a certain extent merely by the change of environmen- 
tal condition under which they grow. According to our finding (80), 
when cultivated in broth for successive generations, typhoid bacilli 
will be reduced in virulence, changed to R-type, and their resistance 
to heat is diminished, becoming liable to die out, whereas when the 
bacteria declined in such a way are transferred on agar-plate, they 
will be changed into S-type, whereby their virulence is enhanced and 
the resistance is increased. However, successive cultivation on the 
same agar-plates also lead to a decline in the virulence. The sole 
change in the temperature at which the bacteria are cultivated has a 
similar effect, the virulence tending to increase when the temperature 
is elevated over 37°C. 

The development of ascaris is accomplished when the eggs are 
exposed to low temperatures outside the host body, a fact which may 
also be an example that rejuvenation is established by the change of 
environment without contact with another individuals. 

It can by no means be considered, however, that such a rejuve- 
nation is perfect and complete. As is generally known, the enhance- 
ment in the virulence of bacteria, which have been decayed by the 
continuous cultivation under the same environmental condition, is 
accomplished usually by passage through proper animals, a phenome- 
non which must be analogous to the sexual conjugation as we shall 
see in the following section. 


2. Rejuvenescence of Microorganisms by Making Use of the 
Sexual Reproduction of the Host 


As seen in the preceding Chapter, the deformation in the pattern 
of viruses may arise if they continue to multiply in the protoplasm 
of the same kind of organisms. This deformation will be repaired by 
the host change with the achievement of the rejuvenation of the 
virus. It is considered that the host change answers not only the 
purpose of rejuvenation, but also the purpose of producing new 
species, since viruses through the host change may be able to get 
possession of the new protoplasm structure of the new host. The 
host change, therefore, is comparable to the sexual reproduction. 

In many parasitic protozoa, for example, the parasitic amoebae and 
the intestinal and blood flagellates, no sexual process has been obser- 


22 IV. THE PRINCIPLES OF LIFE-PHENOMENA 


ved with certainty. Protozoan cells tend to grow old after continued 
asexual multiplication and lose both their youthful vitality and repro- 
ductive power. The habitude of host change, like in viruses, is 
common among these protozoa, and therefore it seems possible that 
the host change can substitute the sexual reproduction in these lower 
animals. However, the fact that the faculty for sexual reproduction 
is fairly developed in certain parasitic protozoa, such as malaria 
plasmodium, indicates that the sexual process is much more advan- 
tageous for their evolution than the host change. 

As already discussed in Chapter X in Part III, besides the re- 
juvenation by the host change, viruses can accomplish rejuvenation 
by taking advantage of the sexual reproduction of the host, that is, 
viruses penetrate into germ cells of the host and are rejuvenated there 
together with the germ cells through the fertilization. As we shall 
see in greater detail in Part V, germ cells of higher organisms are 
considered to have the undeveloped structure of the single celled 
creatures from which the organisms have been evolved. In other 
words, germ cells may be produced by the reduction of the developed 
structure of the somatic cells to their undeveloped pattern. Mean- 
while, the pattern of a virus itself, engraved in the mother germ cell 
of the host, will be also altered during the reduction of the cell to 
the germ cell, and hence, in the germ cell, the altered pattern of the - 
virus cannot behave as the virus. However, the pattern will recover 
its original form with virus activity, when the germ cell gradually 
recovers its developed original structure in the developmental process 
following the rejuvenescence by the fertilization. Such a rejuvenation 
of viruses may be quite perfect, because it is brought about together 
with the complete rejuvenation of higher organisms. 

The stronger the structure of a virus, the longer period of time 
it will continue to exist, so that the viruses will become the stronger 
as they evolve the higher. Consequently, viruses may naturally ac- 
quire the faculty to engrave their pattern even into germ cells as 
they evolve to a certain extent; thus they may be rejuvenated to 
continue their existence still further. 

A virus, which can already be rejuvenated by a host change, may 
still further be rejuvenated by penetrating into germ cells of the host. 
Certain plant viruses may be able to enter the germ cells of the host 
plant, while at the same time they may even engrave their pattern 
into the germ cells of their insect vector. This may hold true also for 
animal viruses. 

Viruses, unable to change the host, will likewise acquire the ability 
to engrave their pattern into germ cells of the host, when their 
pattern is strengthened to a certain extent. Therefore, the rejuve- 


VI. FACTORS INTERFERING WITH GENES 273 


nation by means of the sexual reproduction of the host appears to be 
easily attainable for the viruses and accordingly to be most conven- 
tional for them to continue their existence. 

As for the viruses affecting man, most of them can infect solely 
some particular individuals, though some of them, such as those of 
measles and mumps, are capable of affecting almost all the individuals. 
These latter viruses seems to be able to engrave their pattern into 
the germ cells of almost all the human beings, whereas some other 
viruses appear to do so only in particular persons who have the 
predisposition liable to be afflicted by them. The pattern of the 
viruses will be transmitted to the children of such peculiar persons 
affected by the viruses, and the pattern will be developed into its full 
form when the children grow up to certain ages, leading them to the 
disease or rendering them virus-carriers, who will subsequently scatter 
about the viruses. Some of the viruses thus scattered about may 
affect individuals having the peculiar predisposition, and through such 
individuals they can be rejuvenated and transmitted to offspring. The 
viruses must be extinguished if there are no such predisposed per- 
sons. 

Even in the case of measles, about 5 per cent of man are not 
infected with the virus for life. Such individuals must be completely 
freed from the pattern of the virus, since the virus cannot enter their 
germ cells. Measles would be expelled from this globe if all the 
human beings had such a peculiar character. 

In addition, climatic or topographical conditions may play a con- 
siderable role in the development of virus diseases. Persons living in 
a certain region may have a peculiar disposition according to the 
climatic factors in the region and may be liable to be afflicted by 
some virus which cannot, or scarcely, infect the man living in other 
regions. In such a case the virus disease may be called an endemic 
disease, the pattern of the virus in question being engraved only in 
the germ cells of man living in such a region. This may also hold 
for animals other than man, and so it may occur that certain insects 
or other animals in a certain region always prove to be carrying a 
virus peculiar to the region. 

The development, ina child, of the full pattern of the virus which 
is transmitted from parent appears to be effected mainly by the age 
of the child. However, many other factors are apparently involved 
in the development. It is a remarkable fact, as already discussed, 
that leaf hoppers carrying aster yellow virus lose the virus if exposed 
to a temperature of 32°C. (81). This fact can be interpreted as indi- 
cating that the structure of the virus cannot develop at 32°C. 

The incidence of measles changes regularly with the change of 


274 IV. THE PRINCIPLES OF LIFE PHENOMENA 


season as described in Chapter III in Part III. This may be elucidated 
by considering that the pattern of measles develops in some seasons 
while disappears in other seasons. It seems probable that immature 
pattern of a virus present in some organisms cannot develop to a full 
form unless proper conditions are provided. Phage can be detected in 
chicken feces in warm seasons only, especially in summer, as stated 
already, a fact which suggests that the immature pattern of phage in 
chicken cells develops its complete pattern in summer. Even the virus 
pattern present in lysogenic bacteria can develop into complete form 
only when adequate conditions are provided (82). 

In view of these facts it seems difficult to distinguish a newly 
generated virus from a virus inherited from parent. The discrimi- 
nation of the two forms, however, may not be impossible, as in general 
in contrast to fixed, advanced viruses, the structure of newly generated 
ones must be weak and accordingly they are unable to give rise toa 
long-lasting immunity, that is, they cannot exist for long periods of 
time in host cells, not to speak of the transmission to progeny. Fur- 
thermore, newly generated viruses may be liable to undergo variations 
because of their weak structure, so that their immunological specifi- 
city is inconstant, whereas the pattern of fixed viruses is generally 
invariable, being transmissible unchanged in hosts from generation to 
generation. 

Herpes simplex virus appears to be generated by various factors, 
especially by febrile diseases; the disease is said to occur also after 
artificial pyrotherapy. It seems reasonable, however, to regard this 
virus as a typical fixed virus. Burnet (83) has claimed that the most 
individuals acquire herpes virus in childhood, as in the case of meas- 
les, and carry it for the rest of their lives. After the first infection 
with this virus, individuals develop neutralizing antibodies in their 
serum which remain present for many years. Persons who have been 
infected with this virus can thus be detected by serum neutralization 
test, indicating the constancy of its antigenical specificity as well as 
the long duration of its existence in the host. The causative agent, 
such as fever, may therefore aid only the virus pattern inherited 
from the parent to develop into its complete structure. 


CHAPTER VIII 


REJUVENESCENCE OF MICROORGANISMS 
WITHOUT SEXUAL REPRODUCTION 


1. The Microorganisms Parasitic on Insects 


If viruses can accomplish their rejuvenation in such a remarkable 
way aS above described, it should be expected that the secondary or- 
ganisms evolved from viruses may apply the same tool for their 
rejuvenation... 

It is generally accepted that most of pathogenic Rickettsiae are 
inherited in arthropod vectors from generation to generation. It is 
unreasonable to consider that the secondary organisms choose the 
virus-like rejuvenation mechanism only up to the stage of Rickettsiae, 
and that the organisms evolved higher than Rickettsiae all abandon 
the mechanism. According to the writer’s view the microorganisms, 
such as bacteria and protozoa, which perform no distinct sexual process 
are still succeeding to this remarkable habitude of their ancestors. 

This view is strongly supported by the fact that in a large number 
of insect species, a special structure or organ is present, called my- 
cetome, the principle function of which appears to be that of housing 
various microorganisms, some of which are of fungous nature while 
others are called bacteroid. Most of these organisms cannot be culti- 
vated outside the insect body, only capable of proliferating in the 
host. 

These microorganisms are looked upon as symbiotes, and in some 
cases the symbiotes apparently furnish their hosts with essential 
substances, such as vitamines, which are lacking in their regular 
diet ; in some other cases they supply hormone-like substances which 
aid in the development of the ovaries. It appears quite possible that 
the symbiotes of many insects are capable of fixing atmospheric 
nitrogen. Certain insects cannot maintain their lives if the mycetome 
is mechanically removed, or the symbiotes are killed by drugs, such 
as penicillin, or by maintaining the insects for a prolonged period at 
higher temperatures (84). 

Thus the mycetome, an aggregate of microorganisms, is regarded 
as an indispensable organ for the insects, and consequently the organ 
together with the microorganism are inherited from generation to 


276 IV. THE PRINCIPLES OF LIFE PHENOMENA 


generation through the eggs. The character to produce the micro- 
organisms, therefore, must be the inheritable character essential for 
the insects. This fact strongly suggests that certain fungi and 
bacteria are still now succeeding to the habitude of viruses, their 
ancestors, to be rejuvenated. 

Leach (85) has already expressed. the opinion that insects are not 
merely disseminators of inoculum in the case of pathogenic fungi, but 
that the insect-fungi relationship is highly organized and has broad 
biological and evolutionary significance. The fact that over 200 kinds 
of bacteria have been isolated from insects may show what a great 
number of bacteria are making use of insects for their rejuvenation. 
As we shall see later, spirochaetes are also regarded as microorganisms 
succeeding to this habitude; meanwhile about 20 kinds of them have 
been found in insects. 

Many examples are known, as pointed out already, to show.that 
insects are not afflicted by viruses harbouring in them, in some cases 
the relationship between them being regarded even as a symbiosis. 
Such a relationship appears to be kept on even after the viruses have 
evolved into undoubted microorganisms. It cannot be said, however, 
that insects are always not afflicted by microorganisms. Numerous 
infectious diseases due to bacteria or fungi are well known. 


2. The Rejuvenescence of Tubercle Bacillus 


As already dealt with, a phenomenon related to sexual conjugation 
is occasionally observed with colon bacillus, but it cannot be considered 
that the bacillus is provided with the distinct habitude to be rejuve- 
nated regularly by the sexual process. The observed phenomenon 
should be looked upon as only an extremely primitive form of the 
sexual conjugation, and presumably the bacillus is usually not re- 
juvenated by such a process. 

Here again, it seems probable that this bacillus is also resorting 
to the means of rejuvenation which their ancestors used habitually. 
It has been reported that the normal human individual harbours two 
types of colon bacillus, residents and transients, 7. @., persistent over 
long periods strain and limited to a few days strain, the former con- 
tains typically one or two strains (86). This pattern of bowell bacilli 
is shown by very young babies as well as adults. Babies as intimately 
associated as twins harbour the same resident. 

Colon bacillus, however, may not be so adequate as tubercle 
bacillus to be dealt with concerning this subject. Almost all the 
human beings are infected with tubercle bacillus as with measles 


VIII REJUVENESCENCE OF MICROORGANISMS 277 


virus. The manner of establishment of the infection seems likewise 
very similar to that of measles; new born babies are scarcely infected, 
but the incidence increases rapidly with their growth until almost 
all of them are affected. This fact is usually explained to be due to 
the increase in the chance of infection as infants grow older, whereas 
it may also be possible to regard this as analogous to the phenomenon 
already discussed that larvae hatched from eggs of insects which 
were infected by a certain plant virus produce no virus when very 
young, but that as grow older they become infectious. As we shall 
see in the following, there are many good reasons to consider this 
interpretation to be much more reasonable than the former. 

The concept that the tuberculosis is produced only by the infection 
from outside may be confused by the so-called Liibeck tragedy, in 
which 251 new born infants instead of the avirulent bovine bacillus 
BCG, a living human virulent strain was accidentally substituted in 
the laboratory. The bacilli were given by mouth in so large an 
amount as 400,000,000 microorganisms, on three different occasions 
during the first ten days of life, with the result that 75 of them dead 
and 175 were living, eacaped death or even progressive tuberculosis. 
Moveover, in a study of children in tuberculous families, Puffer (87) 
has shown that of the contacts with sputum-positive parents infection 
came earlier than those of sputum-negative parents, but, by the time 
to come of ages of fifty, contact and non-contact cases with positive 
heredity showed the same percentage of infection. 

According to Pottenger (88), the fact that the apex is not particu- 
larly susceptible in first infection, which so commonly takes place in 
childhood, but shows a particular predilection for reinfection in the 
adult, indicates that something occurs in the time between childhood 
and adult life which changes the localization of the bacilli; he stated 
that it might further indicate that the source of the bacilli and the 
route of invasion might be different. Such a phenomenon should be 
only natural if the bacilli are produced in human bodies. 

For a long time it was known that tubercle bacillus can exist in 
a virus-like filtrable form, and numerous workers have claimed that 
this filtrable form constitutes a special stage in the life-cycle of the 
bacillus. This fact strongly supports the above concept, since, if the 
bacillus is transmitted by human germ cells, it should be reduced, at 
least during the transmission, into the form of a virus, from which 
subsequently bacillary form develops. Moreover, tubercle bacillus is 
known to be one of the acid-fast bacteria, but in young cultures it is 
common to find a certain proportion of non-acid-fast forms. In view 
of these facts numerous workers believe that the life-cycle of the 
bacillus consists in that the virus-like particles develop into acid-fast 


278 IV. THE PRINCIPLES OF LIFE PHENOMENA 


bacilli through the stage of non-acid-fast forms. 

If tubercle bacillus can recover its complete form from such a 
virus-like particle, its penetration into germ cell like a genuine virus 
may be possible. To speak more precisely, the pattern of the bacillus, 
like that of a virus, may be engraved in the mother germ cell of 
man, and the pattern may be transmitted to the germ cell in a reduced 
form as in the case of a virus; as the pattern exists in the germ cell 
in this reduced structure, the original pattern of the tubercle bacillus 
cannot be proved in it, but when the germ cell develops to a human 
individual; the reduced pattern of the bacillus also develops to the 
complete, original pattern of virus-like particle having the original 
pattern, from which the acid-fast bacillus may be formed. The first 
infection may thus be established. The character Of the bacillus thus 
developed may change further with the further development of the 
host individual, with the diminution in the virulence which may again 
increase afterwards to cause the reinfection. 

The percentage of children of varying ages who have become 
tuberculin-test positive usually varies with regions, suggesting that 
the period of the development of the complete pattern varies according 
to the regions. In view of the climatic or topographical effect which 
may vary with regions, it seems natural that the disposition of the 
children also varies with them, resulting in the difference in the 
period of the development of the pattern. The infection from outside 
cannot be disregarded, but most of the first infections may result 
from the development of the pattern in the children as in the case of 
measles. 

At any rate, tubercle bacilli, like microorganisms in insects re- 
ferred to in the precedirg section, appear to be entirely involved in 
the host, z. é., in human beings, with which they can be rejuvenated. 
The consumptive is born to a consumptive said Hypocrates. This 
must be the truth. It can even be said that tubercle bacillus is an 
inheritable pattern of the gene in the majority of human beings. The 
reason why such a mention can be made will become gradually 
evident as the description proceeds, especially in the next Part. 

Since tubercle bacilli are connected with the mankind in such a 
firm way, if any alteration occurs in the latter, the former will be 
changed together with it. Some authorities regard tuberculosis as an 
epidemic disease with epidemic waves; the last one, having reached 
its peak about 70 years ago, is now declining. With the exception of 
war times, there has been an almost universal decline in the mortality 
of tuberculosis throughout the world (89). The writer is of the opinion 
that this may be attributable to the wave of the change in human 
disposition ; it should be a very remarkable fact that human stature 


VIII REJUVENESCENCE OF MICROORGANISMS 279 


has meanwhile shown the tendency to increase steadily with the 
decline of tuberculosis. This very important and interesting problem 
will be discussed in most detail also in the next Part. 


3. The Significance of Filtrable Forms of Bacteria 


It seems probable that almost all the parasitic bacteria are being 
rejuvenated in the same way as tubercle bacilli, although the degree 
of the connection with the host may vary greatly with the species of 
bacteria. For example, the connection appears to be particularly firm 
in lepra as well as in tuberculosis, while in other diseases such as 
typhoid and dysentery it may be loose. 

The filtrable form has been known and discussed for the longest 
time with tubercle bacillus. But the presence of related forms appears 
to be universal with bacteria, supporting the above idea that bacteria 
are generally inherited through germ cells of the host. It has long 
been known that .dysentery bacillus can restore its full form even 
after decomposed into filtrable particles; that is, viable dysentery 
bacillus can be formed from lysed filtrate of the bacteria by phage 
(90). It is said that newly generated bacteria are phage resistant, 
which must be expected from the theory of the writer as already 
referred to. Again, Kasahara ef al. (91) have found with many kinds 
of bacteria that original forms are actually produced from the filtrate 
of bacteria decomposed by ultra-sonic vibration. 

On the other hand, Klieneberger (92) isolated from cultures of 
Strepto-bacillus miliformis a minute organism which she designated as 
L-form. This resembles the virus-like organism of bovine pleuropneu- 
monia. They are isolated from many other bacterial species, and 
transformation into this virus-like forms is brought about apparently 
always by an injury to the bacteria, especially when treated with 
penicillin. Diens (93) is of the opinion that these virus-like organisms 
are part of a life-cycle not only in the streptobacillus, but in other 
bacterial species. In fact, he has succeeded in causing their appear- 
ance in cultures of E. coli, Haemophilis influenzae, Flavobacterium, the 
gonococcus and F. funduliformis. Some workers believed at least some 
of these L-forms to be analogous to the so-called fitrable forms, but 
some others appear to hold the opinion that these two forms are 
different (94) (95). It has been reported that in penicillin-containing 
media P. vulgaris showed L-type growth. It is a note-worthy fact 
that L-forms thus produced could be changed into bacillary forms 
when transplanted to penicillin-free media or passed intraperitoneally 
through mice; the L-form appeared to be anaerobic and non-pathogenic 


280 IV’ THE PRINCIPLES OF LIFE PHENOMENA 


for mice (95a). 

At any rate, there seems no doubt that bacteria can recover their 
normal, complete form even after decomposed into smaller, virus-like 
particles. According to Dienes and Zamecnik (96) the presence of 
high concentrations of glycine in the media induces the transformation 
of some bacteria into L-form, glycin being the most effective among 
a variety of amino acids, although very few bacteriostatic chemicals 
induce the transformation. It is interesting that only one toxic bacte- 
riostatic chemicals among many examined produced L-form, while 
three amino acids which are necessary constituents of the cell pro- 
duces L-form when they were present in high concentrations. This 
fact suggests that the L-form transformation is never a mere mecha- 
nical decomposition of bacterial bodies. but may be associated with a 
peculiar structural transformation like that in spore formation. 

There is little doubt that sporogenesis is associated with consider- 
able changes in protoplasm structure. Hardwick and Foster (97) have 
found that seventeen different enzymatic systems, abundantly demon- 
strable in vegetative cellular extracts of B. mycoides and other aerobic 
sporeforming bacilli, could not be detected in spore extracts tested 
concomitantly. ‘They regarded this fact as supporting their hypothesis 
that sporogenesis occurs at the expense of enzyme proteins preexisting 
in the vegetative cell. However, as stated already, any structural 
change in protoplasm should be accompanied by some change in enzy- 
matic system, and hence this fact must only be indicating the oc- 
currence of the structural change in the protoplasm. It is considered 
that the change leads to a primitive structure of the protoplasm, 
whose return to the original structure is to be accompanied by the 
recovery of the vegetative form, an important concept which will be 
discussed in great detail in Part V. 

In order to accomplish the rejuvenation in germ cells of the host, 
bacteria must exist in a virus-like state at least at the early stage 
of the development of the host, not to speak when present in the 
germ cells. In virus-like state at such a stage, they may be unable 
to exhibit the function as bacteria even when incubated iz vitro in 
much the same way as that the germ cells of higher organisms 
cannot develop into the adult 7” vitro. Therefore, failure of demon- 
stration of the filtrable forms may not necessarily disprove the pos- 
sibility of the bacteria to be transmitted by the host germ cells. 
Nevertheless, as above stated numerous authors believe that the 
various bacteria involve a virus-like stage, supporting strongly the 
above concept. 

Almost all the human individuals seem to be utilized by tubercle 
bacillus. However, bacteria, like viruses, may be unable to utilize all 


VIII REJUVENESCENCE OF MICROORGANISMS 281 


the host individuals for their rejuvenation. Some individuals peculi- 
arly susceptible to a certain kind of bacteria may be infected and 
utilized by them to establish the inheritance of their pattern to the 
offspring of the host, in which bacteria will subsequently develop and 
the offspring will be infected or become the bacterial carrier. 

As in the case of viruses, the susceptibility of the host may vary 
with the regions where the host resides, leading to the establishment 
of endemic diseases. Namely, in a certain region there may live 
animals or man possessing extremely high susceptibility to a certain 
kind of bacteria, which the latter therefore can continue their existence 
particularly in such a region. In India cholera is almost always 
prevalent. The true home of cholera is said to be the delta of the 
Ganges. It spreads occasionally widely from this area, but in other 
areas it usually dies out, though it may be reimplanted from time to 
time. This may be accounted for by the non-existence, in areas other 
than India, of hosts in which the bacteria can harbour. 

In addition to bacteria, a kind of yeasts (Schizosaccharomycetes 
filtrans) and of protozoa (Trypanosoma Brucei) are known to become 
filtrable at a period of their life-cycle. Moreover, spirochaetes have 
been reported to be able to exist in filtrable forms. According to 
Nicolle (98), spirochaetes go’-into a filtrable granular stage produced 
by fragmentation of the adult forms, and in the cycle of spirochaetal 
development a phase seems to occur in which the organism persists 
in the form of small granules. He considered that this form is ap- 
parently resistant and latent, becoming infective when it regenerates 
spirochaetes. Syphilis is caused by a kind of spirochaetes, Treponema: 
pallidum. It is a well known fact that the disease is transmitted 
from parent to infant; it should be noted that some authors believe 
that the transmission is established not only by mother but also by 
father. Numberous viruses have been confirmed to be transmitted 
through germ cells of the host, but in the majority of cases only egg 
cells are believed to be involved. Nevertheless, in the case of mam- 
mary cancer. of mice, which is generally recognized as caused by a 
virus, it has been well established that males of cancer strains of 
mice may transfer the agent through the sperm (99) (100). Treponema 
pallidum may be transmitted in a similar way to the offspring. 

As stated already, fungi in the insect are undoubtedly transferred 
through eggs from generation to generation, whereas many grass 
smuts are known to be seed-born; that is, the pathogenic fungi are 
carried by the seeds to progeny (101). The mechanical transport of 
the fungous spores by seeds may not be an adequate explanation of 
this fact. 

It is worthy of note that in the gut of the termite, protozoa occur 


282 IV. THE PRINCIPLES OF LIFE PHENOMENA 


in so tremendous numbers that the protozoa constitute as much as 
one third of the weight of the termite (102). The protozoa is de- 
finitely benefitable for the termite and without which the insect can- 
not continue to live, suggesting that the termite harbours the protozoa 
as a heritable pattern of the gene. 

As already referred to, phage is found in tissue cells of various 
animals, such as chickens, mice, flies, etc. It may not be impossible 
to regard such phages as the virus stage of bacteria parasitic on the 
animals. The appearance of such organ phages is under the influence 
of season as in the case of the virus of measles. Vincent (103) has 
studied the flora of the small intestine of normal and X-radiated rats 
and found that the coli-form and pathogenic staphylococcus increased 
markedly following irradiation. This effect of irradiation was ob- 
served not only with the bacteria in the intestine but with those in 
blood. Thus, bacteriaemia of enteric origin has been found to occur 
in a high percentage of mice following total-body X-irradiation (103a). 
This may be interpreted as indicating the revelation of the pattern by 
the irradiation. It should be remembered in this connection that 
active phage is readily revealed in lysogenic bacteria following ultra- 
violet irradiation (82). 

In order to utilize the host effectively for the purpose of rejuve- 
nation, microorganisms should not exert too great detrimental effect 
upon the host, and so mostly they may establish a commensal relation- 
ship with the host. This may be the case also with plants; it is 
well known that various non-pathogenic bacteria exist within plants, 
remaining viable and possibly multiplying, but not producing any 
visible disease symptoms (104). 

The writer’s concept discussed so far may seem erratic to some 
authorities. However, no one can doubt the fact that in our bodies 
there are normally produced cells named leucocytes which behave like 
protozoa able to move like amoeba and eat various substances which 
are injurious to us. Moreover, when a man develops to a certain 
extent, cells capable of acting as the gametes which can be regarded 
as an independent unicellular organism are raised in him. It is 
evident that these agents are not brought about by an infection from 
outside, although they have each immunological specificity like par- 
asitic microorganisms. No one can deny the opinion that they should 
be attributed to respective heritable patterns of the gene. 

A pathological agent belonging to this category may be cancer. 
When a man having the predisposition to cancer grows up to a cer- 
tain age, occasionally a strange cell, termed a cancer cell, is produced 
which behaves like an independent microorganism, multiplying vigor- 
ously at the sacrifice of the normal cells until even the host itself is 


VIII REJUVENESCENCE OF MICROORGANISMS 283 


killed. It has actually been known that the mammary cancer of mice 
is induced by a virus-like agent which can be transmitted from parent 
to offspring through germ cells; that is, the predisposition to the 
cancer is inheritable. 

The writer is of the firm opinion that at least the majority of the 
primitive secondary organisms can accomplish their rejuvenation by 
becoming thus patterns of the gene of their hosts, but it seems reason- 
able to suppose that they must abandon such means as they advance 
higher in the course of the evolution. As above considered, primitive 
secondary organisms such as bacteria, fungi, and perhaps protozoa 
can apparently invade the germ cells, but when they evolved into 
higher multicellular organisms, their structure might become too 
complicated to be reduced into a virus state with the result that 
they would become unable to make use of the host germ cells. 


CHAPTER IX 


METABOLISM 


1. Energy Requirement by Extremely Primitive Organisms 


The constant renewal of substances and energy is often believed 
to be the essential feature of life, and consequently life is sometimes 
compared to a fountain or a flame, which has a constant shape but 
whose component is being continuously renewed. 

Such a view, however, may be erroneous. Viruses exhibit every 
principal character of organisms, but there is no proof of metabolism 
involved in them; all the attempts by a number of workers to de- 
monstrate the metabolism in viruses have been failed. Furthermore, 
it seems possible to present the evidence that no significant energy 
may be required even for the multiplication of viruses. Thus, it is 
generaily recognized that viruses can multiply in dead cells. For 
example, aS already stated in Chapter VII, Part II, influenza virus 
can proliferate in chick embryos killed by the exposure to a low tem- 
perature, and phage multiplies in bacteria killed by various agents, 
such as penicillin, mustard-gas, and ultraviolet ray. 

In these cases, killing means to render them non-viable. For the 
proliferation of cells high metabolism of both substances and energy 
should be necessary, and accordingly viability may be lost by the 
destruction of the metabolic systems. The multiplication of viruses 
in the cells devoid of the reproducing faculty, therefore, may indicate 
that at least the energy so great as required for the multiplication of 
cells is not necessary for the rearrangement of protoplasm structure 
to answer the pattern of the virus. On the one hand, it is known 
that neither heat is produced nor energy is consumed when a protein 
is affected by proteolytic enzymes. On the other hand, multiplication 
of a virus may be looked upon only as the structural change of 
protoplasm protein by the action of a virus, which can be regarded 
as a Special type of enzymes. Even in this respect it seems also pro- 
bable that no peculiar energy is required for the multiplication of 
viruses. 

In this connection it should be mentioned that there are numerous 
facts suggesting that virus multiplication is closely connected with the 


IX. METABOLISM 285 


metabolic activity of host cells. For instance, as already described, 
phage is commonly produced in young, proliferating bacteria. Acker- 
mann (105) was able to confirm that the yield of influenza virus was 
apparently directly proportional to the residual oxygen consumption 
of embryonated chick tissu@s. These facts may only show that young, 
active cells possess protoplasm which is particularly suited for the 
virus multiplication. 

Ackermann and Johnson (106) found that 2,4-dinitrophenol inhibits 
completely the propagation of influenza virus in chorioallantoic mem- 
brane, while this reagent shows no virucidal effect zz vitro. In minced 
preparations of chorioallantoic membrane the reagent was shown to 
have a pronounced stimulating effect upon adenosine-triphosphatase, 
and when this reagent was used with intact tissues, an excellent cor- 
relation was found between the inhibition of viral propagation and 
the stimulus of respiration and release of phosphate. From these 
facts they concluded that the energy required for viral synthesis was 
derived from the oxidative phosphorylative activity of the host tissue. 
However, this conclusion might be somewhat rash, since it can also 
be considered that the effect of the reagent upon the protoplasm may 
be unfavourable for the production of the virus pattern though 
favourable for the enzymatic action. Peculiar influences of a variety 
of such reagents upon the production of virus pattern will be dis- 
cussed in detail in the next Part. 

On studying the development of phage in bacteria ‘‘killed’’ by 
ultraviolet or X-ray, Labaw eé al. (107) have found that although the 
phosphorus metabolism of the bacteria is greatly depressed by ‘‘kill- 
ing,’’ it will continue for a short time after the treatment, and it is 
during this time that phage can multiply; the yield of phage is large 
even for completely killed cultures, if infection occurs immediately 
after the treatment, but decreases with the time of incubation of the 
treated bacteria before infection. This fact, however, may not show 
that phosphorus metabolism is indispensable for the virus multipli- 
cation. It may be more reasonable to interpret this fact as indicating 
that for the virus multiplication the protoplasm structure could not be 
so severely damaged as phosphorus. metabolism was completely lost. 

The writer holds the opinion as discussed already that the prim- 
ary organisms were generated and evolved by the mechanism by which 
viruses multiply. If energy is required for this mechanism the theory 
of the writer cannot be held, because no metabolic system is con- 
sidered to have been present in the protoplasm-like masses in which 
the organisms might develop. The fact that no energy appears to be 
particularly needed for the action of the enzyme must be one of the 
main reasons for which life could be raised on this globe. 


286 IV. THE PRINCIPLES OF LIFE PHENOMENA 


As aleady stated, the primary organisms might have been generated 
and evolved by affecting other organisms or protoplasm-like polymerized 
proteins in rearranging their structure just as viruses multiply in the 
host cell. However, when such primitive primary organisms or the 
masses of assimilase evolved to a certaff extent, they might have to 
rearrange the structure of free protein molecules or to synthesize pro- 
tein molecules from smaller components to incorporate them into their 
bodies. If they evolved up to such a stage and were capable of syn- 
thesizing proteins, considerable quantities of energy would be neces- 
sary for the active and incessant structural change of protoplasm pro- 
tein as well as for this function, and the energy required would be 
supplied by the decomposition of high molecular substances which 
might have been either synthesized by utilizing the energy of the sun 
or taken from other organisms. 

Life may be nothing but the manifestation of the function of 
assimilase. If an assimilase was once generated and had to continue 
its existence, as a necessary consequence it would evolve and become 
to have complicated structure until the form and the function of 
undoubted organisms were obtained. It would be unable to become 
more fitted unless it was advancing continuously. Thus, assimilase 
might have to choose between the extinction and the advancement, 
and if the advancement reached to a certain extent the development 
of metabolic system would follow as a natural consequence. 

In both cases of the primary and the secondary organisms, be- 
fore they had enough evolved to synthesize proteins from smaller 
components, they might have to acquire the faculty to rearrange the 
pattern of free protein molecules, the structure of which was only 
slightly different from those of the organisms, in order to incorporate 
the molecules into them. It is well known that primitive secondary 
organisms, such as the organism of bovine pleuropneumonia, can 
sometimes multiply outside the host if blood-plasm proteins are added 
to the medium. Such organisms may be looked upon as having ac- 
quired this faculty. In addition, it seems possible that the cells of 
higher animals existing at present still adopt this easy way of as- 
similation. Thus it has been claimed that dogs can be kept in health 
when the only source of nitrogen is blood-plasm injected in their 
veins (108). The animal must, therefore, be able to form any required 
protein from the protein present in the plasm without preliminary 
hydrolysis by the enzymes of the digestive tract. Only a little energy, 
if any, may be required for such an easy assimilation procedure, in 
which are utilized complete protein molecules that have the structure 
not greatly different from that of the protein composing the assimilase 
itself. 


IX. METABOLISM 287 


However, if the protein which is to be utilized has the structure 
entirely dissimilar to the pattern of the assimilase or the protoplasm 
of the organisms, incorporation following the sole rearrangement may 
be impossible; preliminary decomposition appears to be needed in 
such a case. Presumably reconstruction of a protein from decomposed 
components may be accomplished with more ease than the rearrange- 
ment of the pattern without decomposition; the rearrangement may 
be impossible because the protein would be disturbed in its structure 
so profoundly on the adsorption onto the protoplasm that the decom- 
position would take place before the occurrence of the rearrangement, 
as the pattern of the protein differs so greatly from that of the pro- 
toplasm. 

Such a protein in its turn may exert some disturbing influence 
upon the protoplasm itself because of its foreign structure. This may 
be the reason why foreign proteins are generally toxic. The rapid 
decomposition of foreign proteins, therefore, may be needed even for 
the elimination of their toxic effect. Moreover, decompésed components 
would be favourable owing to their small molecular state to be 
transported to another part where the protein synthesis occurs. The 
function of the reconstruction of specific proteins from foreign ones 
through the decomposition appears to have already been developed in 
relative primitive organisms such as protozoa, since in a certain group 
of protozoa, for example in paramecium, primitive digestive system 
can be found. 

A considerable attention seems to be paid on the fact that for the 
incorporation in vivo and in vitro of any one amino acid into protein, 
other amino acids are necessary (109). For instance, it has been con- 
clusively shown that the essential amino acids are not able to sustain 
growth or even nitrogen balance in an animal when they are fed at 
intervals, whereas they are effective when fed simultaneously (110). 
This may be expected if each amino acid is to be adsorbed to the 
respective pattern of the protoplasm to form a complete peptide chain 
specific to the organisms. 

It has frequently been suggested’ that the most primevai organisms 
might utilize the sunlight energy by chlorophyll. The writer claims, 
however, that organisms never acquired the faculty to yield chloro- 
phyll until they evolved up to a considerably high stage. It is en- 
tirely inconceivable that primeval organism could synthesize an- 
enzyme so highly developed as chlorophyll. Nevertheless, it may be 
possible that primeval organisms could synthesize proteins from car- 
bon dioxide and ammonia or other N-compounds by making use of the 
sun energy, even long before they could yield chlorophyll, but because 
of the non-development of the perfect synthesizing system, the syn- 


288 IV. THE PRINCIPLES OF LIFE PHENOMENA 


thesis of the protein might occur extremely slowly, so that there 
would be no significant flow of energy. In this connection, it should 
be realized that bacteria can, in general, make use of carbon dioxide 
for their growth regardless of their lack of chlorophyll. 

Even before proteins which were synthesized in the primitive 
ocean could form polymerization product capable of acting as typical 
assimilase, carbon dioxide and some N-compounds like ammonia might 
be condensed into protein molecules or amino acids by the sun energy. 
The first proteins might be synthesized in this way, but the primeval 
organisms, entirely disregarding such a synthesizing process, would 
utilize the preexisted proteins, which had been already produced by 
such a tedious way, just as the viruses utilize the protoplasm proteins; 
however, as they evolved higher and higher and the protein structure 
became complicated and advanced, the synthesyzing mechanism being 
gradually arranged until highly differentiated enzyme system, such 
as chlorophyll, were developed with the manifestation of evident flow 
of the energy and substances. 

The organisms appearing to be primitive secondary ones, such as 
lower fungi and some protozoa, can produce chlorophyll to perform 
free-living despite their extreme primitiveness. This may be explained 
by assuming that these organisms have evolved from viruses which 
were parasitic on higher plants containing chlorophyll, succeeding to 
the highly developed enzyme system of the host, that is, chlorophyll, 
so that they might be able to accomplish free-living in utilizing the 
sun energy by means of chlorophyll despite of their most primitive 
nature. Some bacteria also can live independently of their host, sug- 
gesting that they might likewise obtain the enzyme system to utilize 
.carbon dioxide by transfer from the host. 


2. Nucleic Acid as Energy Donor 


The incorporation of various amino acids into protein may require 
considerable. amounts of. energy, the majority of which may be pro- 
duced by glycolysis whereby sugar. is decomposed with. the produ- 
ction of energy. The energy, however, is apparently not given directly 
in the form of heat, but it is preserved in the so-called energy-rich 
phosphate bonds which will be decomposed in case of necessary to 
liberate the energy. Most available energy-rich phosphate bonds are 
believed to be involved in adenosine triphosphate (ATP) which is one 
of the components of nucleic acids. . 

This may be regarded as one of the reasons why the cell prolife- 
ration is always connected with the increase in nucleic acid. The 


IX. METABOLISM 289 


active proliferation of cells should be associated with the vigorous. 
synthesis of proteins. Spiegelman and Kamen (111) have suggested 
that nucleoproteins are specific energy donors which make reactions 
possible leading to protein and enzyme synthesis. They. have found 
that when protein synthesis is occurring in the presence of nucleopro- 
tein containing P*, this latter element is liberated from the protein, . 
while on the interruption of the synthesis by the addition of sodium 
azide or dinitrophenol the liberation of P® is ceased, indicating the 
occurrence of the decomposition of nucleic acids solely in case of 
energy requirement. In addition, many facts are known suggesting 
that a certain correlation exists between the synthesis of proteins, 
particularly fibrous proteins, and the occurrence of phosphatase and 
nucleic acids both in the cytoplasm and in the nucleus (112).. Nucleic 
acid in the cytoplasmic granules is considered to be involved in 
protein synthesis, and it is known from the work of Brachet. and his 
colleagues (113) that these granules contain phosphatase. The tissue 
in which protein synthesis appears to be most active is also particularly 
rich in nuclear phosphatase. 

On the other hand, numerous synthetic reactions driven by an 
influence of energy-rich phosphate bonds are known. Peptide formation, 
the synthesis of urea, transmethylation from methionine, the syn- 
thesis of a— and (keto acids of fatty acids, and the process of 
bioluminescence appears to belong to this group (114). In most cases 
of such reactions ATP is apparently involved; and there are many 
evidences that ribonucleic acid is intimately connected with protein 
synthesis (29). 

The synthesis of proteins can be regarded as the growth of proto- 
plasm, a mixed crystal. Foreign proteins or various amino acids are 
adsorbed to the surface of the crystal or the protoplasm, and after 
the establishment of the rearrangement of amino acids or of polar 
groups in the protein molecules to answer the template pattern of the 
protoplasm, they may be incorporated into the crystal. Such must be 
the synthesis of proteins, whereby the growth of the protoplasm is 
achieved. 

It has been already stated in Part II, Chapter VIII, that nucleic 
acids possess the faculty to strengthen the pattern of the protoplasm. 
Haurowitz (26) claimed that the role of the nucleic acids is apparently 
to maintain the template protein film in the expanded state, a state 
in which proteins do not remain unless exposed to the stress of inter- 
face forces. In addition to such an expanding action, if any, nucleic 
acids may have the hardening action by which the structure of the 
polymerization product of proteins is made firm. 

Thus, the writer has come to possess the opinion that the role of 


290 IV. THE PRINCIPLES OF LIFE PHENOMENA 


nucleic acids in the protoplasm is firstly to strengthen its template 
pattern and secondarily to provide the energy for the protein syn- 
thesis. 

As is well recognized, APT is involved in the energy metabolism 
in the contraction of muscles. The muscle contraction, on the other 
hand, is believed to be caused by expanding and contracting of the 
component proteins, mainly a protein called actin. According to Szent- 
Gyorgyi (115) ATP acts to expand the muscle protein and in case of 
contraction ATP undergoes decomposition, yielding inorganic phosphate 
and energy. Munch-Petersen (116) likewise showed that ATP expands 
monolayers of myosin, another chief muscle protein. Mommaerts (117) 
claimed that ATP is essential for the polymerization of actin. Poly- 
merized actin can be separated from accompanying protein by ultracen- 
trifugation. The separated actin, after dissolution and depolymerization 
in water, does not polymerize again upon addition of salt, unless ATP 
is present during the entire depolymerization process. He has further 
reported that in a pure system a stoichiometric reaction takes place 
between actin and ATP in which 1 mole of globular actin reacts with 
1 mole of ATP, yielding actin in the polymerized form with the 
liberation of energy and inorganic phosphate (118). These evidences 
clearly indicate that ATP, the chief component of nucleic acid, has 
the function to maintain the protein molecules in a definite form 
while it can provide energy on the decomposition. The nucleic acid 
in the protoplasm may behave in a similar way. 

Spicer and Rozsa (119) have found in an electron microscopic study 
that actomyosin tends to form fibrous aggregates in the presence of 
ATP. At a lower pH the tendency to form fibrous structures appears 
even in the absence of ATP, and needle-like or spindle-shaped aggre- 
gates are to be seen, but this tendency is intensified by the presence 
of ATP, showing that ATP contributes to the stretch of the actomyo- 
sin molecules. 

There are two kinds of nucleic acids, z. e., desoxyribonucleic acid 
(DNA) and ribonucleic acid (RNA), but it seems probable that only 
the latter is used as the energy donor, as it is confirmed that the 
nucleic acid which increases in case of active protein synthesis is 
RNA, not DNA. 

Histological and chemical studies on the distribution of RNA in 
different tissues and microspectrophotometric researches have led to 
the recognition of a constant coincidence in space and time of a high 
content of RNA and rapid protein formation. When protein formation 
or growth is taking place the RNA content is high, but when the 
same cells are not producing proteins, there is less RNA (29). 

Davidson and Raymond (112) fed labelled ammonium citrate to 


IX. METABOLISM 291 


pigions and rats, and found appreciable amount of N* in the RNA, 
subsequently isolated from the livers, but only negligible amount in 
the DNA. It has thus been generally ascertained that at least RNA 
is metabolically much active than DNA. Griffin ef al. (120) have 
even concluded that DNA is entirely inert. 

The nucleic acid existing in the gene which always requires the 
most strong pattern appears to be DNA, whereas as pointed out 
already DNA is much more stable than RNA. It may be said, there- 
fore, that DNA is fit for the establishment of strong pattern but unfit 
for the action as an energy donor for which some brittleness should 
be required. On the contrary, RNA is suitable for the purpose of 
providing energy owing to its brittleness although inferior to 
DNA in the action of pattern formation, so that while DNA is present 
in nucleus to provide the strong pattern, RNA exists in cytoplasm to 
deliver the pattern directed by the gene to proteins to be synthesized 
and also to provide the energy required for the synthesis. .Schénhei- 
mer (121) has shown that proteins are continuously being synthesized 
and hydrolysed so that it is quite possible that part of the energy for 
synthesis of proteins is obtained from the hydrolysis of other proteins 
or peptides. Such proteins may possibly be nucleoproteins containing 
RNA. : 

As already considered, in case of protein synthesis, component 
amino acids are to be adsorbed onto the template, which is probably 
involved in cytoplasmic particles containing RNA in rich amount, in 
adapting themselves to the specific pattern of the template, but since 
they have both amino and carboxylic groups in a free state prior to 
condensation into polypeptide chain, they may exert a disturbing 
effect upon the template. This disturbing effect may lead to the 
partial decomposition of RNA with the liberation of the high energy 
phosphate bond. Meanwhile, amino acids adsorbed may also be 
disturbed in their structure and as a result they are activated to be 
readily united into a polypeptide chain. 

It has been shown by using various isotopes that proteins com- 
posing animal tissues are constantly being renewed in a considerable 
speed. For example, liver tissue rapidly takes up amino acids follow- 
ing the injection or feeding, and the time for half replacement of the 
liver protein in the rat has been estimated to be about 7 days (122). 
Such a rapid renewal of tissue protein may partially be dependent 
upon the decomposition of the nucleoprotein as above mentioned, but 
there may be another important reason for the renewal. Since all the 
life phenomena involve the structural alteration of the protoplasm, 
the pattern of the protoplasm may be more or less deformed during 
the life process. As was mentioned earlier, the pattern thus deformed 


292 IV. THE PRINCIPLES OF LIFE PHENOMENA 


will be readjusted by the gene during mitosis, but it seems possible 
that part of the protoplasm or the proteins may be deformed too far 
to be repaired; such proteins must be expelled from the cell. This 
may be the other reason for the rapid exchange of the protein, which 
is known especilly manifest in tissues of active function. 

The infection with viruses «generally results in the nucleic acid 
increase in the cell as referred to already. Similar increase appears 
to be brought about by proper chemical or physical stimuli, followed 
by the ective synthesis of proteins. It is considered that an adequate 
stimulus applied. to protoplasm’may enable the protein to acquire an 
active state leading to the temporary liberation of polar groups, as 
may occur in general in the protein denaturation ; as a result capacity 
of combining with nucleic acids or the function of synthesizing nucleic 
acids may be enhanced. The enhancement in the action of phospha- 
tase is apparently associated with the increase in the amount of 
nucleic acids (112), suggesting that in the active state of protoplasm 
the polar groups acting as phosphatase are also liberated. Phospha- 
tase activity thus enhanced may be favourable for the liberation of 
energy. 

As mentioned previously, it seems necessary to hold constant in 
the protoplasm the amount ratio of nucleic acid to protein for the 
smooth establishment of life processes. If so, the increase in the 
protein amount will follow the increase in nucleic acid content in 
order to maintain the ratio contant. There is ample evidence that 
nucleic acid accumulation does occur before protein synthesis (29). In 
short, the increase in the amount of nucleic acid, ‘which may be 
caused by the temporary liberation of ‘polar groups in protplasm pro- 
teins following a stimulus, may lead to the enhancement of the 
template effect resulting in the active synthesis of protein, whereby 
the constant ratio of nucleic acid to protein is recovered, and thus the 
cells or the organism may be able to hold the state favourable for the 
continuance of their existence. 

There seems no doubt that the constant ratio of nucleic acids to 
proteins is indispensable for organism. Therefore the mechanism to. 
keep the ratio as constant as possible should be highly developed in 
organisms; and those failed to obtain the mechanism would surely 
become extinct. 

There appears no particular réason why mainly RNA is increased 
on the application of certain stimuli which may activate the protoplasm 
structure, but for the rapid synthesis of protein which alleviates the 
unbalance in the ratio of nucleic acid to protein, RNA must be more 
adequate than DNA. ‘Thus the accumulation of RNA would be 
favourable for the organisms if the organisms were brought under 


IX. METABOLISM 293 


some conditions which might act as a stimulus to activate the proto- 
plasm to accumulate or synthesize nucleic acid, and under which the 
vigorous multiplication was desirable for the organism. Therefore, the 
organisms which could accumulate RNA under such conditions might 
become fitter and left as such. 

As is well known, the nucleic acid which will accumulate in 
bacterial cell after the infection with phage is DNA; the RNA content 
of the host remaining constant. This may result in the relatively 
long persistence of the unbalance of the ratio, and at the same time 
this should be favourable for the liberated protoplasm particles to act 
as the virus, if not for the bacteria themselves to act as living or- 
ganisms. This property of bacteria to accumulate DNA on the virus 
infection might contribute not only to the virus multiplication but 
also to the evolution of the bacteria themselves, because the property 
to make the virus pattern rigid is to make the superior pattern of 
others spread easily. 


3. Dimensions of the Mass of Assimilase with Special 
Reference to Protein Synthesis 


The chemical analysis of the cytoplasmic particles, such as micro- 
somes and mitochondria, shows that they contain ribonucleic acid in 
rich amount, and it is evident that these particles account for a very 
large proportion of cytoplasmic ribonucleic acid. It has been suggested 
that protein synthesis is mainly connected with such particles, which 
are therefore recognized as the fundamental units of living organisms 
having the power of selfduplication (8) (26). 

It is evident, however, that each separate particle has no faculty 
of synthesizing protein and accordingly not selfduplicating, because 
as is generally accepted viruses cannot multiply outside the living 
cells. Nor any one has succeeded in making the cytoplasmic particles 
isolated from the cell multiply zm vitro. Even the ability for incor- 
poration of protein molecules is lacking in viruses, an ability which 
is not attained until they reach the stage of Rickettsiae. As already 
mentioned, a certain type of Rickettsiae can multiply outside the host 
if blood plasm is added to the media. 

This ability seems intimately related to the dimension of the 
mass of assimilase or of the microorganisms, for as discussed in Part 
I assimilase action is, to some extent, a function of the size of the 
assimilase or the degree of polymerization of proteins. For the func- 
tion of synthesizing the protein from amino acids may be needed still 
greater dimensions. Rickettsiae, pleuropneumonia-organisms, and re- 


294 IV. THE PRINCIPLES OF LIFE PHENOMENA 


lated primitive organisms can sometimes be cultivated’ in media 
containing blood plasm, but never in media containing merely amino 
acids or other simple protein components. The cultivation can only 
be achieved when the organisms or assimilase masses acquire the 
dimension of bacteria. 

Liver slices are able to incorporate amino acids 7” vitro, but the 
ability is remarkably reduced by the mechanical grinding. According 
to Winnick (123) rat liver slices are 4 to 41 times as active as the 
homogenate in the uptake of methionine and 3 times as active in the 
uptake of glycine, indicating that synthetic ability is markedly re- 
duced by the decomposition of the cell mass. In recent years numer- 
ous reports on the uptake of labelled amino acids not only by tissue 
slices but also homogenates have appeared (124), and considerable 
evidence has accumulated indicating that this uptake of amino acids 
represents a synthesis of peptide bonds (125). 

It was found by Brunish and Luck (126) that the protein fraction, 
containing nucleic acids, in a liver homogenate, can incorporate the 
greatest amount of phenylalanine. On studying the uptake of radio- 
active alanine 7” vitro into the proteins of rat liver homogenate, 
Siekevitz (127) concluded’ that when the alanine was incubated with 
the homogenate, the highest specific activity in the homogenate protein 
was found in the microsome fraction. Significant incorporation did 
not occur when either isolated microsomes or mitochondria were in- 
cubated with radioactive alanine, but when mitochondrial and micro- 
somal fractions were incubated together in the presence of the alanine 
incorporation occurred. 

This finding suggests that the dimension of the assimilase is im- 
portant not only for the reason that the dimension may determine 
the polymerization degree but also for the reason that the great 
dimension may provide the space for the association of a number of 
particles having a variety of properties, because it is conceivable that, 
in order to secure the energy supply for the synthesis of proteins, 
that is, for the uptake of amino acids, a variety of particles may be 
needed to cooperate for the common purpose, whereby each of the 
particles may play each specific part. A single virus-like particle 
may thus be unable to synthesize the protein even if the size of the 
particle is considerably great; only when numerous different element- 
ary-bodies are united, protein synthesis becomes possible. 

In the recent work on the incorporation of labelled amino acid into 
proteins in cell-free systems, Zamecknik and Keller (127a) have shown 
that, in addition to a microsome-rich fraction, a soluble non-dialysable 
fraction and also an ATP generating system are necessary for the 
process. Thus it has been clearly indicated that a single virus-like 


IX. METABOLISM 295 


particle cannot incorporate amino acid, but that it can only be achiev- 
ed in the presence of complete energy supplying systems. 

Since each type of protein molecules may be able to act as each 
type of enzyme systems as already suggested, such cooperation of a 
variety of particles for a common purpose may mean the establishment 
of an enzyme system for the protein synthesis. Rickettsiae are com- 
posed of a number of virus-like particles or elementary bodies, which 
may be different from each other in their physical and chemical pro- 
perties, and hence the enzyme system may start to function and thus 
a certain type of Rickettsiae can proliferate outside the host. 

The fact that some bacteria can recover their complete form 
after being decomposed into virus-like particles, may appear to show 
that each particle shares the faculty of synthesizing proteins. Yet, 
this seems not to be the case. Separated single particle cannot grow 
as above considered. In order to multiply, decomposed particles 
having various properties may have to unite with one another into 
larger masses; only in. this way they may be able to accomplish the 
synthesis. 

It has already been stated. that virus-like protoplasm particles 
can be fused 7 vitvo into cell-like masses. This may be looked upon 
as a restitution of the original cell mass. A remarkable example of the 
restitution of body form and function has been found with sponges 
and hydroids (66). These organisms were cut into small fragments 
and then pressed through the meshes of fine bolting cloth. In this 
way the flesh was broken up into very small fragments. Shortly 
after the operation many of the isolated elements were observed to 
have fused together into lumps or sheets of tissue. Soon larval stages 
arose very Similar to the typical normal larva. Ultimately new com- 
plete adults developed. 

Such a reversible decomposition of cells or organisms is probably 
based upon the general reversibility of protein structure. As repeated- 
ly emphasized, the reversibility involved in the nature of protein is 
of the utmost importance in developing the various reversible charac- 
ters in living substances. This reversible decomposition may be only 
one of these examples. It was found that haemocyanin, the copper- 
containing respiratory chromoprotein of invertebrates, is split into 
subunits of one half or one eighth of the original molecular weight 
by exposure of the protein solution to pH 8.5, and that reassociation 
by exposure of the protein solution to pH 8.5, and that reassociation 
at pH 6.85 to form the original units occurs quite specifically. Frag- 
ments of haemocyanin from Helix pomatia recombine with fragments 
of the same haemocyanin, but not with fragments derived from the 
haemocyanin of Littorina littorea (128). Various evidences of such 


296 IV. THE PRINCIPLES OF LIFE PHENOMENA 


reversibile decomposition have been shown with tobacco mosaic virus 
protein as already referred to. ’ 

The protein molecules of the same type tend to combine with one 
another. The protoplasm or the organism is produced by this property 
of protein, and the protein can reform organism even after the de- 
composition into the protein. However, since the protoplasm is a 
mixed crystal, the component or proteins are not necessarily of the 
Same structure. Proteins having different structures can form one 
and the same protoplasm if the arrangement of their polar groups 
can be subjected to the original pattern of the protoplasm, and thus 
they may exhibit various functions according to their various chemical 
structures to accomplish a common purpose. 

Although the primitive primary organisms were presumably pro- 
vided from the beginning with the size and shape of ordinary cells of 
the present time, they would only be able to affect, like viruses, 
other weaker organisms to transfer their template pattern to the 
weaker. The faculty of protein synthesis might be obtained after 
an immense span of time following their generation, as a result of 
the proper differentiation of particles composing their body. Of 
course, individuals composed of particles thus differentiated might be 
much more fitted for the continued existence than those having un- 
differentiated particles, and so the differentiation of the particles or 
proteins and accordingly of the enzymes would be more and more 
developed. 

The differentiation in the chemical structure, on the other hand, 
would result in the differentiation in the physical structure of the 
mass of assimilase or the organisms, since protein molecules or ele- 
mentary bodies of the related chemical structure would preferably 
combine with one another, and thus various form-elements such as 
nucleus and cytoplasmic granules would be produced. As was already 
stated, even the artificial cells prepared by the writer from plant 
materials occasionally possess a variety of granules some of which 
has the nucleus-like appearance. It can be demonstrated that such 
granules stain particularly well when treated with a proper dye such 
as fuchsin, showing that they are the aggregates of proteins with 
related chemical structures different from those of other parts. 

Thus, in the ordinary cells of the present day organisms, ele- 
mentary bodies containing DNA may agglutinate into a body termed 
a nucleus, and those containing RNA may form various sized bodies, 
iz. €@. cytoplasmic particles such as microsomes and mitochondria, 
according to the different characters of the elementary bodies. Chan- 
trenne (129) emphasized that the separation of the two fractions 7. e. 
microsomes and mitochondria, might be arbitrary, and suggested 


IX. METABOLISM 297 


that there might actually exist a more or less continuous spectrum 
of size and chemical composition. Indeed, there are reports that the 
microsomes and mitochondria may be separated into biochemically 
different fractions. Novikoff ef. al (130) have separated the cytoplas- 
mic particles of rat liver homogenates into as many as eight fractions 
by differential centrifugation, and have found evidence cf biochemical 
heterogeneity among the isolated particles. 


CHAPTER X 


THE CHANGE OF PROTOPLASM STRUCTURE 
AND THE REVELATION OF LIFE 
PHENOMENA 


1. The Oscillation of Protoplasm Structure 


In studying the inactivating progress of phage or of rennin by 
the addition of inactivating agents such as antibody or tannin, we 
have found that the inactivation occasionally proceed in an extremely 
irregular way (131). In Fig. 26 is shown an example of an irregular 


Log of the ratio of the number of 
remaining active phage particles to 


that of the total phage particles added. 


Time in hours, 


Fig. 26. The progress of inactivation of phage protein due to for- 
maldehyde at pn 4,6. Formaldehyde concentrations: 0,022% (I), 
0,0449 (II), 0,08896 (III), 0,175%6 (IV). Concentration of phage 
protein (sample No. 3): 0,05%.— Laboratorary temp. 


progress of phage inactivation in a weakly acid solution by for- 
maldehyde, and in Fig. 27 a similar irregular inactivation progress of 
rennin by tannin. 

In these examples, it is clearly shown that.the inactivation proceeds 
with remarkable oscillations as if it were a physical phenomenon, 


X. THE CHANGE OF PROTOPLASM STRUCTURE 299 


though such a phenomenon was rather rarely observed, in most cases 
only regular progresses being seen. 


— Rennet quantity remaining, % 


—> time (minutes) 


Fig. 27. 

Decreasing progress of the rennet action due to tannic acid. 
Tannic acid: Kahlbaum; used a month after its dissolving. Rennet 
solution (0.0194), 1ec+tannic acid solution, 1 cc+milk (1% CaCl,) lcc. 
Tannic acid concentrations: 

T. 0.003125% II. 0.00625%6 III. 0.0125% IV. 0.025% 
V. 0.05% VI. 0.194 


Even when freed from protoplasm, proteins of the same origin 
exhibit the property to combine mutually. On account of this pro- 
perty, as pointed out already, structural change of a protein molecule 
is likely to spread to other molecules existing in the same system. 
It is probable that such an oscillation is also brought about by a loose 
combination of protein molecules in a solution. If loose combination 
exists between protein molecules, each molecule may be prevented 
from behaving of its own accord, and accordingly all the molecules 
present in a solution may have to act as a single system. On the 
other hand, as stated repeatedly, since protein molecules possess the 
structural reversibility, phage or rennin inactivated to a certain extent 
may be striving to resume its original active structure in resisting 
the inactivating action of the added substance. These two opposite 
actions may cause the oscillation. 

The writer has been able to prove that the turbidity produced in 
a solution of ovalbumin by the addition of tannin shows also an 
oscillation in the progress of time following the addition (132). It has 
also been found that the turbidity of a protein solution oscillates with 
the change of the added amount of sodium chloride (133). 


300 IV. THE PRINCIPLES OF LIFE PHENOMENA 


Such oscillation phenomena would not be revealed if each mole- 
cule changed its structure arbitrarily of its own accord and the 
mutual connection failed to prevent the arbitrary behaviour. Since, 
in the protoplasm, the component proteins are mutually associated in 
most orderly way, the oscillation phenomena must be much more 
manifest than in a mere solution of proteins. Indeed, this seems 
actually the case. Thus, oscillation or rhythmic contractibility appears 
to be an essential. property of all forms of protoplasm as in heart 
muscle, intestine,-the -diaphragma, and in many-unicellular organisms 
such as leucocytes, swimming protozoa, and myxomycetes. Its finest 
demonstration may be seen in the oscillating motion of cilia and 
flagella. 


2. Resorption and Excretion 


The writer has found a series of facts with which vital pheno- 
mena of resorption and excretion appear to be closely connected (134). 
In this section a discussion will be made on experimental results 
concerning the facts carried out with sugar and virus particles. 

A heavy water suspension of vaccinia virus particles isolated by 
our isoelectric-point precipitating method is mixed with the same 
volume of sugar solution (glucose and fructose) of varying concentra- 
tions, and after left for 30 minutes at laboratory temperature the 
particles are precipitated by centrifugation. With the supernatant 
fluid the sugar concentration is measured, and at the same time a 
similar manipulation is carried out with the sugar solution mixed 
with the same volume of water instead of the virus suspension, and 
then from the difference between these two measured concentrations 
the rate of the penetration of the sugar into the particles is es- 
timated. 

There seems to exist in the particles a space into which sugar 
fails to penetrate, and this space is designated as sugar-insoluble 
space. Relations between such spaces and the sugar concentrations 
are shown in Fig. 28. Usually curves like GI or FI shown in this 
Fig. are obtained when virus particles isolated by an ordinary way 
are used, showing that both glucose and fructose can penetrate, to 
some extent, into the particles when their concentrations are high, 
the rate of penetration being directly proportional to the concentra- 
tions. As the water quantity combined with dried virus particles 
may be about 10 times as great as the latter, the sugar-insoluble space 
will be about 10 when no penetration occurs, and hence values smaller 
than 10 may indicate the occurrence of the penetration of sugars in 


X. THE CHANGE OF PROTOPLASM STRUCTURE 301 


the hydrated particles. 

It seems natural that sugars can penetrate when their concentra- 
tions are high as shown in curves GI and FI; but very strange 
results are obtained if virus particles are used to which peculiar 
manipulations were preliminarily added. Ordinary curves such as GI 


. dry weight 


Sugar-impenctrable space (c.cm.) pro gr. 


-yl 6 G25% 425% 05% 


Conc. of the sugars, %. 


Fig. 28. 
Sugar-impenetrable space of vaccinia particles. G=glucose, 
F= fructose. 
Roman numerals indicate the No. of the preparations. 


and FI are secured when virus particles are used which were prepared 
by merely washing with the water of a weakly acid pH without 
neutralizing the acid after each precipitation. However, when the 
experiments are carried out with the particles ‘‘purified’’ by repeated 
precipitations with acetic acid, which is neutralized by NaOH after 
each precipitation and centrifugation, the rate of penetration of sugars 
is not always proportional to the concentration as shown in many 
curves in the figure. Similar irregular results are yielded when the 
particles are preliminarily heated to an adequate temperature. 

A mention will be made as regards the curve GIII in the figure, 
which shows that when the sugar concentration is (.06 per cent the 
sugar-soluble space takes a negative value, indicating that the sugar 
concentration in the particles is higher than that of the outer medium, 
whereas at the concentration of 0.125 per cent this is not the case. 
Thus, the sugar is absorbed when the particles are present in the 
sugar solution of alow concentration but excreted in that of a higher 
concentration. Similar results are obtained with both glucose and 
fructose, but not with lactose. This may account for the non-absor- 
bability of the disaccharide. 


302 IV. THE PRINCIPLES OF LIFE PHENOMENA 


Such a phenomenon is not confined to virus particles, but is 
generally recognized with protoplasm particles without concerning 
virus activity. Even with coagulated casein particles similar results 
are obtained. In addition,-not only with sugars but also with various 
inorganic salts related phenomena are demonstrated. 

In view of these facts, it may be concluded that the absorption of 
a substance by a cell may be raised by a physicochemical combination 
of the substance with the protoplasm protein of the cell. The com- 
bination may be called adsorption, which may be established according 
to the structure of the protoplasm protein, so that absorption and ex- 
cretion may take place in response to the change of the structure. 
On the other hand, as the protoplasm proteins are arranged so as to 
easily achieve structural changes, the absorption and excretion of a 
certain substance, if occurred in this manner, could be accomplished 
with ease. 7 

The writer (131) once expressed the opinion that the oscillation 
phenomena referred to in the preceding section might be accounted 
for by such absorption and excretion of the virus inactivating agents 
in question, but it must be more reasonable to consider that this 
phenomenon of absorption and excretion is rather based upon the 
oscillation of protein structure; that is to say, the structure of the 
protoplasm particles, including virus particles, used in the experiments 
were presumably highly disturbed by the preliminary treatment, and 
the disturbed structure would be striving to resume its original state 
in an oscillating way during the experiments, taking a certain struc- 
ture at a period and another structure at another, so that even so 
slight a manipulation as the addition of sugar or other substances 
might be sufficient to effect the phase of the oscillation according to 
the concentration, and in conformity with the change in the structure 
absorption or excretion might take place. 

In any case, it may be said that a certain substance is adsorbed 
by, or eluted from, a protein, if the structure of the portein is pro- 
perly changed. Absorption and excretion of substances by a living 
cell must be attributed to such adsorption and elution. 

Being consistent with this concept, it has been found by klotz ef 
al. (135) that there are structural specificities in the interactions of 
some organic ions with serum albumin. Human albumin undergoes 
major configurational changes with changes in pH, making new sites 
available for interaction with ions of specific structural properties. 
Thus, it is obvious that albumin can absorb and excrete some organic 
substances when pH of the solution is changed. Moreover, it has 
been shown that human serum albumin combines preferentially with 
chloride ion in sodium chloride solutions, even when the net charge 


X. THE CHANGE OF PROTOPLASM STRUCTURE 303 


on the protein would be expected to favour the absorption of the 
sodium cation. Modification of the albumin by treatments which 
weaken the basic groups generally decreases the adsorbing properties, 
and it is generally accepted that the preference which albumin shows 
for anions is connected with the activities of the lysine and arginine 
residues, both of which possess single positive charges at physiologi- 
cal pH (136). 

In the customary consideration of the absorption mechanism the 
existence of the so-called semipermeable membrane seems to be essen- 
tial. The membrane is believed by some workers to be present even 
in viruses. For example, sedimentation of virus. particles in’ solu 
tions of low molecular weight, such as sucrose, sodium chloride, and 
glycerol, has revealed a dependence of sedimentation rate on solute 
concentration, and further the rate was found to vary with time of 
contact with the solute. These facts seem to be often interpreted 
as giving evidences of the existence of a semipermeable membrane 
susceptible to osmotic effects of solutes of low molecular weight (137). 
However, these facts should be accounted for by the change in the 
rate of “‘absorption’’ responding to the concentration of the substances 
to be absorbed and responding to the time of contact. Membrane-like 
images shown in electron micrographs of some viruses may, as already 
pointed out, be interpreted as only an artificial product produced by the 
surface-protein molecules fused into a membrane-like aggregate through 
the heat of electron bombardment. 

Bergold and Wellington (138) claimed that they could isolate from 
certain insect-virus particles particulate protein complex without DNA, 
which they believed to be the membrane of the virus. However, it is 
possible that this ‘‘membrane’’ may be the particles, which contain no 
DNA, contaminating the “‘virus particles’’ that contain a large amount 
of DNA. Insect virus particles were separated from inclusion bodies 
which had been formed in the insect after the virus infection. Ac- 
cording to Bergold (139) the inclusion bodies consisted of about 95 per 
cent of a homogeneous protein and about 3-5 per cent of virus particles; 
these virus particles which contained DNA in rich amount could be 
liberated by dissolution of the inclusion bodies in weak alkali and 
separated from other component proteins by centrifugation. This fact 
suggests that the ‘‘virus particles’? were the DNA rich elementary 
bodies which could withstand the alkali treatment owing to their 
high contents of DNA; the other component bodies might be decom- 
posed and dissolved in the alkali so that virus activity might be left 
only in the undissolved particles containing DNA. It should be noted 
that the ‘‘membrane’’ was reported to dissolve in alkali like the main 
component protein of the inclusion body, suggesting thus strongly that 


304 IV. THE PRINCIPLES OF LIFE PHENOMENA 


it might be the main component itself. 

To sum up, in order to elucidate the phenomenon of absorption 
and excretion the assumption of a semipermeable membrane seems not 
only unnecessary, but it is obvious that even if such a membrane 
could exist, the phenomenon would not be explained by the mem- 
brane. 

If absorption took place always through the semi-permeable mem- 
brane, any penetration of macromolecular substances .into..cells would 
be impossible. However, this seems the case. Thus, according to 
Coons ef al. (140) various protein injected intravenously into mice 
were clearly demonstrated in the unaltered form in the nuclei of 
certain normal cells, often even in higher concentrations than in the 
cytoplasm. If proteins were adsorbed by nuclei, they would naturally 
be found in the nuclei. The specific pattern of the protoplasm is 
expected to be strongest in the nucleus, and so the proteins, if the 
adsorptive force is involved in the specific pattern, will be most 
strikingly adsorbed by the nucleus. Again, there are evidences, as 
mentioned already (Part II, Chapter VII) that virus nucleic acid or 
its components can readily penetrate into the host cells. In this case, 
the specific pattern of the host protoplasm must involve the specific 
adsorptive force directing to the virus, and the pattern may be most 
powerful in the nucleus or in some cytoplasmic particles. Hence, the 
penetration into the cell of the nucleic acid in which the specific 
structure of the virus is retained should naturally follow. 

Anderson (141) found that phage could be inactivated by suspending 
the particles in high concentrations of sodium chloride, and rapidly 
diluting the suspension with water. The inactivated phage was visible 
in electron micrographs as tadpole-shaped ‘‘ghosts.’’ Since no inacti- 
vation occurred if the dilution was slow, he attributed the inactivation 
to ‘‘osmotic shock’’ and inferred that the particles possessed a semi- 
permeable membrane. However, this phenomenon should be interpreted 
as based upon a structural derangement of phage protein due to-the 
rapid release of adsorbed ions. As a result of this derangement, the 
association between nucleic acid and protein may be destroyed, as it 
is reported that DNA is released by this procedure (142). It is a note- 
worthy fact that the ghost could adsorb to bacteria and lyse them, 
though it cannot act as the active virus. 


3. The Movement of Organisms 


Protein molecules in protoplasm are mutually associated and orderly 
arranged, and can readily change their structure as a single system 


X. THE CHANGE OF PROTOPLASM STRUCTURE 305 


under the influence of various effects. It may only be natural, there- 
fcre, that such a change is sometimes revealed as the movement of 
organisms. 

Amoeboid movement is said to be established by the sol-gel change 
of the protoplasm (143), thereby amoeba can move in pulsatory motion 
(144). If we assume that the protoplasm protein in amoeba are usually 
in a state somewhat contracted and that both the extension and 
contraction of the protein threads tend to occur too far, not exactly re- 
sponding to the degree of the stimuli causing the change, the amoe- 
boid movement can be easily explained. Thus, when a stimulus which 
may cause the extension of the protein threads is given at a site of the 
protozoan, the protein molecules at the site will be extended with the 
projection of the protoplasm, but as this extension may go too far, a 
compensatory contraction will follow. This contraction likewise may 
occur excessively, spreading to other proteins, with the successive 
coagulation of protoplasm into elementary particles; combining force 
between the proteins may then be increased and the coagulated elemen- 
tary bodies will be attracted and drifted towards the site of the stimulus. 
If the stimulus extending the protein continues to be applied to the 
same site, the protein in the coagulated particles, thus attracted to 
the site, will be extended, and the protoplasm will be projected, followed 
by the contraction of the protein and subsequently by the gathering 
of coagulated particles. 

On the contrary, if a stimulus causing a contraction is applied, 
the protein molecules at the site of the stimulus may undergo con- 
traction, which may spread to the opposite side, where no contracting 
stimulus is present, so that soon the extension will follow at this 
side, with the projection of protoplasm. But this extension may be 
temporary if contracting stimulus continues to exist, so that contration 
may follow which will attract the coagulated particles to the side 
where no stimulus exists, as a result the protozoan can move to 
escape the stimulus as shown in Fig. 29. 

If the protoplasm protein is stretched by the benevolent stimulus 
and contracted by the detrimental one, the movement will be said 
purposeful and the organisms having the protein of such a property 
can continue existence and well prosper. Since intense and unusual, 
physical or chemical stimuli generally cause the protein to coagulate 
or to contract, it should naturally follow that organisms will achieve 
the movement to get rid of such stimuli. However, occasionally the 
same stimulus may give rise to the different reactions among varying 
organisms, indicating that some stimulus is favourable for some 
organisms but unfavourable for others. The property to achieve such 
a purposeful movement must have been raised because only the 


306 IV. THE PRINCIPLES OF LIFE PHENOMENA 


Extensive | WWW Ww Contractive| “Wy ryv~v cr 
: MDA NN An : Mn Wau) 

stimulus stimulus Sher AAA 
ce ceed 


si— 


> 
> 
2 


23823338 
3323338 


aN 
nw 
wu 
nu 
wy 
VAC aN) 
avavay) 


seeseees 
38383883 
33385358 


Fig. 29 
Diagram of amoeboid movement. 


organisms which would move purposefully were able to continue their 
existence. i 

The property of protoplasm protein to contract and expand exces- 
sively not precisely responding to the degree of stimulus must be 
required for the movement, such as amoeboid one and oscillation of 
cilia or flagella, and organisms which need movement must have the 
protein having such a property. If movement was favourable for 
certain organisms, this property would be developed in them, since 
the more developed the property, the organisms would become the more 
fitted and the more easily win the struggle for existence. 

Monné (145) claimed that folding and unfolding of polypeptide 
chain seem to be the essential features of the protoplasm, and he 
stated that mobility and contractibility, which are due to active fold- 
ing of polypeptide chains, are no doubt the characteristic life pheno- 
mena of all living fibrils. This view is quite cempatible with the 
writer’s. 


X.. THE CHANGE OF PROTOPLASM STRUCTURE 307 


On studying the X-ray diagrams of bacterial flagella, Astbury (146) 
has found that films produced from the flagella show fine a-keratin 
reflection and, upon squeezing, this goes over into the §-keratin pattern. 
Since it is known that a-keratin pattern is formed when keratin 
molecules are expanded, while §-keratin pattern is revealed when the 
molecules are contracted, it is obvious that the movement of the fla- 
gella is brought about by the extension and contraction of the protein 
molecules. 

However, whether or not the movement of flagella can lead to 
the movement of the bacteria themselves is another question. The 
writer holds the opinion that bacterial movement is not brought about 
by the flagella, but that bacteria can move by making use of the 
thermal motion of water molecules. As is well known bacteria show 
a very active Brownian movement which is caused by the bombard- 
ment of water molecules in thermal motion, it should therefore be 
expected that bacteria will be able to move in a definite direction if 
they have a specific form by which the bombardment of water mole- 
cules become effective only in one direction. It may not be difficult 
for bacteria to acquire such a form. Presumably flagella act as rodders 
and perhaps, in addition, play a role in making effective the energy 
of the water molecules. It is inconceivable that contraction and 
extension of the flagella, only frail bands of protein molecules, result 
in the active movement of the bacteria. a 

In this connection, it should be noted that pijper (147) claimed that 
bacterial flagella are not the locomotive organs of the bacteria, but are 
merely mucous twirls trailing from the cell surface. It seems, however, 
possible that the flagella themselves can achieve some motion and have 
some connection with the bacterial movement, and so it seems more 
reasonable to consider that though bacteria do not move by flagella, 
flagella contribute to the movement. 

The writer has reached such a conclusion mainly from an obser- 
vation on a kind of bacteria, perhaps a strain of water vibrio, which 
accidently contaminated a protein solution stored in an ice box. This 
strain of bacteria could vigorously proliferate at the low temperature 
in ice box, and move very briskly in a definite direction at a full speed 
as if flying in the air. Under the microscope of dark field illumination 
the speed appeared to have no connection with the temperature; they 
moved in icy water apparently at the same velocity as in water of 
laboratory temperature. If the movement is raised by flagella composed 
of protein molecules being enabled to move by the interposition of 
lipids, it should be very sensitive to environmental temperature just 
as the movement of cold-blooded animals. On the contrary, if the 
thermal motion of water molecules is directive, the temperature 


308 IV. THE PRINCIPLES OF LIFE PHENOMENA 


differences involved in the usual environments of animals should be 
insignificant, since the molecular motion is directly proportional to 
the absolute temperature not to the ordinary one. 

Of course, organisms which may be able to utilize the molecular 
motion of water should be very small in size, and perhaps not larger 
than bacterial size. Animals larger than bacteria may be making use 
for their movement of the reversible contractibility of protein molecules. 
Muscle contraction of higher animals must also depend upon this pro- 
perty of protein molecules. Actin molecules, the main protein com- 
ponent of muscles, can exhibit even 7m vitro the reversible contraction 
as already pointed out. 

The remarkable readiness with which impulses can be transmitted 
along nerve fibres may be based upon the most suitable arrangement 
of protein molecules for the spread of their structural changes. The 
transmission of an impulse must be the spread of the wave of the 
structural change, although this wave cannot be seen as a formal 
change such as contraction. It is shown in electron micrographs that 
protein threads are orderly arranged in nerve axon along the long 
axis of the fibre (148). Chambers (149) injected oil drops into the 

xoplasm of giant nerves of the squid and found that the drops assumed 
ovoid shapes along the long axis of the nerve. After agitating the 
axoplasm with a microneedle near the oil, the drop assumed a spherical 
shape, suggesting the occurrence of a contraction of protein molecules. 

It has long been known that nerve tissues are especially rich in 
lipids, which may serve as lubricating oil enabling the protein molecules 
easily to alter the structure. It may be considered that phospholipids 
are involved, in addition, in the energy metabolism for the impulse 
transmission. Anaestetics or narcotics are generally lipid-soluble; the 
action of the drugs may depend upon their effect on the lipids, the 
lubricating oils, rendering the spread of the structural change im- 
possible. 

Neurotropic viruses, such as poliomyelitis virus, are said to travel 
preferably along neural paths. According to Sanders, mouse encephalo- 
myelitis virus travels at about 0.2mm. per hour (150). If structural 
changes of the protein are readily transmitted in nerves, the structural 
change induced by a virus must likewise be easily spread in them. 
The spread of the structural change induced by a virus may be nothing 
but the travel of the virus itself. 

Since the structural change of protoplasm proteins should accom- 
pany a change in polar groups, the spread of the change must be fol- 
lowed by an electric disturbance, which is known as action current. 

On stimulation of muscles remarkable diffusion of K ion takes 
place. In nerves, as in muscles, there is a difference between the 


X. THE CHANGE OF PROTOPLASM STRUCTURE 309 


concentration of K ion in the inside and on the outside of the nerve 
fibre. The ratio of K ion in the inside to that on the outside is given 
as 29: 1. However, during excitation this ratio is changed, the ion 
present in the inside being liberated. The occurrence of such an ex- 
change of ions by stimulus can be demonstrated with the help of 
various isotopes (151). The exchange may be recognized as action 
current when examined electrically. ae 

The existence of similar ratio of ions is also known between red- 
blood cells and the serum: In the cells, K ion is present in a much 
higher concentration than Na ion, but in the serum the ratio is reverse 
as in the sea water. K ion seems present in such high concentrations 
generally in the cell protoplasm, a fact which may depend upon the 
property of the protoplasm protein to adsorb preferably K ion. This 
adsorbed K ion, on the excitation, will be eluted by the structural 
change of the protein, giving rise to an electric disturbance. 

It may, therefore, be concluded that action current is raised by 
the ‘‘absorption and excretion’’ of inorganic ions. On the other hand, 
it is known that action current is associated with the true absorption 
and excretion, a fact which can be expected, since this phenomenon 
may also be brought about by the structural change of protoplasm as 
discussed earlier. 

The electric disturbance occurring in the brain may be called brain 
wave. The function of brain must likewise be given rise to by a 
similar structural change of the protoplasm protein. The fact that 
brain current is generally oscillating and wavy, suggests that the 
structure of the brain protoplasm tends to oscillate regularly. 

The structural pattern of brain may be more or less disturbed 
and deformed during the constant change of the structure. Its mending 
may be accomplished by sleep during which no stimulus is given and 
sufficient time is provided for the recovery of the original pattern; 
presumably the reversibility of the protein structure accounts for this 
recovery. 

Since all the life phenomena must be raised by the structural 
change of protoplasm, which is always to be accompanied by the 
electrical disturbance, this latter phenomenon should be “‘the sign of 
life’’ as Waller proposed a long time ago. 


4. The Mechanism of Blood Coagulation. 


The theory of the writer on life phenomena discussed so far is 
based upon the basic assumption that the structural change of proteins 
is transmissible. This assumption was made for the first time by 


310 IV. THE PRINCIPLES OF LIFE PHENOMENA 


taking into account of the view presented by Fischer (152) who found 
that the blood coagulation can be transmitted indefinitely in blood plasm. 
He found that the addition of coagulating agent, which is. being pro- 
duced in a coagulating blood plasm, to another plasm, leads to the 
coagulation of the latter, in which cogulating agent in turn is produced, 
thus the agent multiplying indefinitely in the plasm. This coagulating 
agent, however, can be demonstrated only during the progress of 
coagulation, and so Fischer considered that the active radicals of the 
agent diappear by the mutual saturation with the completion of the 
coagulation, 

It seems reasonable to consider that blood coagulation is caused 
by a kind of denaturation of plasm proteins, thereby as was previously 
mentioned protein molecules may unfold their polypeptide chains; the 
active groups of the coagulating agent may be produced by such an 
unfolding. Since the polypeptide chains are to be refolded upon the 
completion of denaturation with the disappearance of polar groups 
liberated by the unfolding, also the active groups must disappear 
with the finish of the blood coagulation. 

According to the writer’s concept, denaturation of proteins is gen- 
erally infective because of such polar groups liberated during the 
denaturation process. ‘The fact that the infection is particularly dis- 
tinct in blood coagulation may suggest the presence, in the blood plasm, 
of the mechanism by which the transmission of the denaturation is 
readily brought about. Now, we shall consider in detail this mechanism. 

It seems a general concept that the process of the blood coagulation 
consists of the following two phases (26): 


Prothrombin ----- 4 pee oe eens sao — Thrombin. 


FUDrimO gen nme eens —- Pubiin. 


The factor termed thromboplastin or thrombokinase is usually 
found in blood platelets, and this factor appears to be vius-like protein 
particles containing both RNA and phospholipids (153), presumably a 
kind of elementary bodies of the protoplasm. When blood platelets 
are disintegrated into such particles, the particles will combine with 
prothrombin, a kind of serum globulin. By this combination the protein 
is disturbed in its structure just as host cells are disturbed by the 
combination of a virus. As a result prothrombin undergoes a denatu- 
ration, with the unfolding of polypeptide chains and the liberation of 
polar groups. The prothrombin affected by the thromboplastin particles 
can combine with other prothrombin molecules through these polar 
groups, causing the denaturation in the latter molecules and thus the 


X. THE CHANGE OF PROTOPLASM STRUCTURE 311 


denaturation spreads as a Chain reaction as above mentioned. 

The denaturated prothrombin is called thrombin, in which active 
group or groups are being produced as a result of the structural change 
due to the denaturation. Thrombin, the denaturated prothrombin, can 
combine with another kind of serum globulin, called fibrinogen, through 
these active groups to produce the structural disturbance in the latter. 
The fibrinogen thus denaturated is termed fibrin. During this change 
polar groups are likewise temporarily liberated, whereby the change 
is transmitted to other intact fibrinogen molecules. This is the general 
concept of the writer as regard the mechanism of blood coagulation, 
by which all the phenomena known concerning blood coagulation may 
fairly be elucidated. 

Thus it may be clear that the blood coagulation as a whole is a 
chain reaction given rise to mainly by temporarily lberated polar 
groups. However, there are ample evidences that active group of 
thrombin is retained without disappearing long after its formation. 
Therefore, it must be considered as follows: The active group of 
thrombin is different from the polar groups which are liberated tem- 
porarily during the process of the denaturation, but their group may 
arise as a result of the denaturation and so can remain without dis- 
appearing. The reason why active group can be produced following 
denaturation will be discussed in the next Part. 

Further it should be added that thromboplastin may not be present 
as such in the platelets, but that active groups capable of acting as 
thromboplastin may be produced upon the disintegration of the cells 
into elementary bodies just as the active groups of thrombin are raised 
by the denaturation of prothrombin. In fact, it is said that thrombo- 
plastin is present in cells as thromboplastinogen or prothromboplastin 
(154). This latter may be the natural state of elementary bodies 
present in platelets. During the conversion of prothromboplastinogen 
into thromboplastin transient liberation of polar groups may take place, 
leading to the transmission of the denaturation to accelerate the change; 
thus the phenomenon may appear the more complicated (155). 

Throboplastin seems to be usually produced from platelets, but it 
can also be isolated from a variety of other materials such as brain, 
lung, and blood plasm, showing that it is elementary bodies not peculiar 
to the platelets. [ts action is lost by the extraction of lipids; the 
lipids may be involved in the combination of the thromboplastin with 
prothrombin. ‘Thromboplastin fails to act without Ca ion. As was 
already mentioned, Ca ion is generally necessary for the transmission 
of structural change of proteins because of its faculty to favour the 
liberation of polar groups in proteins, and hence the need of Ca ion 
is mever peculiar to blood coagulation. 


312 IV. THE PRINCIPLES OF LIFE PHENOMENA 


Various denaturating agents involved in blood coagulation may be 
called enzymes in a wide sense. However, since it has been found 
that thromboplastin reacts with prothrombin in stoichiometric amounts 
(156), at least thromboplastin cannot be regarded as an enzyme in a 
narrow sense. On the other hand, it has been claimed that thrombin 
is not involved in the coagulation of the fibrinogen molecules, indicat- 
ing that thrombin acts as a true enzyme, because a protein which can 
disturb the structure of another protein and which is not consumed 
in this reaction should be termed a true enzyme. Nevertheless, the 
fact that papain clots fibrinogen just as thrombin does is not sufficient 
to prove the enzyme nature of thrombin (157). Trypsin is also known 
to be able to convert prothrombin into thrombin. These enzymes may 
act only to disturb the structure of the protein as do the blood clotting 
agents such as thromboplastin and thrombin. 

At any rate, blood coagulation may be regarded as a chain reaction 
brought about by a series of enzymes in a wide sense. The concept 
that a protein having a stronger structural influence overcomes a weak- 
er by disturding the structure of the weaker, or, in a particular case, 
by converting the structure of the weaker to be identical with that 
of the stronger, has led the writer to the theory of enzyme function 
and further of the mechanism of the generation of life. It may be 
considered that life phenomena are based upon the combination of 
various kinds of proteins, that possess this fundamental property, 
joining together orderly as in this case of blood coagulation for a 
definite purpose in making an “‘enzyme system’’. 

Presumably, enzyme systems present in living organisms are not 
necessarily composed of enzymes in a narrow sense. Protein molecules 
which can act as true enzymes may be rather exceptional, mostly 
behaving as enzymes in a broad sense as does thromboplastin in the 
blood coagulation. 


5. The Mechanism of Mitosis. 


In case of the cell division, the nuclear substances capable of 
determining the inheritable character are divided into two equal parts, 
each of which are subsequently distributed to daughter cells, by an 
apparently very complicated process termed mitosis. Though this pro- 
cess appears very mazy, since it is a basic phenomenon generally 
taking place in orgnisms, it must be a process raised by some simple, 
fundamental properties of protoplasm. 

In the writer’s opinion, as described in Part II, the protoplasm is 
composed of polypeptide chains, which make up elementary bodies in 


X. THE CHANGE OF PROTOPLASM STRUCTURE 313 


forming bundles; these elementary bodies are arranged in a regular 
manner associating loosely with each other. The bodies must be ar- 
ranged in the protoplasm in an equal direction as otherwise the polarity 
of the cell or organism may not be raised. If the end-to-end association 
of the elementary bodies is tighter than the side-to-side association, 
the protoplasm may appear as if it were composed of protein threads 
in a parallel alignment, as shown in Fig. 30, but such threads may 
be invisible under the ordinary microscope. 


& A B c 


Fig. 30 
Diagram of mitosis 


When the cell, after its full maturation, is brought under the 
influence of a certain stimulus, the protoplasm particle termed centriole 
may be activated by the liberation of some active groups, resulting 
in its vigorous growth until it becomes so large that it fails to exist 
any more as a Single particle which therefore is to be divided. Through 
the division the structure of the proteins in the particle is disturbed 
severely, and a kind of denaturation follows with the temporary liber- 
ation of strong active groups which can attract the elementary bodies 
present nearby. The elementary bodies thus pulled and combined by 
the centrioles drag in their turn other elementary bodies successively, 
and moreover the denaturating process of the centriole is transferred 
to them, acting as a stimulus to contract the protein threads; as a 
result they are coagulated and thus the coagulation is spread centrifu- 
gally from the centrioles, so that radiating system of fibers, the so-called 
_ asters, become visible at least in some animal cells as shown in B in 
Fig. 30. This denaturating process is transmitted also to the nucleus 


314 IV. THE PRINCIPLES OF LIFE PHENOMENA 


which accordingly loses its normal appearance with the coagulation 
of nuclear substances. 

Now, the centrioles and coagulated particles of nuclear substances, 
i.e. chromosomes, may have the-same electrical charge since they 
are composed of proteins of a similar nature. If the surface substance 
of the cell is equally charged, the centrioles and chromosomes will be 
situated as far as possible both from the cell surface and from each 
other. It is known that beneath the surface of cells there is a thin 
layer of gel called cortex, which may be the charged surface substance, 
since both the centrioles and chromosomes are considered to be also 
the gel of the same category. 

At any rate, the divided centrioles are thus provided with the same 
charge, so that they have to be separated from each other when the 
division is accomplished ; at the same time the chromosomes, 7. é. the 
coagulated nuclear substances are shifted to acquire the most stable 
position in the field of the electrical force, and therefore finally chromo- 
somes are distributed in a plane exactly between the two centrioles 
and to right angle to a line connecting them. e 

Meanwhile, this process may activate the chromosomes to increase 
in their mass until the division is unavoidable just as the centriole is 
activated by some stimulus to undergo the division. Through this 
division chromosomes are likewise disturbed in their structure with the 
production of free polar groups, at least a certain point of each particle, 
which can attract the protoplasm threads and thus each thread present 
close to each divided particle of chromosomes is attracted and combined 
with the particle and subsequently contraction occurs in the thread 
as the active group of the chromosomes acts as a stimulus to produce 
the contraction. Thus, each set of divided chromosme is pulled toward 
each centriole as shown in E in Fig. 30. The separation of the divided 
centrioles may be caused not only by their mutual expulsion but also 
by the contraction of the protoplasm threads between the cortex and 
the centriole. Now, between the pulled chromosmes a space is pro- 
duced where no protoplasm substance is present, so that the cell is 
divided by the ‘‘furrow’’ formation around the equator of the cell. 

Since all the changes above described are initiated by the temporary 
liberation of active groups following the denaturation of the protein, 
the normal state of the cells must be recovered with the disappearance 
of the active groups, owing to the reversibility of protoplasm structure, 
and accordingly two new cells with the quite normal structure are to 
be produced. 

Mitosis, an apparently very complicated vital phenomenon, can be 
in this way easily explained by the theory of the writer. However, 
the assumption of existence of the specific particle termed centriole may 


X. THE CHANGE OF PROTOPLASM STRUCTURE 315 


be surplus, because it may be reasonable to consider that the elementary 
body of the protoplasm which by chance is situated in the center of the 
cell may somewhat be disturbed in its structure, because of its special 
position, by the physicochemical influence coming from the cell surface 
upon which a certain stimulus is added; the disturbence in the struc- 
ture of the body may give rise to the liberation of free groups which 
can fuse other proteins or protein components into the body leading 
to its vigorous growth until the division takes place. The growth 
and the division of chromosomes distributed between the two centrioles 
may be likewise attributed to their special position, 7. e. the center 
of the electrical field produced by the centriole particles in a stimu- 
lated state. 

The long effective distance of the charge of centrioles may be ac- 
counted for by the layer of water molecules attracted and regularly 
oriented around the centrioles as considered in Chapter II in Part II. 
The effect of the charge of both cortex and chromosomes must be 
likewise of the same nature, and therefore the pattern of the equili- 
brium of the effect at the phase when the separation of the centrioles 
is established may be as shown in Fig. 31. Hence, chromosomes are 


, Chromosome 
Za 


_Cortex 


, Centriole 


Higa 
Pattern of equilibrium of electrostatic forces in mitosis 


not to be regarded as being distributed in a circular plane between 
the two centrioles, but distributed on the circumference of the circle. 
The protoplasm threads passing through the inside of the circum- 
ference may be either combined with the chromosomes or broken off 
at the middle point by the extensive pulling, thus contributing to the 
occurrence of the division of the cell mass. 


316 IV. THE PRINCIPLES OF LIFE PHENOMENA 


It is known that the mitotic spindle is birefringent and that the 
birefringency decreases when the chromosomes are separating (158). 
This demonstrates that the protoplasm threads between the two cent- 
rioles are pulled extensively so that they are arranged quite regularly 
in a parallel alignment, thus the birefringency revealing in the spindle, 
but that when the contraction of the threads takes place causing the 
separation of the chromosomes, the regular alignment is to be lost 
followed by the disappearance of the birefringency. 

The fact that mitosis is the general, fundamental feature of or- 
ganisms must depend upon that it is the mechanism easiest and most 
favorable for the organism to achieve the cell division. ‘Thus the 
division of chromosomes without the preliminary fission of centriole 
may tend to cause only the increase of chromosomes without the cell 
division, because the divided centrioles only can produce the space 
where the essential protoplasm substance is absent, making the cell 
division possible. As is well known colchicine prevents the formation 
of the mitotic spindle, but do not affect the chromosomes,so that in the 
presence of colchicine chromosomes are increased without cell division, 
a fact suggesting that colchicine inhibits the growth or the division 
of the centriole. g 

The reason why the division of centrioles and chromosome is initi- 
ated normally is not clear. It may depend upon some basic property 
of proteins. Protoplasm-like masses prepared in vitro by the writer 
also cannot grow unlimitedly, suggesting that the fusion of elementary 
bodies can occur only to a certain limit. Both centrioles and chromo- 
some may be the aggregates of elementary bodies, so that if they grow 
over a certain limit they may be bound to split. Colchicine may exert 
some influence on physicochemical property of the protein composing 
the centrioles to make the centrioles unable to grow or to divide if 
the growth is not inhibited. | 

An interesting phenomenon associated with neutron- or 7—irradiation 
of maize seeds has been found by Schwartz (159). The seedling irradi- 
ated at the higher dose levels were not only taller but also much 
healthier in appearance than those at the lower levels. These seedlings 
could not be differentiated from normal unirradiated young seedlings 
except that there was a cessation of farther growth after approximately 
5 days. The finding concerning the phenomenon which must be noted 
here is that cytological examination of the root tips from plants which 
received high doses of irradiation revealed a complete absence of cell 
division. In other words, the growth was due entirely to cell elon- 
gation in these seedlings. Thus the conclusion drawn by Schwartz 
was that at high radiation levels the seeds were killed in the sense 
that no farther cell divisions occurred. According to the writer’s view 


X. THE CHANGE OF PROTORLASM STRUCTURE 317 


this may be attributed to a structural change in the protoplasm protein 
induced by the radiation, whereby presumably the division of either 
centrioles or chromosomes or of both becomes impossible. 


CHAPTER XI 


THE SUMMARY OF PART IV 


1 


Organisms generated from the protoplasm of preexisted creatures 
are designated as the secondary organisms and those generated originally 
in the primeval oceans without living substances as the primary 
organisms. The former were evolved from viruses, while the latter 
were presumably developed from protoplasm-like masses, a model of 
which could be prepared by the writer using plant materials especially 
castor beans. 

In general, coagulated elementary bodies are produced by the 
decomposition of protoplasm. These bodies yielded from plant materials 
are liable to fuse into a larger homogeneous mass when stand in a 
weakly acid solution. 

Elementary bodies possess a character to aggregate readily in a 
weakly acid water solution. Under this condition, aggregated particles 
appear to liberate their folded polar groups to combine with one another 
and fuse into a homogeneous mass. In coagulated elementary bodies 
protein molecules may be in a contracted, folded state, while ina 
solution of weak acid, the isoelectric point of the protein, the 
stretching or unfolding of the molecules may take place, leading to 
the mutual combination of the elementary bodies. 

Such a stretching appears to occur readily in plant elementary bodies 
in contrast to animal ones. Elementary bodies prepared from castor 
beans manifestly exhibit this property and occasionally produce fair 
protoplasm-like masses, which may be termed artificial cells. The 
most primitive feature of the primary organisms may be seen in such 
a mass. 

In chemical composition the mass resembles the protoplasm, being 
composed of globulin-like protein and lipids, while also in its form it 
bears a striking resemblance to a kind of protozoa, having a globular 
form with a diameter of scores of 4, granules of various sizes being 
included, some of which have frequently a nucleus-like appearance. On 
the application of a proper physical or chemical stimulus, the mass 
coagulates into minute particles just as the usual protoplasm, but may 
recover gradually its original, homogeneous state following the removal 


XI. THE SUMMARY OF PART IV 319 


of the stimulus, a fact showing that it has a primitive feature of 
irritability. Furthermore, it can grow by incorporating the minute 
particles of lipoprotein yielded from castor beans, and the growth will 
be followed by fission if a suitable environmental change is provided. 
Thus it can accomplish both the growth and multiplication. 

Such cell-like masses can be prepared not only from elementary 
bodies, but also from the constituents of the elementary bodies into 
which the latter was decomposed. Merck’s preparation of ricin which 
is composed of globulin of a molecular state, combining with lipids, can 
form elementary-body like particles when its water solution is made 
weakly acid (pH 5.5) by addition of acetic acid and left in ice box for 
several days. The particles thus formed can further fuse into proto- 
plasm-like masses, when stand in the water of pH 5.5 at laboratory 
temperature. Thus, under a Suitable condition, globulin with lipids can 
form protoplasm-like masses following the formation of elementary-body 
like particles. 

Since the assimilase, or the protoplasm including viruses, can be 
regarded as a kind of liquid crystals composed of globulin-like proteins 
polymerized with lipids, the fact just mentioned indicates that a liquid 
crystal closely resembling the protoplasm can be produced 7 vitro and 
that it is not entirely unreasonable to regard such a crystal as a very 
primitive organism. 


2 


The oceans of the primitive age might contain various substances 
as there were no microorganisms to devour them up, and the sea water 
might be weakly acid on dissolving carbon dioxide which might be 
present in rich amount on the surface of the earth. Consequently, 
when globulin-like protein having the property to sediment readily at 
a weakly acid reaction were produced in the ocean, they would be preci- 
pitated accompanied by lipids, and the sediments would form elemen- 
tary body-like particles which in turn would fuse into protoplasm- 
like masses. These masses would be endowed with assimilase action 
by their specific structure and thus. primeval organisms might appear 
on this globe. 

Such masses of a liquid crystal would accumulate on the bottom 
of the primeval ocean, and if a certain structural change capable of 
producing a structure stronger than the original one occurred in some 
of the masses, the newly formed structure owing to its stronger charac- 
ter would spread to surrounding masses. The structure thus could 
multiply just as viruses can in the protoplasm of host cells. The 


320 IV. THE PRINCIPLES OF LIFE PHENOMENA 


generation of a new Structure would be brought about by an environ- 
mental change or a stimulus just as the generation of a new virus. 
The change of the structure might occur repeatedly, and whenever a 
stronger structure was produced it would spread and multiply. Thus 
the structure would become stronger and stronger, thereby the primi- 
tive organisms would evolve higher and higher with the enhancement 
of the assimilase action. 

Some of the masses of the assimilase or the primeval organism 
might be carried to the shore by a tidal current, and in the active 
motion of the wave they might be able to come to contact with other 
masses generated in other regions. If they could then combine with 
each other, the individual with stronger structure would overcome 
the other and thus the stronger one would assimilize and devour up 
the weaker. Through such a struggle for existence, the structure 
would be more and more strengthened and the masses of assimilase 
would approach nearer and nearer the domain of the undoubted or- 
ganisms. 


3 


When two different protein molecules can combine with one another, 
they would mutually exert influences arising from the different struc- 
tures. On such a combination, if one of them is stronger in its 
structure than the other, only the structure of the weaker is disturbed, 
and if by this disturbance are lost the polar groups through which the 
weaker can combine with the stronger, this latter can be freed from 
the weaker and combine with another weaker molecule. In such a 
case the stronger protein is called an enzyme in a narrow sense. If 
the structure of the one is overwhelmingly stronger than the other, 
the structure of the latter would be changed to become identical to 
that of the former, that is, the latter would be assimilized by the 
former. In such a case the stronger one is called assimilase. The 
polymerization of numerous protein molecules is considered to result 
in the development of the strong structural effect and hence the assimi- 
lase is usually the polymerization product of protein molecules. Thus 
all the protein molecules may be enzymes in a wide sense, and the 
enzymatic nature may therefore be one of the characteristics of the 
proteins. 

The protein molecules produced in the primitive oceans would be 
polymerized orderly owing to the weakly acid reaction of the water, 
with the appearance of assimilase action. However, even before they 
could polymerize thus orderly and act as a fair assimilase, protein 


XI. THE SUMMARY OF PART IV 321 


molecules might have structural influences upon each other and mole- 
cules with stronger structure would more or less assimilize others. In 
this way the evolution of the protein structure itself would have had 
to continue to proceed for a dreadful span of time before the globulin- 
like protein which could polymerize orderly to exert strong structural 
effect could be produced. 

Lipids are not necessary for the production of the strong structure, 
but if lipids are not inserted among protein molecules, these latter 
would not be able to change the structure freely so as to be assimilized 
by the stronger assimilase. The evolution of proteins as well as or- 
ganisms would never be established without labile structures yielding 
to assimilation. In addition, free changes of the structure must be the 
essential feature of life, and thus where there is no lipid there is no 
life. 


4 


In order to strengthen its pattern and to become stable, the as- 
similase needs nucleic acids which may raise rigid structure in the 
polymerization product of proteins. On the other hand, the protein 
molecules in the assimilase are required to move or change freely for 
the achievement of various life phenomena, for which the rigidness of 
the structure is obviously unfavourable. 

The organisms seem to escape fairly from this dilemma by mixing 
lipids in the greater part of the protoplasm, thereby the protein molecules 
being rendered easily movable, whereas in its smaller part nucleic 
acids are inserted instead of lipids to make the structure rigid. Since 
this smaller part of the protoplasm owing to its rigidness in the struc- 
ture can remain unchanged even when the greater part containing 
lipids is altered in its structure, it enables the changed structure to 
return to its former state, as it can exhibit extensive structural effect 
by acting as a strong assimilase because of its rigid structure. 

Such part of protoplasm rich in nucleic acid is in higher organisms 
usually located in nucleus and acts as the standard template of the 
cell, and is composed of minute particles termed genes. Thus the 
genes are a kind of elmentary bodies capable of preventing the deforma- 
tion of the protoplasm pattern. As the pattern of the gene is, in 
general, unchangeable, the properties of the organisms can be retained 
unchanged. 

Various genes are present in the cell of higher organisms and each 
gene seems to direct each specific, restricted structure of the protoplasm 
protein. In other words, a gene is composed of protein molecules, only 


322 IV. THE PRINCIPLES OF LIFE PHENOMENA 


a certain restricted portion of which is of strong structure, and it 
can accordingly direct only a restricted portion of the protoplasm 
protein corresponding to the restricted strong structure, other portions 
being governed by other genes. 


5 


The protoplasm is a kind of mixed crystals composed of a variety 
of molecules or molecular aggregates, but the polar groups of the 
molecules must be arranged in an equal way in order to coexist in one 
and the same protoplasm. The pattern of this arrangement is subjected 
to the genes, and therefore the “‘crystal shape’’ of the protoplasm is 
determined by the genes. 

Principal components of this mixed crystal are elementary bodies, 
the chemical composition of which can not therefore be uniform even 
in one and the same protoplasm. Because of this different chemical 
composition, some elmentary bodies can behave as genes and some others 
as specific enzymes. 

There are two types of nucleic acids in protoplasm; one is des- 
oxyribonucleic acid and the other ribonucleic acid. The former seems 
stronger than the latter in their stabilizing action of protoplasm and 
so the elementary bodies containing the former can behave as genes, 
whereas those containing the latter are present in cytoplasm to be 
controlled by the genes, and apparently participate in the synthesis of 
proteins to endow the specific pattern directed by the genes to newly 
formed proteins. 

The genes thus control the pattern of all the proteins in the pro- 
toplasm, whereas all the proteins can be regarded as enzymes in a 
wide sense. All the enzymes, therefore, including enzymes in a narrow 
sense present in the cell, are to be subjected to the genes. 

Hormones are looked upon as substances produced physiologically 
by organisms for the purpose of interfering with the action of genes. 
They may be capable of directing the pattern of the protoplasm in a 
certain direction according to their specific structure, thus giving rise 
to the shape and function, favourable for the organisms, which cannot 
be attained by the genes. 

Various inorganic ions, like hormones, can exhibit similar effects 
upon the pattern of the protoplasm. However the ions, unlike hormones, 
are not the substances produced by organisms, but are the normal pro- 
toplasm components usually present there in large quantities, so that 
their effect must be extensive if not so strong in themselves as hormones. 
Organisms can develop various shapes and functions which vary with 


XI. THE SUMMARY OF PART IV 323 


the various parts of the body, presumably by making use of inorganic 
ions as well as hormones and related substances, even if the genes 
were Similar througout the body. 

The so-called antibiotic agents, such as penicillin, and synthetic 
chemotherapeutic agents, such as sulphonamide compounds, may be 
effective because they can interfere with the action of genes of the 
pathogens as do hormones. However, in contrast to hormones, the alter- 
ation of the pattern induced by these agents is unfavourable for the 
pathogenic organisms, presumably because of the extensive and pro- 
found disturbances occurring in a variety of enzyme systems as a result 
of the deformation of the pattern. Similar changes in enzyme systems 
may occur in the cells following the infection with some virus which, 
as a free gene, can interfere with the original gene. 


6 


The primeval organisms generated in the oceans were advanced to 
certain extents with the complication of their structure, meanwhile the 
elementary bodies composing the organisms might differentiate to 
possess various properties, and thus some of the elementary bodies might 
become to contain nucleic acid in rich amount so that they would 
behave aS primitive genes. Since such bodies were provided with 
stable and strong structures owing to the nucleic acid, they might 
be able to transmit their structure to other organisms even when 
liberated from the organisms. Thus such bodies would act as viruses 
or free genes. 

Moreover, Since the structure of such bodies might be reversible 
owing to the rigidness, they would not be deprived of the revérsibility 
even when present in a weak individual, which had been assimilized 
or devoured by a stronger individual, although thereby the transient 
loss of their original pattern could occur, so that they might be able 
to recover their original structure when freed from the influence of 
the bodies or the genes of the stronger individual, and would be able 
in turn to affect individuals which were still weaker than themselves. 
On such a Stage, the primitive organisms would be said to have only 
a pair of genes. 

Further developments of the organisms would lead to further differ- 
entiations in the protein structure of genes, resulting in the development 
of strong structure in restricted portions of protein molecules; thus 
different genes would come to be composed of different proteins which 
have each strong structure at a different restricted portion of the 
molecule. If two organisms containing each of such genes combined 


324 ' IV. THE PRINCIPLES OF LIFE PHENOMENA 


with each other, the different strong structures of each gene would be 
left in the combined individual instead of the complete disappearance 
of one of them. The same would result if the gene itself, after 
liberated from an organism, combined with the other organism. 

Thus, the splendid mechanism by which the characteristics of both 
individuals are combined and left in.the progeny would be developed, 
and since such a mechanism would be the most favourable for the 
evolution of the organisms the differentiation of genes would be exten- 
sively advanced until the highly complicated gene system of the present 
higher organisms was attained. 

The phenomenon in which two individuals having different genes 
combine or conjugate to produce new individuals that are endowed with 
the characteristics of the two is called sexual reproduction. In this 
case, one of them can achieve its function in a state of free gene and 
termed male gamete or sperm, whilst the other involves considerable 
quantities of cytoplasm in which combined pattern is to be spread or 
multiply and is designated female gamete or egg. 


7 


Viruses fail to retain their pattern unchanged, and consequently 
undergo senescence, when continue to multiply in the same kind of 
cell protoplasm because of their continual affliction by the assimilase 
action of the host protoplasm, though they are stronger than the pro- 
toplasm in the action. In entirely the same way, primeval primary 
organisms would be unable to keep their pattern when continue to 
multiply in the same group of protoplasm-like masses or primeval 
organisms. Therefore, they would need rejuvenescence for the con- 
tinued existence. This rejuvenescence would be achieved by the contact 
with another group of the masses just as viruses are rejuvenated by 
the host change. The contact with another group is regarded as nothing 
but the sexual conjugation; the sexual process would arise for this 
purpose of rejuvenescence, but in addition to this, through this process 
a new character would be obtained by the combination of two different 
structures, and hence the sexual process would become essential for 
the evolution of the organisms both primary and secondary. 

The secondary organisms, however, would apply a peculiar method 
for the rejuvenescence before they are enough evolved to achieve the 
sexual reproduction. The method consists in their taking advantage 
of the sexual process of the host with which they themselves are to 
be rejuvenated. 

With the advancement in their structure, viruses become, as a rule, 


XI. THE SUMMARY OF PART IV 325 


strong enough to be capable of spreading their pattern to the germ 
cells of the host, and thus they can be transmitted to the progeny. 
Primitive secondary organisms such as bacteria and protozoa which 
cannot as yet achieve the evident sexual reproduction, may succeed to 
the habitude of their ancestors, 7. e. viruses. If the pattern of such 
secondary organisms, say, bacteria is transferred to the mother germ 
cell of the host, the pattern will be transmitted by the germ cell to 
the progeny of the host in a form of virus-like particles. It is considered 
that the pattern exists it the germ cell in an altered state, but with 
the development of the germ cell into a full individual, the pattern in 
the virus-like particles also develop into its original form, followed by 
the fusion of the particles into the bacterial form. 

When the bacteria thus produced are injurious to the host, infection 
will follow, and in case of the continued production of the bacteria, 
the host will become bacterial carrier. On the other hand, it is possible 
that some produced bacteria exhibit favourable effect upon the host. 
In such a case the individuals harboured by the bacteria will be more 
fitted for their existence than those without the bacteria, so that the 
property to produce the bacterial pattern within its body will become 
an essential inheritable character of the host. The example belonging 
to this category is seen particularly in the relationship between insects 
and microorganisms. 

Primitive secondary organisms can be rejuvenated in such a manner 
to continue their existence, and at the same time can evolve higher by 
taking advantage of the new structure which the host has acquired 
through the sexual process. 


8 


Life is often compared to a fountain or a flame, and it is claimed 
that life isa constant flow of both energy and substances. The advan- 
cement of the assimilase with complication and enhancement in its 
function would be followed by the requirement of energy for the 
active function, and since this energy must be provided by the expendi- 
ture of certain substances, active life phenomena should be always 
accompanied by the flow of both energy and substances. Such a 
flow is, however, only a by-product of life, never the life itself; 
presumably the distinct flow was revealed only after the assimilase 
had considerably evolved. 

Viruses are known to be able to multiply in ‘‘dead’’ cells, that 
is, in the cells which have lost the faculty of multiplication. This 
fact indicates that for the multiplication of viruses at least not so 


326 IV. THE PRINCIPLES OF LIFE PHENOMENA 


much energy is required as for the multiplication of the cell, since 
the cells are ‘“‘dead’’ presumably because the mechanism, whereby the 
energy required for the cell multiplication is produced has been de- 
stroyed. It is generally known that the action of proteolytic enzymes 
is not associated with neither significant expenditure nor recognizable 
production of energy, whereas viruses are regarded as a kind of enzymes 
capable of changing the protein structure. Also in view of this fact, 
it can be concluded that energy is not, or scarcely, needed for the 
mutliplication of viruses. This conclusion is compatible with the 
assumption that the primary organisms were generated by the mech- 
anisms of virus multiplication in the protoplasm-like masses in which 
no energy-producing system was present. 


9 


The progress of inactivation of phage or of rennin by inactivating 
agents such as tannin or the respective antisera is occasionally found 
to be oscillating as if it were a physical phenomenon. Since the protein 
molecules of the same kind tend to combine with each other to make 
a Single system, each protein molecule ina solution is unable to behave 
of its own accord but only permitted to act in concert with other 
molecules. This may be the main reason for the occurrence of oscil- 
lation. 

Even if molecules in a free state oscillated in their chemical 
reaction, the oscillation would not come out as such because of their 
arbitrary behaviour. Protein molecules have a structural reversibility, 
that is, the changed molecules are striving to recover their original 
structure; on account of this property they may rebel against the 
action of inactivating agents resulting in the reaction which may re- 
veal itself as the oscillation owing to the mutual combination of the 
molecules. 

In the protoplasm, the association between the protein molecules 
is complete, so that oscillation is especially evident in the organisms. 
The protein molecules are able to freely change their structure owing 
to the existence of lipids in spite of their orderly association. 

Protoplasm particles, including virus particles, when suspended 
in a sugar or inorganic salt solution after a manipulation which may 
cause their structural disturbance, can sometimes absorb and sometimes 
excrete the solute. This may depend upon the adsorption and elution 
of the solute according to the change in the protein structure. In this 
Way, organisms can achieve absorption and excretion by changing the 
structure of protoplasm protein. 


XI. THE SUMMARY OF PART IV 327 


When the change of the protoplasm protein is raised by in the 
contraction and extension of the molecules. the oscillation comes out 
as a rhythmic motion. In general, the movement of organisms may 
be achieved by such a contraction and extension of protein molecules 
even when it is not oscillating or moving rhythmically. 

Not only the motion but also all the life processes are presumably 
given rise to by various changes of protoplasm protein. Since the 
changes should be associated with the adsorption and elution of a varie- 
ty of ions and perhaps also with the appearance and disappearance 
of polar groups, life phenomena are always accompanied by electric 
changes as for example action current. 


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PART V 


THE NATURE OF EVOLUTION 


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CHAPTER I 
THE THEORY OF MEMORY 


1. The Faculty of Protein to Memorize Its Structure 


As was described repeatedly, many evidences can be presented 
suggesting that protein molecules can return to the structural pat- 
tern, which they once possessed, when their structure is disturbed by 
some adequate agents. The writer designates such a,phenomenon as 
the reversibility of protein structure or the memory ef,protein. Since 
this phenomenon is of the most importance to elucidate the mechanism 
of both the individual development or ontogeny and the organic evolu- 
tion, it will further be discussed in this chapter in great detail. 

In general, the reversible inactivation of physiologically active 
proteins such as enzymes and toxins can be looked upon as based upon 
this character of proteins. With viruses many phenomena apparently 
due to this character are also known. For example, a virus which has 
been changed in its property by an environmental change may recover 
its original property when brought under another environmental con- 
dition. 

This phenomenon can be interpreted as indicating that a protein 
which can take two structures, A and B, attains one structure, A, 
under a condition where A-structure is stable, while under another 
condition where the other structure, B, is stable it is shifted to the 
structure B as follows: 


b 
A-structure B-structure. 

A-structure is stable under the condition of ‘‘a’’ and B-structure 
under ‘‘b’’, but in most cases A-structure seems not to be shifted to B 
by a mere change of the environmental condition, @ +b, unless some 
proper stimulus disturbing the protein structure is provided. This may 
be analogous to the phenomenon that water can be coold under 0°C. 
without changing into icc, but that the super-cooled water will be 
suddently frozen over on the application of an appropriate stimulus. 
This phenomenon was already discussed as we dealt with the denatu- 
ration of proteins (Chapter X, Part III). 


336 V. THE NATURE OF EVOLUTION 


It has been known that bacteria or phages damaged by ultraviolet 
light can be fairly reactivated by the subsequent exposure to visible 
light (1). This fact may be explained if we assume that reactivation 
occurs because reactivated structure is stable under the exposure to 
visible light which may cause the proteins to return to their original 
structure. Bawden and Kleczkowski (2) have found with Phaseolus 
vulgaris that irradiation for two to three minutes with ultraviolet 
light had not immediately obvious effects on the leaves, but that when 
kept in the dark for twenty-four hours after irradiation, became se- 
verely bronzed within the next two days; this was prevented when 
the irradiation was succeeded by exposure for some hours to day light. 
In this case day light presumably acted upon the leaves to make its 
protoplasm structure, which had been changed by the ultraviolet light, — 
return to the original structure before the symptoms came to the forth. 


It has been found that a strain of ultraviolet irradiated E. col 
can be reactivated also by heat (3); the reactivation has been reported 
to be likewise possible by the addition of various metabolites (4). Fre- 
quently we observed that phage inactivated by unknown cause is re- 
activated by heating to an adequate temperature or by the addition of 
inorganic salts (5). Heat or salts in these cases may disturb non- 
specifically the protein structure to initiate the returning change. 
Visible light may act in a similar way, but in addition it may exert 
a specific effect in favour of the original structure. 

Herpes may occur following febrile diseases and poliomyelitis fol- 
lowing tonsillectomy, and usually common cold is associated with 
chilling. These phenomena may be based upon the reversion of virus 
structures rather than upon the generation of the viruses themselves. 
Vago (6) has claimed that a virus which causes polyhedron disease is 
present in the majority of the insect, Bombyx mori, remaining latent 
through several generations, but it is activated and accordingly the 
disease is developed by the administration of fluorine compounds or by 
the feeding of insects on the leaves of the Maclura. 

The recovery from virus diseases is possibly ascribed to this 
memory phenomenon. ‘Thus, when an environmental condition, under 
which the structure of a virus is stable, is provided in the protoplasm 
of a certain organism, the virus will be generated or the organism 
will be readily infected by the virus, whereas if the environment is 
changed and a new environment is favourable for the original structure, 
the virus structure will be expelled with the cure of the disease. 

On the one hand, as already mentioned, seasonal effect is striking 
on the incidence of virus diseases, while on the other hand functions 
of hormones are under the influence of seasonal factors. This sug- 
gests strongly that the hormones are involved in the environmental 


I. THE THEORY OF MEMORY 337 


condition directing the virus structure. 

In some particular cases, however, it seems that temperature alone 
can act as the directing factor. According to Kunkel (7) aster yellow 
virus is lost when the leafhoppers carrying the virus are exposed to 
32°C. The insects exposed to this temperature for a day lose the 
virus, but on lowering the temperature to 24°C., the virus appears in 
a few hours, whereas those heated for a week regain the virus only 
after two days or longer at the lower temperature; the virus is never 
regained when the insects are heated for more than 12 days. This 
fact indicates that the structure of the virus is stable at 24°C. but 
unstable at 32°C. at which the normal protoplasm structure is stable. 
It is highly suggestive, however, that the prolonged exposure to the 
high temperature renders the virus structure irreversible. This is 
considered to be dependent upon the “‘oblivion’’ of the virus structure, 
an extremely important phenomenon which should be discussed later 
in great detail. 

Numerous facts indicating that the structure of plant viruses are 
apparently unstable at higher temperatures have been shown. For ex- 
ample, Vinca rosea and Nicotiana rustica infected with aster yellow 
virus are freed from it after growing for two weeks at 40°C. and V. 
vosea is also cured by immersion in water at 45°C. for a few hours. 
This V. vosea is also freed from potato witches’ broom and cranberry 
false blossom virus by growing for 14 days or more at 42°C. Potato 
tubers with diameters up to 2 cm., infected with witches’ brooms 
virus, are cured by storing at 36°C. for 6 days (8). 

This seems to be the case also with animal viruses. We may 
generally become feverish following the infection with a virus, a_phe- 
nomenon which may be of significance from the point of the expulsion 
of virus structure. It has customarily been believed that sweating 
treatment with a sudorific or with a hot drink is effective to common- 
cold. It cannot be said, however, that high temperatures are always 
effective to make virus structures unstable. Since herpes febrils is 
generally raised accompanying with feverish diseases, the structure of 
herpes virus must be stable at higher temperatures in contrast to 
that of common-cold virus. 

Thus, a virus structure may be lost when the environmental con- 
ditions become unfavourable for its existence, resulting in the cure of 
the diseases, but as the virus structure itself is also reversible the 
disease will return when the conditions become again favourable for 
the virus structure in so far as the structure is not ‘‘forgotten’’. 

At present, chemotherapy is winning a brilliant victory over the 
campaign against some bacterial diseases. The effect of the drugs 
on virus diseases, however, appears to be insignificant, presumably 


338 V. THE NATURE OF EVOLUTION 


because of the likeness of the virus structure to the normal structure 
of the protoplasm and because of the concealment of virus structure in 
the protoplasm, not existing as an utterly independent entity. This 
may also hold for bacteria; namely, chemotherapy may be ineffective 
against bacterial pattern which is present in the protoplasm in a latent 
state or in a state not yet developed into their full pattern. For in- 
stance, chloromycetin, according to our observation, can for a period 
of time expel dysentery bacteria from the carrier but the bacteria be- 
come soon demonstrable in the feces, indicating that the antibiotic is 
ineffective to the undeveloped pattern of the bacteria. Streptomycin 
may likewise be effective only to the tubercle bacilli having the com- 
pletely developed pattern, so that the secondary infection by the bac- 
teria themselves may be well effected by the drug, but with the in- 
complete pattern or the latent bacteria in the tissues from which the 
bactéria are being produced it may be powerless. 

An accomplishment of radical therapy by physical or chemical 
agents, however, may not be impossible if it is attempted from the 
view point of the reversibility above suggested. Red-ray therapy is 
an old practice for small-pox, that is, red-ray exhibits beneficial effect 
upon the disease which will be cured readily if the patient is exposed 
to red-light. This should be comparable to the fact that bacteria or 
phages damaged by ultraviolet-ray are activated by visible rays with 
the recovery of the original structure. Similarly, tobacco-mosaic is 
known to be cured by the exposure to red or blue light, whereas some 
other plant virus diseases are said to be intensified by the visible 
ray (9). 

Chemical agents may also be efficacious in this respect. The 
agents must have hormone-like action in order to expel the unfavour- 
able structures. It has been found by Wright (10) that coli bacteria 
which underwent a mutation can recover their original character in 
the presence of certain organic acids having a particular configuration. 
Tartaric acid has this particular configuration and therefore proves 
to be effective, but it should be noted that only natural p-form is 
active, the L-form being inactive, suggesting its hormone-like action. 

Again according to Hamre ef al. (11) benzaldehyde thiosemicar- 
bazone reduced the fatality rate of mice inoculated intranasally with 
vaccinia virus. This chemical agent does not appear to inactivate the 
virus as a result of prolonged contact im vitro. Derivatives with sub- 
Stitutions in the para-position of the benzene nucleus or in the 4-posi- 
tion of the thiosemicarbazone molecule produce little or no protection 
against vaccinia infection in mice (12). The =N—NH—CS—NH:z group, 
as such, appears to be essential for antivaccinal activity (13). Acker- 
mann (14) found that administration of sodium fluoroacetate to mice 


I. THE THEORY OF MEMORY 339 


infected intranasally with influenza virus inhibits the growth of this 
virus in the lungs of the mice; further he (15) discovered that three 
a-aminosulfonic acids cause marked inhibition of multiplication of in- 
fluenza virus in tissue of the embryonated egg. Again, it has been 
reported that 2, 4-dinitrophenol inhibits completely the propagation of 
influenza virus in cholioallantoic membrane (16). These reagents 
showed no virucidal effect i” vitro. 

As stated above, the so-called antibiotics may affect directly the 
complete pattern of pathogens and accordingly their effectiveness seems 
to be restricted. Shope (17) has shown recently, however, that a cer- 
tain mould can produce an agent which may provide an unfavourable 
condition against the production of a virus pattern. He has isolated 
an antiviral substance from penicillium funiculosum, which is effective 
upon infection of mice with viruses such as SK encephalomyelitis 
virus. He believes that the agent causes an inhibition or interruption 
of multiplication of the virus presumably by interference with some 
stage in the developmental cycle of the virus. 

However, not all the agents which prevent the virus-reproduction 
can be regarded as being capable of specifically acting to inhibit the 
development of a certain virus pattern. Some may non-specifically al- 
ter the protoplasm structure to render it inadequate for the establish- 
ment of the structural change due to some viruses. 

Mirick eé al. (18).claimed that urethane, given paraentrally or oral- 
ly to mice, increased the severity of the infection with pneumonia 
virus of mice. Not only were the lesions more extensive but mice 
could be infected with smaller inocula of virus and the multiplication 
of virus in the lung was enhanced. It should be naturally expected 
that certain reagents are able to inhibit the development of a virus 
pattern, while others promote it as in this example. 


2. Training Effect and the Oblivion of Memory 


The reversibility of protein appears to be especially manifest in 
the protoplasm. Meanwhile, it should be noted that in the protoplasm 
the reversible change from the normal to a certain other structure 
appears to be facilitated by the repetition of the change. The so- 
called anamnestic reaction (anamnesis=recollection) may be cited as an 
example of this phenomenon. This reaction consists in that an animal, 
which previously produced antibody in response to a specific antigenic 
substance, will begin anew to produce the antibody vigorously when 
a minute quantity of the antigenic substance is injected. For the 
establishment of the reaction the repeated preliminary injection of the 


340 V. THE NATURE OF EVOLUTION 


antigenic substance is generally necessary; this reaction may depend 
upon the recovery of the former pattern which was provided and 
strengthened by the repeated injection of the antigen, as the pattern 
produced by the antigen can be regarded as the changed structure of 
protoplasm protein of the antibody-producing cells. 

An elastic substance, as for example a rubber tube, may become 
easily bent at a definite pertion at which a bending was previously 
applied. If the bending is repeatedly applied at the same portion, it 
will become more supple, and the bend will occur at the portion even 
when a force is applied at another portion. Anamnetic: reaction is said 
to occur likewise even when a proper substance different from the for- 
mer antigen is injected. The structural reversibility of protoplasm pro- 
tein can thus be analogized with the elasticity of elastic substances. 

On the other hand, there are many evidences to prove the presence 
of elasticity in the protein molecule itself. Organisms are without 
exception composed of protoplasm whose main components are proteins, 
while protoplasm itself is elastic and accordingly all the organisms 
are elastic, a fact which may be based upon the elastic nature of the 
protein. Strong elasticity can actually be demonstrated in isolated 
proteins such as gelatin or fibrin. As is well known, casein can pro- 
duce an elastic paste if treated with alkali. Such an elasticity may 
be attributed to the thread-like form of the molecules which can com- 
bine end to end to form a long chain as in the case of other elastic 
substances. The elasticity of proteins appears to be not only revealed 
against physical forces but also against chemical agents. 

A splendid application of this elastic nature of proteins, 7. e. the 
reversibility, can be seen in the memory of nervous cells. When an 
animal is repeatedly subjected to the same kind of stimulus, the re- 
action to the stimulus will become more rapid and more exact, thus 
resulting in the training effect and in the strong memory. The proto- 
plasm structure of our brain cells may undergo a change in response 
to a stimulus; the change will soon fade away, but when afterwards 
a related stimulus is given, the change will be revived owing to the 
reversibility of the structure. This must be the recollection of the 
stimulus. Every one knows that recollection is strong when the 
stimulus was previously given repeatedly or severely, and the recol- 
lection of acertain affair, when its ‘‘impression’’ was strong, is given 
rise to by some stimulus which has no direct connection with the 
affair. 

Some memories are long-lasting, strong impressions being generally 
held in the brain cells for life. Since the protoplasm protein of the 
brain is to be renewed repeatedly during such a long period of time, 
the memory should be transmitted successively to newly formed pro- 


I. THE THEORY OF MEMORY 341 


tein molecules. The renewal of the protein in the brain cells may be 
tedious as compared with that of other cells, but it seems little doubt 
that even the brain cells have to change occasionally the old protein 
molecules with new ones. If the pattern of the memory was not 
transferred to the newly synthesized protein, the memory of the cells 
would be lost on every renewal of the protein. Thus, the protein 
holding a memory must involve a specific structural pattern cor- 
responding to the memory. In other words, the transmissible pattern 
of memory must be a structure produced in the protoplasm protein 
by the ‘‘impression’’. 

Now, if we assume that structural factors which may prevent the 
change of a protein from A to B-structure are removed when the pro- 
tein achieves the change A-—B, it may be expected that the protein 
having recovered the original A-structure, will achieve the change 
more readily than before, and since the repetition of the change may 
result in the more complete removal of the preventing factors, the 
repetition of the change will facilitate the reaction the more. The 
protein structure resulting from the removal of such factors can be 
considered as the pattern of the memory. This may be comparable to 
a path beaten across a wilderness overrun with grass. If a man 
traverses the pathless wilds for the first time, he must have a hard 
walk, but on repeating the traverse the grass will be gradually re- 
moved until at last an easy path will be made. The above cited in- 
stance that the rubber tube is easily bent at a certain part on the 
repetition of the bending at that part, may also depend upon the re- 
moval of the structural factors which prevent the bending. 

The path beaten across the wilds by repeated walk will be lost 
by being overgrown with weeds if the walk is given up for a long 
time. In a similar way, oblivion may occur in the brain by the reap- 
pearance of the preventing structural factors which were once removed. 
Thus it can be said that oblivion of the memory is also a result of 
the structural return to the original form. 

The concept thus far formed of the memory phenomenon is illus- 
trated in Fig. 32. Protein-I which is folded in the form-A under a 
certain environmental condition is converted to the form-B when the 
environment is changed in favour of the establishement of B-form. 
During this change the protein is temporarily to be unfolded into the 
polypeptide chain; the side chains represented by © and © is assumed 
to prevent the change, AB. The repetition of this change leads to 
the removal of these preventing side chains, and as a result protein-I 
is changed in its structure into II. 

When such a change is once established, the conversion to B-form 
will occur with facility, training effect being thus attained. Since the 


342 V. THE NATURE OF EVOLUTION 


protein molecule having synthesized asa replica corresponding to such 
a template protein possessing the structure of a memory should have 
the same structure of the memory, the memory or the training effect 
can be “‘inherited’’. However, if the training is neglected, the side 
chains, © and ©, will gradually reappear, with the recovery of I- 
structure, on account of the reversible character of the protein struc- 
ture, thus occurring the oblivion of the memory. 


Fig. 32. Diagram of memory and oblivion in proteins. 


The situation may be somewhat different with B-form; thus, when 
the protein long stands without returning to A-form, under the en- 
vironment which favours B-form the side chains will reappear but 
not in the same arrangement as in I; the arrangement occurs so as 
to be favourable for the protein to take the B-form. As a consequence, 


I. THE THEORY OF MEMORY 343 


the protoplasm structure is converted to III. Thus, both forms, A 
and B, possess each specific structure. If the protein acquires III- 
structure B-form becomes the normal state of the protein, so that the 
return to A-form becomes difficult, that is, when the protein stays for 
a prolonged time at B-form it will forget A-form. 

The newly production of the rearranged side chains in the protein 
left long under a new environment must be the result of the rear- 
rangement of polar groups so as to become adaptive to the new form. 
The same rearrangement of polar groups will take place when the 
protoplasm is affected by a virus, in response to whose structure the 
polar groups of the protoplasm protein are rearranged. Such adaptive 
rearrangement of the structure appears to be a common character of 
proteins, but the character is never peculiar to proteins, since similar 
adaptation to a new environment can occur with usual elastic sub- 
stances other than protein. A piece of elastic thread, for instance, 
whether it is composed of protein or steel, after standing in A-form 
for a prolonged period can be changed to B-form only with difficulty 
at first, but the repetition of the change will make the change easy 
and a long maintenance of the B-form will render the form normal, 
the return to the original form becoming difficult. Also in such a 
case, the adaptation may occur owing to the modification of the inner 
structure of the thread to adapt itself to the new form. 


3. The Cause of Aging 


It has been known for a long time that generally colloids includ- 
ing proteins grow old like livings. In the opinion of the writer, the 
process of just considered adaptation of the protein to environments 
leads in the long run to aging. During the adaptation process the 
fixation of the inner structures may be advanced so far that the forms 
of the protein molecules become hardly changeable to other forms. 
Such a hardening of the structure is considered to be aging. 

We shall consider in detail of this phenomenon with Fig 33. Now 
it is assumed that a polypepide chain can take two forms, @ and 8, 
according to the environment, A and B, under which it stands. If 
the protein is left for a prolonged period under the same condition, 
the inner structure will gradually be fixed by the rearrangement or 
the shift of polar forces, until it attains the forms @, or b,. The pro- 
tein in such a form is ina well adapted state so that the change from 
one form to the other will be brought about only with difficulty, but 
if the change is repeatedly raised by the repetition of environmental 
change, the arrangement of the polar forces which may hinder the 


344 V. THE NATURE OF EVOLUTION 


change will be removed or rearranged to return to the original state; 
as a result the change will come readily to occur. 


Environment A Environment B 


Fig. 33. Aging of protein molecule. 


On the other hand, further progress of the fixation due to the per- 
sistent staying under the same environment will result in the es- 
tablishment of the form @, or b;. In such a state the polypeptide 
chain is folded very firmly by the mutual attraction of polar forces of 
opposite signs, leading to a shrinkage accompanied by a deformation 
and the loss of elasticity as well.. This must be aging. 

It is a well known fact that young tissues are tender and elastic, 
while old ones are hard and fragile. Similarly, as is generally recog- 
nized, colloids tend to change into gel when become aged, thereby the 
component particles becoming attached firmly and the contained water 
is pressed out as known as syneresis, a phenomenon which is also well 
acknowledged with living tissues. These phenomena may be expected 
from this figure. Instead of the long thread of polypeptide in this 
figure, many long colloidal particles may also be considered which are 
attached to one another in a colloidal system. 

In the previous Part the writer has assumed that the senescence 
of viruses or of primeval organims may be mainly attributed to the 
effect of the assimilase action of the host or of the surrounding mas- 
ses with which they are to be evolved. However, it has now become 
clear that another important reason for the senescence is involved 
in the nature of the protein itself. The senescence due to the 
assimilase action of the host is brought about because the viruses are 
persistent on the same host, that is, persistent on the same environ- 
ment. Thus, it may be said that in both cases senescence, whatever 
the cause may be, results from the persistent staying under the same 
environment. 

The senescence therefore, whether it be raised by the adaptive 
nature of the protein itself or by the assimilase action of the host 


I. THE THEORY OF MEMORY 345 


will be prevented by the host change or in general by environmental 
changes. The host change as well as the sexual reproduction may be 
effective for the rejuvenation, as the individual is able to come into 
contact with other individuals by these processes, whereby the internal 
structures which are becoming fixed are unloosened and unfolded, and 
in addition the intermixing of the structural component of another 
individual can contribute to prevent the fixation. 

As already stated, according to the writer’s view, the cytoplasm 
is rejuvenated, presumably at every cell division, by the template 
action of the gene. The cells, however, must become senescent as 
above considered, a fact which seems to contradict with this view. 
But it should be realized that the gene itself gradually grows old. 
The cytoplasm recovers its proper form by the template action of the 
gene, but the template itself has to become aged with which the 
cytoplasm also must grow old, as the cytoplasm is to be endowed with 
the gradually aging pattern of the gene. 

Individuals are thus bound to become old. As the aging is the 
general fate of the elastic substances composed of thread-like constitu- 
ents, the senescence is the unavoidable fatesof livings. Elastic sub- 
stances such as gum, just like the living protoplasm, exhibits the 
phenomenon of memory and forgetting; at the same time they can 
adapt themselves to the environments until finally undergo senescence. 

On the other hand, senescence makes a great contribution to the 
evolution of organisms; the evolution might be even impossible with- 
out senescence. In order to remove the old-patterned individuals for 
the promotion of more-advanced juniors, senescence is of course indis- 
pensable. But in addition, senescence of a proper degree plays an im- 
portant part in the evolution. Since rigid structure was necessary for 
the continuance of the existence of the organisms, in order to achieve 
the evolution, to become rigid in their structural pattern would be 
required for viruses and primitive organisms including even primeval 
protein molecules. The writer considered previously that this rigidness 
might generally be acquired through the struggle for existence; but 
without this they would surely be able to become stronger in their 
structure, for the protein is provided with the nature in itself to be- 
come thus rigid in its structure. 

Newly generated viruses are as arule weak, but they may be able 
to be stabilized gradually without the struggle for existence owing to 
this nature. Thus, viruses and primeval organisms can acquire auto- 
nomically the rigid structure necessary for acting as strong assimilase; 
the rigid structure is raised without even increasing the amount of 
nucleic acid, the stabilizing agent of the protein. 

Organisms can achieve the evolution thanks to this character of 


346 THE NATURE OF EVOLUTION 


the proteins, a character leading to the adaptation and finally aging, 
and also to the recollection and forgetting, without which the evolu- 
tion would be entirely impossible. The mechanism of the evolution 
can be fairly elucidated by taking into account of this character of 
the protein as will be fully discussed in later chapters. 


CHAPTER II 
ONTOGENY 


1. The Principle of Individual Developmen: 


The development of a single cell into a complex, highly differ- 
entiated organism is one of nature’s most marvelous pageants, a series 
of events that occurs in such an orderly fashion as to fill the observer 
with awe, and yet the writer believes that this marvelous event con- 
sists of nature’s tricks only based upon the reversible nature of pro- 
tein above mentioned. 

According to his concept, there is a mutually reversible relation 
between the protoplasm structure of germ cells and that of somatic, 
and somatic cells are converted to germinal cells under a series of 
proper stimuli under which the structure of the latter is stable. The 
ontogeny or the individual development is considered to be no more 
than the return of. the germinal cell to the original, differentiated 
structure. Since the germinal cell can function independently of the 
somatic as a unicellular organism, its protoplasm must have the primi- 
tive structure of unicellular stage of the organism from which it has 
evolved. and the structure must have attained by the reversibility. 

The spores of unicellular organisms may be analogous to the ger- 
minal cells of multicellular organisms and may be produced when 
unicellular organisms, such aS some bacteria or protozoa, are reduced 
in their protoplasm structure to more primitive structure under a 
change of environmental conditions. A new environment under which 
the primitive structure is brought about must favour the reduction of 
the structure; as a result of the reduction the cells may be decomposed 
into virus-like particles, the more primitive forms of the organisms, 
thus producing spores, which, however, can return to the original 
structure under certain other environmental conditions, with the re- 
covery of the form of unicellular organisms. As the protoplasm is 
looked upon as a kind of liquid crystals of proteins, the shape of the 
protoplasm should be determined by the structure of the protein. 
Therefore, the return of the structure to the original should be fol- 
lowed by the recovery of the shape. 

As stated in the previous chapter, organisms are bound to grow 
old when they stand always under the same environment, but they 


348 THE NATURE OF EVOLUTION 


can be rejuvenated by adequate environmental changes. The spores 
or germinal ceils must be the splendid rejuvenated specimens produced 
by the environmental change. The spores or germinal cells may be 
comparable to the form @ in Fig. 33 which was cited in the previous 
chapter, and somatic cells to b,, and accordingly J, is the differentiated 
structure of @, to which Bb, can be transformed if the environment is 
changed from B to A. And further the form @, the spore or the ger- 
minal cell, thus recovered from b, can again be changed to b,, the 
somatic cell or the vegetable form, when the environment is again 
changed to B, and if b, can take cell form while @ only virus-like 
particle, the cell composed of 8, will be decomposed to the virus-like 
particles when brought under the condition A. Thus the spores or 
the germinal cells are looked upon as the completely rejuvenated form 
of the organisms. 

As for higher plants, it seems generally accepted that germ cells 
can be directly originated from somatic cells, whereas in animals the 
cells, from which germ cells are to be produced, are usually believed 
to be distinctly separated from the somatic cells at their early stage 
of the development. It should be born in mind, however, that the 
cells which seem to be thus separated from somatic cells are not the 
germ cells themselves but cells from which germ cells are to be 
produced. Not only gonads but also other organs are generally de- 
veloped separately from an early stage of the development. At present 
the majority of embryologists hold the view that the cells constituting 
gonads in which the germ cells become differentiated are not essen- 
tially different from usual somatic cells. 

Many species of planarians have great power of regeneration, and 
when an individual of them is divided by cut into a head- and a tail- 
piece, each of which heals the wound and forms the missing parts by 
cell division, whereby germ cells are subsequently produced from the 
individual regenerated from the head-piece in which gonad was origi- 
nally absent, indicating clearly that germ cells are produced from 
somatic cells. 

In short, the germ cell thus produced from the somatic cell by the 
reduction in its structure to: primitiveness can return to the differ- 
entiated structure of the somatic cell, if a proper stimulus is given 
under suitable conditions favourable for the development of the differ- 
entiated, original. structure. This return process in itself is on- 
togeny. 


II. ONTOGENY 349 


2. The Mechanism of Regeneration 


Organisms can regenerate in general their lost parts, and some- 
times even the whole individual is formed from a small piece. This 
regeneration process is particularly conspicuous in plants, although 
some animals such as sponges and hydroids can regenerate new indi- 
viduals from cuttings, as do many plants, but in many animal species 
the restoration of lost parts is more limited. 

It is a note-worthy fact that, in the case of organisms able to re- 
generate, sectioning of a structure is followed by a brief period in 
which the ‘‘blastema’’ forms, undifferentiated cells accumulating in the 
region of the wound (19). Thus, it appears that certain cells in the 
region of the wound are reduced in their structure to the primitive, 
undifferentiated, from. which subsequently the complicated, differ- 
entiated structure is anew recovered. Regeneration must be, there- 
fore, a reversible structural change of protoplasm just as ontogeny. 

Anaerobic glycolysis is the metabolic process peculiar to undiffer- 
entiated, primitive cells and was discovered by Warburg in cancerous 
and in embryonic tissues. It shoud. be noted that this primitive meta- 
bolic process was found in normal cells after injuries, indicating the 
occurrence of functional reduction in the wound regions which must 
be based upon the structural reduction (20). 

Such a reduction of the structure to a primitive state may be 
caused by the stimulus of injury, and in each cut piece a primitive 
structure like that of germ cells may be produced, from which the 
original, differentiated structure will subsequently be restored, if each 
piece has the capacity to form a normal adult. 

The remarkable process of reassociation of cells after dissociation 
occurs in some sponges and hydroids. For example, pieces of Micro- 
ciona prolifera, the common red sponge of the Atlantic coast, can be 
squeezed through silk bolting cloth so that the cells are separated or 
dissociated. If these fragments are allowed to settle upon the bottom 
of adish of sea water and remain undisturbed, they will become reas- 
sociated in small spherical masses within 24 hours and then develop 
as thin encristations upon the bottom (21). Attention should be paid 
to the fact that masses arising first are of a larval stage similar to 
the typical normal larva. This phenomenon may be also based on the 
formation of primitive structures by cutting. 

Thus there is no doubt that somatic cells of certain organisms can 
recover their primitive structure when they are stimulated by such a 
simple factor as cutting. Germinal cells may develop into somatic, 


350 V. THE NATURE OF EVOLUTION 


probably because the environmental condition under which the germi- 
nal cells were produced is changed to become favourable for the for- 
mation of somatic cells. However, the condition or the factor which 
formed the somatic cells may usually be removed when the somatic 
cells are once completed, so that somatic cells are existing normally 
under the condition favouring the primitive form, but without return- 
ing to the original, just as water can sometimes be present as such 
under 0°C. without being changed to ice. Therefore, the completed 
somatic cells may be in a metastable state with a great tendency to 
change into the primitive structure. In this train of thought it is easily 
explained that a simple stimulus such as cutting is sufficient to cause 
the recovery of the primitive structure just as super cooled water 
is readily changed into ice on the application of a slight stimulus. 

The capacity of regeneration seems to be especially great in plants. 
This indicates that some somatic cells of plants have particularly 
strong tendency to return to the primitive state. On the other hand, 
in higher animals regeneration occurs only to a small extent and even 
with difficulty. This may be due to the presence of some mechanism 
preventing the recovery of the primitive structure. In the majority of 
animals, gonad-forming cells are differentiated for the purpose of the 
production of primitive cells, 7. e. germ cells, whereas, in plants, 
primitive cells including germ cells appear to be generated with ease 
from usual somatic cells not differentiated for the purpose. This fact 
also shows that in plants the structural reduction to the primitiveness 
can easily take place. Again, it is known that a variety of physical, 
chemical and biological agents readily induce pathological growth in 
plants; in general, cells of primitive structure have the capacity of 
vigorous growth, while pathological growth is associated with the 
primitive charactcr as with cancerous cells. Of physical agents in- 
ducing such a pathological growth mechanical injury is well known. 
Diseased growth considerably beyond that necessary for healing fre- 
quently occurs in plants following injury. A large number of chemi- 
cals are capable of inducing more or less diseased growth. This is so 
common that one can expect diseased growth to follow the application 
of almost all chemicals at a concentration which induces injury but 
does not kill. Also living agents, such as bacteria, fungi and nema- 
todes, cause various kinds of pathological growth (22). 

Thus, it may be concluded that plant cells are strongly inclined 
to be reduced to primitiveness following the application of a stimulus 
disturbing the protoplasm structure, in the same way as the super- 
cooled water tends to be readily frozen on the application of a disturbing 
stimulus. This character is especially manifest in plants because it 
is a beneficial for them; namely, since this character enables plants 


II, ONTOGENY 351 


to regenerate and the individuals having the stronger regenerative 
capacity might be able to survive the more, the character would be 
more and more advanced until the present status of plants was reached. 

The occurrence of proliferative changes following injury, however, 
is observable also in higher animlas though not so manifest as in 
plants. Observations of Cooke ef al. (23) on the changes which oc- 
curred in excised rabbit and human skin after injury have shown 
that even after removal from body certain tissue elements may retain 
the ability to react with proliferative changes in response to tissue in- 
jury if kept under artificially stimulated physiological conditions. 


3. The Formation of Organs 


Unlike the growth of usual crystals, in the development of organ- 
isms different shapes are developed on the different parts of the body 
with the formation of a variety of organs. Fundamental principles to 
induce such a dissimilarity was already discussed in Part IV, Chapter 
VI, stating that the main factors involved in it are considered to be 
hormones, which provide different stimuli to different parts of the 
body, according to which different restricted structures are developed, 
with the production of the complicated ‘“‘crystal shape’’. 

If 5-stimulus is added to a germinal cell having A-structure, the 
structure will be converted to B with the formation of the ‘“‘crystal 
shape’’ corresponding to B-structure. In the same way, c-stimulus 
will induce C-structure and its “‘crystal shape’’. In this case, B and 
C are considered to be organs generated from A, the germinal cell, 
and b and ¢ are the hormones inducing the respective organs. A has 
the ‘‘predisposition’’ to be changed into both B and C in responding 
the respective stimuli, b and c. In other words, A recovers B-struc- 
ture under b-stimulus, and C-structure under c-stimulus. As will be 
mentioned later A is a structural complex, not a single structure. In 
the following the matter will be considered in a less abstract way. 

As was mentioned already, protein structure is greatly influenced 
by environmental factors. On the other hand, the surface of an egg 
cell may not be similar with environmental factors, such as gravita- 
tion, light, and temperature. Whitaker (24) stated that in the egg of 
the brown alga, Fucus, the differentiation is clearly influenced by en- 
vironmental factors before the first nuclear division occurs. Thus, 
gradients of visible light, ultraviolet radiation, metabolic products, 
hydrogen ions, and temperature are capable of determining polarity of 
an egg; even mechanical elongation of the egg influences its polarity. 
In addition, since the protoplasm consists of thread-like protein mole- 


352 V. THE NATURE OF EVOLUTION 


cules arranged in parallel alignment, the cell itself should have a 
structural polarity. Harrison (25) believes that the polarity is based 
upon the dipole character of protein molecules. 

Such a polarity, due to the asymmetrical character of protein 
molecules and numerous environmental factors as well, may control 
the manner of distribution of some components, such as yolk and nu- 
cleus, causing a dissimilarity of the cell mass, resulting in different 
structures according to different parts of the cell. The differentiation 
of a fertilized egg cell may thus start. At the beginning, however, 
the differentiation appears to be insignificant and easily reversible. 
As described already a newly formed structure is at first reversible 
and labile, but if the structure remains long under the same environ- 
ment favouring the structure, it will ‘“‘forget’’ the former structure 
becoming irreversible and stable with the establishment of the ‘‘adap- 
tation’’ to the environment. 

For example, at an early stage of development, when a piece of 
the tissue which is to develop into A-organ is transplanted to another 
part where B-organ is to be developed, the piece will develop into B 
not into A. Thus specific differentiation seems not developed at an 
early stage, every cell of the embryo appearing capable of producing 
every organ according to the region into which it is planted. That 
is to say, each piece of tissue develops into the kind suited to the new 
region, and not in accordance with the place of its origin. The lack 
of specific differentiation is well shown by the fact that, if the first 
two cells of the frog embryo are separated, each gives rise to a whole 
embryo, although in some other animals, each of the first two cells 
will develop only half an embryo if separated. 

However, as development progresses, certain cell masses are local- 
ized and differentiated into specific organs, and after localization is com- 
pletely established in a region, differentiation is apparently independent 
of the influence of another region onto which it is grafted. At such 
a stage, many embryonic tissues, once started on their course of 
specialization, will continue the specialization as self-differentiating 
structures even when removed from the body and grown in artificzal 
cultures. 

Such a fixation of structure should be compared to the evolution 
of viruses. Newly generated viruses are weak and labile, but gradu- 
ally become stronger and stable, acquiring a faculty not only to retain 
their pattern firmly, but to transfer their structural pattern to the 
surrounding protoplasm. 

A group of cells, such as those of dorsal lip of the blastopore, 
known as an organizer, can be regarded as part of embryo which may 
first acquire a highly stable structure comparable to a strong virus. 


II. ONTOGENY 393 


A piece of this part, when transplanted onto another part of embryo, 
is able not only to retain its specific structure but also to exert its 
structural influence upon surrounding tissues until a new embryo is 
formed around it. The ectoderm of the ventral and lateral surfaces 
of the frog embryos normally develops into epidermis, while ectoderm 
of the mid-dorsal region gives rise to the neural plate; however, when 
the dorsal lip of the blastopore, the portion called organizer, is trans- 
planted beneath the ectoderm on the ventral or lateral regions of a 
frog at the gastrula stage, the ectoderm coverning such a transplanted 
dorsal lip gives rise to a neural plate, not to epidermis. 

Such an action of organizer to induce a nervous system is appar- 
ently brought about by a hormone-like factor, which has proved to be 
a steroid. It has been shown that a variety of lipids induce experi- 
mentally a similar effect, but it cannot be said that the physiologically 
acting factor is entirely identical with the substances which may ex- 
perimentally exhibit a similar action. 

In addition to this inducing action, the organizer like viruses is 
capable of transmitting its structure to surrounding protoplasm. Thus, 
a portion of embryo which has no inducing action in itself, if trans- 
planted onto a region near the organizer, will be endowed with the 
inducing action, becoming itself an organizer, indicating that the 
structural pattern of the organizer is transferred to the transplanted 
region. 

In short, the organizer having developed under a specific influence 
of environment has a strong structure comparable to fixed viruses, and 
can transmit its structure like viruses. In addition it can producea 
substance, a kind of hormones, which can induce peculiar ‘‘crystal 
shape’’ in its surroundings. This hormone may assist the virus-like 
action of the organizer, providing the specific structure to surrounding 
cells; as a consequence these latter cells may become the second or- 
ganizer which in turn produces another hormone. Thus, differentiated, 
strong structures may be successively induced. 

These successively induced organizers, however, may not be iden- 
tical with the first organizer in their structure, even if they were 
induced by virus-like agents, for each portion of embryo is under each 
different environmental condition; each portion adapts itself to each 
environment to attain strong structure; and if a stronger structure is 
present near it, it will be influenced by the structure, but without be- 
coming entirely identical with the stronger structure which may exert 
its influence as a virus. According to their different structures, each 
organizer may produce each different hormone-like substance, thus ex- 
tremely complicated “‘crystal shape’’ being formed. 

Since the substances behaving as hormones, whether steroid or 


354 V. THE NATURE. OF EVOLUTION 


protein, are determined in their structure by the protoplasm from 
which they were produced, they will vary in their structures if.the 
cells from which they are to be produced are being changed as the 
embryo is developed. It has been found that the press juices of chick 
embryos of different incubation times acted differently on the growth 
of cultures of fibroblastic cell (26), indicating that the composition of 
the press juices which may act as hormones undergo changes during 
development of the embryos, and so it can be concluded that the com- 
position of the body fluid which can exert hormone-like effect is gradu 
ally changing during ontogeny. 

In a study on growth and mortality effects produced in early 
chick embryos by antiserum, results were obtained which point to the 
possibility that there are different protein complexes which make their 
appearance in the chick embryo concurrent with the embryo’s develop- 
ment (26a). Crystals of the haemoglobins of the human adult and of 
the human foetus have different shapes (27); the difference is also de- 
monstrable in their X-ray interferences (28), showing likewise that the 
protein structure is changing during the course of development. ‘The 
change in protoplasm structure, which may vary with different re- 
gions of the body, will thus be advanced with the formation of vari- 
ous shapes and functions. 

In higher organisms, hormones are, of course, distinct from virus- 
like agents, but at an early developmental stage of embryos or at an 
extremely primitive stage of organisms, the difference between the 
two might be subtle. If the protein, the main component of the proto- 
plasm, is freed from the protoplasm in an orderly polymerized state, 
it may function as a virus, but when the protein or other components 
such as steroid having the pattern of the protoplasm are liberated 
from the protoplasm, not forming orderly polymerized masses and 
accordingly in a state unable to act as a virus, they may be able to 
act as hormones and are capable of exerting their specific structural 
influences, although unable to produce the exact replicas as do vi- 
ruses. Consequently, primitive hormones may be regarded as incom- 
plete viruses, only being capable of exerting on other protoplasm un- 
certain influences specific to their structures. At such a primitive 
stage, there might entirely be no essential differences between hor- 
mones and enzymes (see Part IV, Chapter V). The specialization of 
these factors must be advanced as the organisms are evolved or the 
embryos are developed. 

The differentiation of the protoplasmic structure, which may var- 
with different parts of the embryo, may not only lead to the produc- 
tion of different hormones or enzymes as well as of different virus-like 
agents, but also may cause the different distribution of inorganic salts 


II. ONTOGENY t 355 


or other substances which may further promote the differentiation. 
As mentioned already in Part IV, Chapter VI, inorganic salt ions ex- 
hibit hormone-like function. 

Since the famous researches of Vogt, numerous facts have been 
found by a number of workers to show the occurrence of considerably 
rapid movement of component cells in developing embryos (29). 
Through this movement some proteins with a certain pattern may be 
able to contact with certain other proteins having other patterns lead- 
ing to the establishment of ‘‘rejuvenescence’’; thus the cells may con- 
tribute to make the development the more complicated. 

As discussed in the previous section, the mechanism of regenera- 
tion is identical with that of ontogeny, so that phenomena observed in 
ontogeny are also proved in regeneration. If, for example, the re- 
generation bud which is replacing the lost leg of a newt is grafted 
onto the base from which a part of the tail has been removed, the bud 
becomes tail instead of leg. That is, the bud tissue is in the state of 
very early gastrular cells which can readily conform themselves to the 

‘environments under which they are placed. Such a submission to a 
new position is based upon the juvenile character of the bud structure, 
weaker than that of the protoplasm upon which it is transplanted, ac- 
cordingly it may be said that the bud is subjected to the organizer of 
the part. 

However, the position effect is, of course, not always attributable 
to an organizer. Thus, if Tubulavia, a simple marine animal, is de- 
capitated it will regenerate its head, that is, the portion bearing mouth 
and tentacles. If, however, the animal is cut off at both ends and 
that which bore the head is buried in the sand, a new head is regen- 
erated at the free or foot end and a foot at what was originally the 
head end. If both ends are buried in the sand none of them regener- 
ates a head, and if the body is suspended free in the water both ends 
develop heads (30). Here, evidently, some external condition such as 
contact, or some other external chemical or physical factors determine 
whether the regenerative cells make a head or a foot. 

In both cases of regeneration and ontogeny, the first direction of 
differentiation may be determined by external conditions and subse- 
quently specific structures are developed, which, if developed into a 
certain extent, will become stabilized so as to be able to act as 
organizers. 

To sum up, the individual development including regeneration ap- 
pears to be a phenomenon in which the property of protein capable of 
changing its structure in conformity with the environment is revealed 
to the utmost; the changed structure is unstable at first but gradually 
becomes stronger and capable of exerting its structural influence upon 


356 V. THE NATURE OF EVOLUTION 


others according to the general property of protein concerning memory 
and forgetting. The change will be continued until the differentiated 
structure is recovered from which the primitive structure of the germ 
cell was induced, thus the reversible change of the germ cell to the 
somatic being established. 

Now, we have to consider this problem in connection with genes. 
Since protoplasm structure is controlled by genes, the change of the 
former should be involved in the change of the latter. It may be 
said, therefore, that genes are changed during ontogeny in every direc- 
tion according to the various parts, or organs, of the body, so that 
every organ or tissue in a fully developed individual must have its 
own specific genes. 

This conclusion may seem unreasonable to the majority of authors 
who believe firmly that genes are never changed except by mutation. 
Nevertheless, the evidences strongly suggesting the ununiformity of 
genes among various organs even in one and the same individual have 
generally been known. Namely, on the one hand, the immunological 
fact that organs have their antigenic specificity, that is, that organs 
contain their own specific proteins is well known; whereas, on the 
other hand, it is generally accepted that the protein structure ina cell 
is governed by the genes. It should naturally follow from these two 
facts that cells of every organ and tissue share their specific genes, 
never similar to each other. 

The necessity of stressing the importance of chromosomes in the 
organization of plants and animlas, and their decisive role in inherit- 
ance, caused O. Hertwig in 1918 to formulate the ‘“‘Law of constancy 
of chromosome number”’ which implies that all cells of a given organ- 
ism contain the same somatic number of chromosomes. A great num- 
ber of observers have, however, shown that his concept is in need of 
revision. Especially in insects and in plants, it has been established 
that certain cells and even whole tissues may represent different de- 
grees of polyploidy. It has been found that a similar variation occurs 
also in the somatic chromosome number of man (31). This may be re- 
garded as another evidence indicating the ununiformity of genes. 

Thus it may be said that ontogeny is a phenomenon brought about 
by a complicated change of the gene in the germ cell, as is illustrated 
diagrammatically in Fig 34. In this diagram, A represents the gene 
or the gene-complex in the germ cell, A’ and A” the genes in somatic 
cells developed from the germ cell, and A” the gene of the mother 
germ cell developed also from the germ cell. 

A can be converted to A’, A” and A” respectively, by the action 
of the environmental factors, a’, @’ and a’. A’ has the property to 
be readily reduced to A by the effect of x, Also between A’” and A’ 


II. ONTOGENY 357 


or A”, there is an intimate correlation, and both A’ and A” continu- 
ously exert structural influence upon A”, an important assumption 
which will be discussed in great detail in later Chapter. 


Oo \ 
\ 
9 \ 
GG +) 
A = 
7 f 
/ 
/ 
! 
A 


eg. 34. Relationship between germinal and somatic cells. 
A: gene of germ cell. <A’, A’’: genes of somatic cells. 
A’'’: gene of mother germ cell. 


Fi 


That A can reveal various structures under varying environmental 
conditions must indicate that A consists of different proteins, each of 
which has different memory, Since a single protein may have commonly 
only a single memory. Protein molecules having a similar memory may 
form a gene and the memory may be involved in a restricted, specific 
structure of the gene. Therefore, A must be a gene-complex, consisting 
of many different genes. It is actually known that a single germ cell 
is provided with numerous different genes. A can be converted to A’ 
under the environment of @’, because there exists in A a gene having 
the memory to recollect A’-structure under @’-environment. If a gene 
in A thus recovers the proper structure A’, other genes remaining in 
their undeveloped structures must be subjected to this developed gene, 
and accordingly the protoplasm of the cell must have the structure 
of A’. Ina similar way, #’-structure is established by another gene. 
‘‘Predisposition’’ of the organism must be such a memory of genes. 

A gene can change its structure in such an easy manner, but the 
direction of the change is generally fixed, and a changed gene should, 
as arule, resume a definite structure under a given condition. There- 
fore, the structure of a gene is generally constant under a given con- 
dition. In this respect, the concept of the auther conflicts in no way 
with the customary view of the constancy of genes. 


353 V. THE NATURE OF EVOLUTION 


4, The Significance of Fertilization 


The germ cell with primitve structure returns to its differentiated 
state through the development and this developmental process is 
generally initiated by fertilization. Fertilization is comparable to the 
application of a stimulus to super-cooled water, which will be frozen 
by the stimulus; super-cooled water corresponds to the egg cell and 
the stimulus to the sperm. 

Somatic cells or mother germ cells are reduced to their primi- 
tiveness to produce egg cells, but when factors are removed which 
caused the reduction and the eggs are brought under the condition 
which favours the differentiated structure, the egg cells may be ina 
metastable state, tending to return to the original, differentiated state. 
This is the reason why germ cells are compared to super-cooled 
water. 

The sperm cell may act as a stimulus to initiate the recovering 
change because of its structure somewhat different from that of the 
egg. Similar effect can be provided by numerous forms of artificial 
means, such as subjection of the egg to certain chemicals, to changes 
in temperature or in density of the surrounding water, or even to 
mild shaking, or to the prick of a needle; all these can initiate fertili- 
zation as does the sperm. Natural parthenogenesis, which is common 
among invertebrates, must be caused by a proper stimulus provided 
by a natural process. 

The development occurs in general only from the egg cell because 
the egg, in contrast to the sperm, contains a plenty of cytoplasmic 
substances, which will provide the energy and substances required for 
the development. Sperm cells like eggs must be in a metastable state, 
tending to return to the original specialized state, so that they can 
develop like eggs if cytoplasmic substances are provided. It is actual- 
ly known that if a sperm is inserted into an egg cell from which the 
nucleus was preliminarily removed, the development will take place 
from the sperm, a phenomenon known as merogeny. 

If the returning process of a germ cell to its original state is 
once inititated by a stimulus of a sperm or of another factor, the gene 
in the germ cell will start on a gradual change towards the developed 
structure, and the protoplasm or the embryo will be compelled to take 
one form after another in succession, in response to the structure ap- 
pearing successively in the returning process, since the form of an 
organism is determined by the ‘‘crystal shape’’ of protoplasm, which 
in turn is governed by the structure of the gene. The cell division 
may be required for the acquisition of the compelled form. 


it,” “ONTOGENY 359 


A number of facts have been found that striking disturbances are 
raised in the egg cell following the fertilization like in super-cooled 
water or in super-saturated solution to which vigorous shaking is ap- 
plied. Thus, unfertilized egg behaves like a cell in a “‘latent’’ state, 
all the metabolic exchanges being extremely slow, but on the fertili- 
zation they are suddently elevated. This may chiefly be attributed to 
the temporary liberation of polar groups. It should be remembered 
that a protein molecule in a metastable state will change intoa stable 
state if a suitable stimulus is provided to initiate the change, and 
that at the first stage of the change the protein molecule is to be un- 
folded with the temporary liberation of free polar groups. 

On the one hand, as was previously stated, the combination be- 
tween proteins and lipids in the protoplasm becomes occasionally loose 
when the protoplasm structure is disturbed by the infection with a 
virus. On the other hand, it has been reported that lipids which can- 
not be extracted from unfertilized egg become easily extractable fol- 
lowing the fertilization (32). It has been claimed, however, that a 
part of cephalin will combine firmly with protein after the fertili- 
zation (33); this may depend upon the liberation from the protein of 
polar groups capable of combining with cephalin. Such a liberation 
of free groups may cause the mutual association of proteins leading to 
the coagulation, which has been observed with eggs of the sea urchin 
to begin three minutes after insemination, obtaining its maximum in 
‘ seven minutes, and being maintained thereafter unchanged for two 
hours (19). If the eggs of the sea urchin are frozen at —77° C. and 
then desiccated at —25°C., one obtains a fine powder that may be 
extracted at low temperatures with KCl solution at pH 7.3. Under 
this condition, 82 to 85 per cent of the proteins from the unfertilized 
egg dissolve, while for the fertilized egg this becomes 69 to 72 per 
cent (19). A portion of the egg proteins, more than 10 per cent of the 
total protein, thus becomes insoluble at the time of fertilization, 
presumably because of the mutual combination or the coagulation of 
the proteins due to the liberated polar groups. 

It is generally known that a variety of enzymes are activated 
following fertilization. This activation may be attributed to the liber- 
ation of active enzymatic structures due to the disturbance by the 
sperm. During the coagulating process of blood a series of enzymes 
or enzyme-like factors are successively produced as stated in the last 
Chapter of Part IV. In the protoplasm of the egg the situation may 
become the same on the fertilization as in the coagulating blood plasm. 

Serum protease, also known as plasmin and fibrinolysin, has been 
shown to be present in an inactive state as a natural constituent of 
mammalian blood. Numerous activating agents have been recorded 


360 V. THE NATURE OF EVOLUTION 


for this enzyme. The treatment with chloroform or the addition of 
bacteria or starch paste are sufficient to activate the enzyme. More 
recently activators from tissue and serum have been recorded. The 
most rapid and potent activators have been prepared from bacterial 
sources, notably the so-called streptokinase from streptococcal culture 
filtrates, and staphylokinase from staphylococcal cultures. These ac- 
tivators may be comparable to a sperm or artificial stimuli that can 
initiate the development of the egg cell; the action of all these agents 
consists in the faculty of disturbing the protein structure. 

The sperm cell may thus initiate the development of the egg cell 
as a mere stimulus, but besides this the sperm will play a decisive 
role in the establishment of rejuvenescence as already discussed in 
Part III. It must be desirable for the rejuvenescence that the sperm 
is as different as possible in structure from the egg. This may be the 
reason why hybrids are generally robust. However, two protein mole- 
cules cannot combine if their structures are entirely different from 
each other, so that for the establishment of the fertilization the sperm 
cannot be different from the egg too extensively. The development 
of the egg of a species of frogs or newts may sometimes be initiated 
by the sperm of some other species, but the embryos will perish earlier 
or later (34), presumably resulting from the difficulty of the mutual 
fusion on account of the too different structures. 


CHAPTER III 
THE DEVELOPMENT OF CANCERS 


1. The Reduction of Protoplasm to Primitive Structure 


Since all the somatic cells have their origin in germ cells, all 
kinds of somatic cells, if their structure is reversible, should be re- 
duced to primitiveness of germ cells when a suitable stimulus is given 
under a proper condition. In fact, as pointed out in Chapter I, the 
reduction tends to occur without difficulty in some lower animals and 
generally in plants, so that regeneration is common among these or- 
ganisms. In higher animals, however, the reduction is not so common 
and it appears that some mechanism must have been developed be- 
cause the reduction is unfavourable for these organisms, but occasion- 
ally this mechanism appears to get out of order, resulting in the 
occurrence of the reduction in some somatic cells. 

The reason why cancer cells are developed from the somatic can 
be explained in this way. It is a well known fact,.as will fully be 
described below, that cancer cells are of a primitive nature comparable 
to that of germ cells. The production of cancer instead of germ cells 
may be due to the incompleteness of the reversion of the primitive 
structure probably because of the presence of the mechanism to pre- 
vent the reversion. 

Hormones, especially sexual ones, appear to be involved in the 
production of germ cells; sexual hormones may be the main factor 
providing the environment under which the reversible change of the 
somatic cells to gametes is established. In short, hormones may be 
factor x which converts A” to A in Fig. 34. In this connection it 
should be mentioned that, as is well known, sexual or other related 
hormones are intimately connected with the development of cancer cells. 
This suggests strongly that the production of cancer cells is carried 
out by the same principle as that of germ cells. It is believed by the 
majority of workers that hormones or hormone-like substances may 
be produced in the wound regions of plants where the primitive cells 
are induced to achieve the regeneration. It is well conceivable that 
mechanical injuries will result in the formation of certain hormones 
which cause the production of the primitive structures comparable to 
cancers. 


362 V. THE NATURE OF EVOLUTION 


The structure of a cancer cell or of a cell present at a pre- 
cancerous stage is sometimes transmitted by virus-like protoplasm 
particles which are often called cancer viruses. The change of a 
somatic cell to a cancer must be associated with a change of the gene, 
and accordingly, if a change of a gene is to be calied mutation, it 
may be said that cancer is raised by a mutation. 

The primitive nature of cancer cell has been well established. 
For example, glycolysis peculiar to embryonic tissues is remarkable in 
cancer cells. The capacity of embryonic tissues to incorporate amino 
acids is much stronger than that of the ordinary ones, and being 
similar to that of cancer cells (35) (36). Again, it has been shown 
that in the conversion of normal hepatic cells to cancer there is 
almost total loss of certain highly specialized functions, including 
synthesis of fermentable carbohydrate from pyruvic acid and forma- 
tion of acetoacetic acid from caprylic acid (37). The pattern of en- 
zymes of foetal rat liver is said to be more closely related to that of 
malignant rat hepatoma than that of normal rat liver (38). As al- 
ready pointed out, primitive structure is so unstable that it may easily 
be affected by viurses as evidenced by the fact that chick embryos 
are commonly used as the most suitable tissues for virus cultivation. 
It is note-worthy that cancer cells also allow viruses, such as those of 
poliomyelitis (39), St. Louis encephalitis (40), and influenza (41), to pro- 
pagate in them. 

Greenstein (42) has stressed the fact that all cancerous tissues 
show a strong resemblance to one another in their chemical pattern, 
and that the more autonomous they are, the more do they deviate 
from the chemical pattern of their tissues of origin, and the closer do 
they approach an apparently functionally undifferentiated type of 
tissue. 

A series of hormones appear to be required in succession for the 
development of a germ cell to an adult organism as already mentioned. 
This must also be the case with the production of cancer cells as well 
as of germ cells, as the production of these cells is only the reversible 
reaction of the former, the ontogeny. Sexual hormones may be re- 
quired at the finishing stage for the completion of the change. Sub- 
stances commonly termed carcinogenic may be regarded as a type of 
hormones or hormone-like substances to initiate the change. Many 
carcinogenic substances are known to exhibit an inducing action like 
that of the organizer (43), suggesting that they are related to the 
primitive hormones produced by the organizer. 

Rous and Kidd (44) stated that when carcinogenics are applied to 
the skin of man, the rabbit, or the mouse, they nearly always elicit 
benign growth some while before cancer appears, and the latter 


lil. THE DEVELOPMENT OF CANCERS 363 


frequently takes origin from one or another of them. Tar cancers 
usually come about by successive, step-like deviation from the normal, 
and soalso do the cancers which derive from virus-induced papillomas 
as well as many human cancers. After cells have become cancerous 
they frequently undergo further changes, some apparently step-like in 
character, and all taking the direction of greater malignancy. 

Sexual hormones may presumably play a role at the last stage of 
such step-like deviation, although most carcinogens are in themselves 
closely related to sexual hormones in the chemical structures. It has 
been stated that acute fowl pox develops in practically every bird 
painted with methylcholanthrene, one of the well known carcinogenics; 
if one continues to paint the bird with this carcinogen, chronic 
inflammatory reactions develop, as well as angiomas and discrete, 
poliferative, epithelial lesions. Upon inoculation of the bird with 
testosterone, a Sexual hormone, all of the lesions flare up and the 
epithelial growths become squamous-cell carcinomas (45). 

Further, if a single application of 3,4-benzpyrene, a carcinogen, is 
made to the skin of mice, it does not induce cancer, but if the single 
application is followed by repeated treatment with croton oil, then 
tumours develop (46) (47). The croton oil treatment alone will not 
induce tumours. Cholestrol exhibits a similar accelerating effect; in 
contrast to croton oil, cholesterol itself has a feeble carcinogenic ac- 
tivity (48). In such a case, croton oil and cholesterol may behave like 
sexual hormones. 

Greenstein (49) believes that carcinogens lower the threashold of 
spontaneous malignancy in the tissues, so that malignant growth is 
brought about by the sexual hormones, which as such are not carci- 
nogen. There are numerous evidences showing that estrogens enhance 
the development of mammary tumour of mice. The inhibitory effect 
of testrosterone in this connection is also well known. According to 
Kirkman and Bacon (50) male golden hamsters will produce almost 
constantly renal cortical cancers if they are treated with estrogen. 

If cancer cells are produced by the structural reduction of somatic 
cells, and if the structural change of the protoplasm is reversible, 
cancer cells must return to the original, differentiated structure as 
do germ cells. In fact this seems actually the case. Thus, if renal 
carcinoma of the frog, Rana pipiens, is transplanted onto the base of 
the fore limb of a newt, and the greater part of the limb being cut 
off, when the cancer cells begin to multiply, leaving some of the cells 
in the tissue of the newt, then the cells are changed to normal frog 
cells during the regeneration of the limb. The frog cells can be easily 
detected owing to the small size of the nucleus (51). Thus the return 
of the cancer cells to their original state is established under the 


S64 V. THE NATURE OF EVOLUTION 


peculiar environment of the regeneration, in which strong factors are 
at work to develop the primitive structure into differentiated. Simi- 
larly, chicken sarcoma cells implanted onto chick embryos are reported 
to be developed into normal cells under the strong influence of 
organizer. 

The recovery of the original, normal structure is common in 
benign tumours, but the normal structure which was recovered from 
the tumour appears to have the memory of the tumour, tending to 
return again to the tumour structure. Thus, according to Mackenzie 
and Rous (52), where tumours once were one finds only epidermis of 
ordinary appearance, yet if tarring is resumed some of them not in- 
frequently reappear, even after months, and by reapplying tar at in- 
tervals they may be made to recur again and again, clear proof that 
some cells in the apparently normal epidermis have retained their 
neoplastic potentialities, that is, their memories of the tumour struc- 
ture. Usually each successive period of tarring brings out more 
growth than before, and they appear very soon, sometimes almost at 
once instead of after several months. It is as if new tumour cells 
had been merely waiting for encouragement. The stimulus of wound 
healing will suffice to make some of them multiply and form tumours. 

The malignancy of the cancer may depend upon its primitive 
nature associated with the character of the vigorous multiplication at 
the sacrifice of the host. It seems strange, however, that germ cells, 
inspite of their primitiveness, fail to exhibit such a malignancy. This 
is presumably because there is some mechanism by which the ma- 
lignancy is inhibited, since the malignancy is, of course, unfavourable 
for the organisms, but the mechanism may be destroyed by the fertili- 
zation to the revelation of the malignancy, that is, the character of 
vigorous multiplication. Leucocytes may be looked upon as another 
group of primitive cells whose malignancy is usually inhibited; the 
destruction of this inhibiting mechanism may lead to leucosis. The 
structural pattern of leucocytes which thus acquire the malignancy 
can sometimes be transmitted by protoplasm fragments, and conse- 
quently the disease is considered to be induced by a virus as in avian 
leucosis. 


2. The Predisposition to Cancer 


Cancers are liable to develop in certain individuals, that is, certain 
individuals possess the predisposition to develop cancers. This predis- 
position is inheritable. As mentioned already, predisposition is a 
memory of the gene, or a genic structure capable of recovering a 


II. THE DEVELOPMENT OF CANCERS 365 


former pattern. ; 

Animals and plants, infected with viruses, appear to transmit 
frequently the viruses to offspring through germ cells. Avian leucosis 
is looked upon as a kind of malignant tumours and considered to be 
induced by a virus. It has been established that the virus is likewise 
sometimes inherited to progeny through the eggs (53) (54). In such a 
case the pattern of the virus is memorized by the germ cell and re- 
covered when the egg develops into the adult. The pattern of a 
virus, which has been impressed into a mother germ cell, must be 
changed during the reduction of the cell to a gamete, so that the pat- 
tern is present in the gamete only as a ‘“‘memory’’ not as a complete 
pattern. However, when the gamete develops to a certain extent fol- 
lowing fertilization, the pattern is also developed to the revelation of 
the virus. In such a case, the pattern of the virus may be impressed 
in, and carried by, a certain gene, just as a normal pattern which 
determines a normal character of the organisms. 

Every morphological and functional character, including organs 
and tissues, are developed by numerous patterns memorized by each 
gene in the germ cell. But peculiar environmental factors are re- 
quired for their development. In the same way, a certain factor or 
factors must be necessary for the development of a virus pattern. In 
the case of cancers, sexual hormones may be at least one of the 
factors. It was already stated that seasonal factors are important for 
the development of the virus of measles (Chapter III, Part III). The 
coincidence of the seasonal incidence of measles with that of the con- 
ception of man, may suggest the involvement of hormones in both 
cases. As previously detailed, hormones are, as a rule, required in 
ontogeny for the development of organs and tissues. 


The predisposition to cancer must also be a memorized pattern of 
a certain gene present in the germ cell, and the full pattern will be 
raised in the individual developed from the germ cell when the proper 
conditions are provided. When the pattern is revealed in the proto- 
plasm structure of a cell, the cell is called cancer cell, and when the 
pattern is stable enough to be retained in protoplasm fragments or 
elementary bodies whereby the transmission of the structure to an- 
other protoplasm is possible, the fragments may be called cancer 
virus. It is considered, as above mentioned, that cancer cells may be 
raised by the destruction of the mechanism by which the reduction of 
somatic cells to primitiveness is inhibited, If this is the case, the 
character tending to lose the mechanism are also to be borne by some 
gene in the germ cell asa memorized pattern which will later develop 
into the full pattern with the revelation of the character to lose this 
inhibiting property in favour of the development of the proper pattern 


366 V. THE NATURE OF EVOLUTION 


of cancer. 

The property to produce germ cells must likewise be impressed in 
a gene as a pattern, for whose development sexual hormones are re- 
quired. This property is thus inherited, and is, of course, one of the 
most important characters of the organisms, in contrast to the proper- 
ty to produce cancer cells, notwithstanding that cancer cells are also 
of a primitive nature and sexual hormones apparently are likewise 
concerned in their production. 

Not only germ cells but also various other cells or organs should 
be determined and produced by respective pattern transmitted through 
the genes of the gamete. If some microorganisms succeeded in en- 
graving the patterns into some of the genes carrying such various 
normal patterns, the organisms would develop as one of the patterns, 
and if the microorganisms were favourable for the host, they would 
become organs or cells indispensable for the host as the mycetoma in 
insects. 

Symbiosis or commensarism between microorganisms and insects 
is well known. An example of this is the commensality betweén the 
corn borer, Pyvausta nubilaiis, and a flagellata discovered in this 
insects by Paillot (55). The protozoan occurs in the Malpighian tubes 
of the corn borers as well as in the alimentary tract. In the midgut 
the flagellates attach themselves by their anterior ends to the epi- 
thelium, standing side by side in a regular fashion as if they were 
ciliated epitheliums of higher animals. It may be not quite impossible 
that symbiotic microorganisms become more and more indispensable 
for the host until they come to be the essential cells or organs, such 
as leucocytes and ciliated epitheliums, appearing as if they were be- 
longing to the host from the beginning. 

The pattern of the microorganism, however, must be very strong 
and must be continuously applied to a host in order to become the 
inheritable pattern of the gene of the host. Unless a new pattern 
becomes normal and the previous one is forgotten, the new pattern 
will not become a predisposition of the host. 


CHAPTER IV 


THE MANNER OF THE GRADUAL 
CHANGE OF GENES 


1. The Explanation of the Biogenetic Law or the 
Theory of Recapitulation 


The biogenetic law or the theory of recapitulation states that each 
organism tends in its individual life history (ontogeny) to recapitulate 
various states through which its ancestors have passed in their racial 
history (phylogeny). This law or the theory should be expected from 
the above concept that the germ cells are the reductive form of the 
somatic and that the change is reversible. 

To begin with, we shall consider of the case of primitive unicel- 
lular organisms such as Sporozoa. If a primitive organism like Sporo- 
zoa is brought under a certain environment, the structure of the cell 
may be changed to produce spores, whereas this latter can recover the 
original cell structure if the environment is again properly changed. 
Since the structure of protoplasm is always governed by the elemen- 
tary bodies, rich in nucleic acid, also the change between the cell and 
the spore must be subjected to such elementary bodies, which can 
be looked upon as the genes. Thus, the change can be expressed as. 
AZB, in which A is the structure of the gene of the cell and B that 
of the spore. 

Now, if this primitive organism advances to have A’-structure,. 
the change is: AA’. Since the evolution of both morphological and 
functional characters of an organism is given rise to by the advance- 
ment of gene structure, the advancement of the gene structure from 
A to A’ should be accompanied by the evolution of the characters 
of the organism corresponding to the structural advancement in the 
gene. 

On the other hand, as the genes possess the structural reversi- 
bility, when A’ is reduced to B-structure of the spore, or of the germ 
cell, it must take the course: A*-+A-B, whereas when B is developed 
to A’ the course should be: B>A-A?. This course is to be repeated 
in every change from the adult form to the spore, or the germ cell, 
and from the latter to the adult form. Through this repetition the 


368 THE NATURE OF EVOLUTION 


reversible course will become a beaten track; in other words, the 
memory of the course will be held completely by the repetition as dis- 
cussed in Chapter I of this Part. Since the memory is a type of the 
structural pattern of the gene, it should be inherited generation after 
generation. 

If the organism evolves further and advances successively to A’, 
A’, and so on, the course will become longer and longer, but as the 
repetition is to be continued without stopping, the track continuously 
being beaten and the memory is as sure as ever. In such a manner, 
the organism can evolve upto an extremely high state as A™. Even 
in this case, in its reduction to the germ cell, it must take the course: 
An—> An-}....., A? A?—>A1>A-B ; and B, the germ cell, must take the 
entirely reverse course in its development to A". It is impossible to 
take another course. Thus, the germ cell must take the course of 
phylogeny when it develops into the adult form. 


2. The Liberation of Active Groups 


Proteins may consist of two types of structures, one of which is 
stable back-bone structure and the other may be involved in polar 
forces in side chains which are to be liberated by the unfolding of 
polypeptide chains. There are numerous evidences that each of two 
structures determines each different character of the proteins (71). For 
instance, as already discussed, immunological character of viruses is 
determined by the stable back-bone structure, while virus action 
cannot be developed without the labile structure. 

In a similar way, the manner of changes in the genic structure 
governing both ontogeny and phylogeny may also consist of two types 
due to these two structures. The main change of chief importance may 
be involved in back-bone structure, but the development of some func- 
tional and morphological character responding to the back-bone pattern 
may not take place without the transient, unstable structure. 

This view is supported by the following fact: Lysogenic bacteria 
‘do not liberate phage by a mere disruption, while an adequate change 
in their environmental conditions, for example, irradiation with ultra- 
violet light results in phage production (56). And the susceptible 
bacteria infected with this virus become lysogenic and at the same 
time resistant to the phage, but usually they do not produce the virus 
unless proper stimuli are given. This fact can be readily explained 
by the assumption that the virus consists of two types of structures, 
stable and unstable. Namely, bacteria may acquire the immunity 
against a phage following the infection with the virus, because they 


IV. THE MANNER OF THE GRADUAL CHANGE OF GENES 369 


are endowed with the back-bone structure of the virus. However, in 
order to act as the virus unstable structure is necessary, which may 
consist of polar forces that will be liberated by the unfolding of the 
protoplasm protein. 

The manner of arrangement of these temporal polar forces may 
be determined by the back-bone structure. In other words, bacterial 
protoplasm proteins are normally in a partially folded state so that 
polar forces necessary for the virus action are also enclosed in the 
folding, but the protein in this folded state can retain its specific 
pattern which determine the immunological character. In order to 
produce the virus, however, an appropriate stimulus is necessary 
whicn can cause the unfolding of the protein. 

Actually it has been proved that after irradiation with ultraviolet 
light lysogenic B. megatherium shows increased capacity in its res- 
piration; the synthesis of desoxyribonucleic acid is blocked during 
approximately the first 1/3 of the latent period, but rapid synthesis 
then commences and continues until the moment of lysis (57). 

When the virus is produced the bacterium is always bound to 
undergo lysis. This fact may be interpreted as due to the virus 
action itself. In the opinion of the writer, however, lysis has no 
concern with the virus. Polar forces liberated by the unfolding of 
the polypeptide chain may subsequently combine with great quantities. 
of water molecules leading to the quelling of the bacterial body and 
accordingly to the dissociation of elementary bodies, thus resulting 
in the lysis of the bacteria. The bacterial lysis due to phage is also 
attributable to the liberation of polar forces attracting water mole- 
cules; the stimulus given by the virus may cause the unfolding of 
the peptide chain. 

The phages released upon lysis of bacteria infected with a cofactor- 
requiring strain possess a nascent activity, which makes possible their 
adsorption in cofactor-free medium. It has been shown that this 
nascent activity is lost gradually, indicating the temporary liberation 
during lysis of active group which enables the phages to combine 
with the bacteria without cofactor (58). 

Many evidences are known that lysis can occur without producing 
any virus. According to the writer’s view, any bacteria can produce 
virus which affects other bacteria having weaker patterns. However, 
if the bacteria cannot liberate the free active group, no virus would 
be produced. Lysogeny may therefore be no more than the bacterial 
character of unfolding the peptide chain readily. Usually the irradia- 
tion of lysogenic bacteria with ultraviolet ray is followed by lysis 
and by phage formation, but the bacteria would undergo no lysis 
when they became non-lysogenic, showing that lysogeny is nothing 


370 THE NATURE OF EVOLUTION 


but the property of readily liberating the polar forces. 

Lysogenic bacteria, having the property to unfold the polypeptide 
chain easily on the application of ultraviolet light, cannot produce 
phage following the irradiation if the bacteria are not present under 
the appropriate environment. For instance, a culture grown in yeast 
extract and irradiated will produce phage and lyse if left in yeast 
extract, whereas it will not produce phage if suspended in broth after 
irradiation. Again certain bacteria in a manganese deficient medium 
will not produce phage after irradiation, but if supplemented with 
manganese after the irradiation the virus is produced. Furthermore, 
the inducing effect of ultraviolet light is inhibited by visible light 
(59). These evidences may show how important are the environmental 
conditions even for the liberation of the polar forces. 

The so-called latent viruses may, in the majority of cases, be the 
virus pattern lacking this polar forces. For example, almost every 
man shares the pattern of herpes virus but the virus is developed only 
after some febrile diseases or after heat application which may cause 
the unfolding. As considered already, common-cold is a disease whose 
‘main causative factor appears to be chilling, but sometimes chilling is 
not sufficient to cause the disease; only persons who are under an 
appropriate condition seem to become common-cold following chilling. 
Chilling is therefore comparable to the irradiation, and the peculiar 
condition or X-factor in common-cold may be comparable to the yeast 
extract in the case of phage production. Persons apart from the 
peculiar condition may not catch cold even if exposed to chilling; in 
a similar way, bacteria suspended in broth may not produce the virus 
even after the irradiation. 

If common-cold is a disease which occurs in such a manner, its 
virus must be a fixed one and accordingly it must cause a long- 
lasting immpnity; but no immunity appears to be left behind the 
infection. This must be attributed to the virus structure tending to 
readily liberate the active group which, if liberated, makes the cells 
fall into a severe pathological state as in the case of herps virus, a 
phenomenon comparable to that concerning the phage against which 
bacteria can become immune but the bacteria are occasionally shocked 
by the liberation of the active group. If a man carrying the pattern 
of common-cold virus is chilled under a peculiar condition, the active 
group of the virus will be unfolded resulting in the severe shock in 
the cells in which the unfolding takes place, thus common-cold being 
developed. 

The pathological symptoms designated iuflammation may be caused 
by the liberation of the polar forces; the liberation is caused not only 
by microorganisms including viruses but also many other physical or 


VI. THE MANNER OF THE GRADUAL CHANGE OF GENES 371 


chemical factors; if caused by a virus, active group for the virus will 
be raised. In short, the liberation of polar forces may result in the 
shock to the cells, and the shock is revealed as inflammation. No 
virus can usually be found following the inflammation due to stimuli 
other than viruses, a fact which must be attributed to the transient 
and labile nature of the polar forces. But normal structure of the 
cell may sometimes become capable after inflammation of acting as a 
virus upon other cells having weaker patterns. 

Lysogeny can be raised by the infection with phage, but certain 
bacteria must originally be lysogenic without infection. The pattern 
determining the lysogeny is controlled by the back-bone structure, and 
so it should also be formed under the appropriate conditions and be lost 
under some other conditions. For example, it is possible to ‘cure’ at 
will a certain strain of bacteria from its lysogeny by serial transfers 
in synthetic media (59). 

Human children may produce measles virus presumably because 
the pattern of the virus inherited from the parent may become com- 
pleted as the individual development is coming to its completion. The 
individual development must be brought about step by step by the 
gradual change of back-bone structure of the gene; the development 
of a virus pattern also must be raised entirely in the same way, but in 
order that the complete pattern acts as a virus the liberation of the 
active group is needed. The seasonal effect upon the occurrence of virus 
diseases is probably due to the liberation of the active group which is 
effected by some hormones or a hormone whose secretion is under the 
influence of seasonal factor. As the active group is of a transient 
nature, it will disappear when the season is over; but will reappear 
in the next year when the same season comes. 

Such a transient group may likewise be involved in the revelation 
of the pattern of cancers and also of that of parasitic bacteria. The 
fact that the virus demonstration of Rous tumour of chickens cannot 
be achieved constantly may be mainly based upon the inconstancy of 
the liberation of active group rather than that of the maintenance of 
the pattern in the particles. As already stated, animals irradiated 
‘with X-ray may excrete in feces and in blood many types of bacteria 
in abundance. This fact may be interpreted as due to the liberation 
of active group of bacterial pattern by the irradiation as in the case 
of the pattern of phage which will be activated by the ultraviolet ir- 
radiation. The release of active group may probably be needed also 
for the activation of normal pattern, that is, organisms may not deve- 
lop a function or a shape corresponding to a certain back-bone struc- 
ture of the gene unless the active group is released. 

Successive appearance of enzyme-like factors in the case of blood 


372 V. THE NATURE OF EVOLUTION 


coagulation and the rapidly occurring activation of a variety of enzyme 
systems after the fertilization of egg may be ascribed to the activa- 
tion of latent enzymes by the liberation of active group. In these 
cases the unfolding of the active group must be caused also by proper 
stimuli. 

The long lasting immunity against viruses is based upon the per- 
sistence of the virus patterns in the host but in animals only the 
back-bone structure is remaining, so that virus detection in the host is 
hardly possible. In contrast to animals, however, the virus is detected 
without difficulty in plants having become resistant to the virus after 
the infection, showing that there is a considerable difference between 
animals and plants in their protoplasm protein, that is, plant proto- 
plasm protein may exist usually in an unfolded state, or a slightly 
folded state to be readily unfolded unlike that of animals. 

In this respect bacteria are similar to higher animals, a fact which 
may depend upon the parasitic nature of the bacteria on higher animals 
from which they are inherited the protein. If so, the bacteria para- 
sitic on plants may have the protein similar to that of the plants, 
although the writer cannot find any report corroborating this suppo- 
sition. The view that plants are thus different from animals in the 
character of protoplasm protein is also supported by the already men- 
tioned fact that plant protein can fuse readily into protoplasm-like 
masses and that plant viruses are inclined to be obtained in crystal 
shapes, indicating the strong tendency of the protein to combine 
mutually. This is probably due to the presence of free polar forces 
mutually attracting. Further, there is another evidence corroborating 
this view. Namely the reduction to young primitive structure is 
readily raised in plant tissues, that is, cancer-like proliferative cells 
and germ cell-like regenerative ones and even germinal cells them- 
selves seem to be produced in every tissue of plants, thus their re- 
generative faculty being striking and no peculiar cells being needed 
for the production of germinal cells, whereas in animals specific cells 
are necessary for the germ-cell production and the regenerative faculty 
is insignificant. 

In order that the differentiated pattern is reduced to the primitive- 
ness the unfolding of the protein structure is indispensable. Therefore, 
it may naturally follow that the return to the primitive structure 
takes place only with difficulty in animals in which the unfolding of 
the protein hardly occurs. 

In the preceding chapter the writer assumed the presence of the 
mechanism in animals which can prevent the return to the primitive 
structure; the character of difficult-unfolding must he regarded as 
being this mechanism. The predisposition to cancers, therefore, must 


IV. THE MANNER OF THE GRADUAL CHANGE OF GENES 373 


mean the possession of the protoplasm protein tending to unfold. Pre- 
cancerous stage is commonly associated with inflammation, which as 
above pointed out means the unfolding of the protein. 


3. The Establishment of the Strong Reversibility 
in Gene Structure 


The biogenetic law would never be established if the gene failed 
to have strong reversible character. In this section, the reason for 
the establishment of such a strong reversibility in genic structure is 
considered. 

If the combination of two primitive organisms, A and B, takes 
place, and the strongest elementary body in A is stronger than that 
in B, then the strongest body in B as well as other elementary bodies 
will be changed to become similar to A. But if the change is rever- 
sible and the strongest body in B can recover its original structure 
when freed from A, the body can behave as a recessive gene. Thus, 
in order to become a recessive gene, the elementary body in B must 
have the complete structural reversibility, that is, it must have the 
faculty to recover completely its structure when the stress is removed. 
This holds true also for dominant gene, since if it had no structural 
reversibility, it would lose the independent character when it fell in 
with a gene stronger than itself. The reversibility must, there- 
fore, be the indispensable character for elementary bodies to become 
genes. 

If the reversibility of a gene is strong, the organisms having the 
gene can recover its original characthr even when it has been changed 
to primitive structure under some environmental conditions. The 
reproduction by means of germ cells cannot be achieved without this 
reversible character of genes. When the organisms were advanced 
in their evolutional course upto a certain degree with the acquisition 
of complicated forms and structures, the mutliplication by fission 
would become very inconvenient or rather impossible. Consequently, 
the organisms having evolved to a certain extent would find it neces- 
sary to fold their complicated structure into a mass as small as posi- 
sible in order to discharge this folded structure out of their bodies to 
expand again the orginal structure from this small mass. 

This small mass is the germ cell, and the expand of the folded 
structure, a process termed ontogeny, can be achieved by the reversi- 
bility of the gene. Therefore, the reversibility is the indispensable 
character for organisms in so far as they continue the reproduction 


374 V. THE NATURE OF EVOLUTION 


by means of germ cells, and accordingly only the individuals having 
the genes, which can exactly expand the original structure after the 
reduction to primitiveness, could survive and continue their existence. 
Thus the reversibility of the gene have been amazingly advanced. 


CHAPTER V 


GRADUAL ALTERATION OF GENE AND 
ITS RELATION TO MUTATION 


1. Orthogenesis 


If the miniature copy of phylogeny is seen in ontogeny, phylogeny 
must have been raised by the same continuous changes as ontogeny. 
In ontogeny the destination is settled preliminarily, the destination 
bound to reach is the original, advanced structure, and since the deve- 
lopment is only the recovery of the original structure by means of 
the reversibility, it can occur rapidly, whereas in phylogeny as the 
destination is unsettled and a new course has to be made, its progress 
must be very tedious. 

Nevertheless, the change of genes in phylogeny, like in ontogeny, 
would occur under the influence of environmental factors, wherein 
hormones would play an important part as will be considered later. 
This change of genes, as shown in ontogeny, would proceed in a 
certain direction and therefore organic evolution would occur gradually 
along a certain course. In fact, it is stated that evolution has occurred 
in straight lines or definite directions as indicated by the term 
“orthogenesis.”’ 

The term ‘“‘orthogenesis’’ has been used to indicate a straight 
genesis or evolution directed by internal factors. It is claimed that 
lines of evolution were not haphazard but were determined, not by 
natural selection but by law of organic growth. True, in some cases 
evolution seems to have proceeded along a certain course. For example, 
the increasing size of such forms as the horses, elephants, and dino- 
saurs, the reduction of digits in the horse series. Many fossil series 
suggest that types begin simply and evolve into complexities, such as 
overgrown spines and tusks, the overdevelopment being followed by 
extinction. Some zoologists and a larger number of paleontologists 
claim that evolution of this general sort has occurred so often in 
widely different types of animals that it can be explained only as a 
result of fundamental forces inherent in a race and carrying it along 
a straight or orthogenetic course. 


376 V. THE NATURE OF EVOLUTION 


Such evolution along an orthogenetic course must have depended 
upon the gradual change of a gene in a certain direction. If some 
gene began to change its structure gradually, the organisms having 
the gene would be compelled to change the character according to the 
change of the gene. However, as seen also in ontogeny, the direction 
of the change would be not always straight, although in the majority 
of cases it appears to have occurred in straight lines. 

This supposed property of genes to alter the structure gradually 
must be of the utmost importance .for the establishment of evolution. 
Full discussions on this problem will be made next. 


2. Phage as a Free Gene 


The gradual alteration of genes must be reversible since the indi- 
vidual development from the germ cell is given rise to by this change, 
‘owing to the reversibility the developed structure can be reduced 
again to the germ cell. On the other hand, genes may frequently 
undergo a sudden, irreversible change. The change of organisms 
due to such an abrupt, irreversible alteration in a gene is called 
mutation. There seem, therefore, two types of genic changes, one of 
which is this irreversible, abrupt change, the mutation, and the other a 
gradual, reversible change. The nature of these two types of changes 
will be considered below. 

Phage among many viruses can be regarded especially as a free 
gene, for it contains a large amount of desoxyribonucleic acid, the 
nucleic acid peculiar to genes. Here, it should be noted that phage 
thus possessing a free gene nature will change gradually in a certain 
direction under the influence of formalin as shown in Fig. 35. If 
phage is regarded as a gene, formalin should be compared to an en- 
vironmental factor having an influence upon the gene. In so far as 
the factor is present the change appears to proceed in a certain 
direction. 

The evolution along an orthogenetic course may thus be attributed 
‘to the existence of some environmental factor or factors which, like 
formalin, caused the gradual change of a gene in a certain direction. 
Orthogenesis, therefore, would come toa stoppage, if the environmen- 
tal factor in question was removed. Thus it may be concluded that 
for the establishment of orthogenesis continued existence of a certain 
stimulus or an environmental factor which caused the change of a 
gene would always be needed. 

The proteins of our somatic cells are bound to undergo senes- 
cence since the time of birth, a phenomenon which may be due to the 


(So) 


V. GRADUAL ALTERATION OF GENE 


ing active phage particles to that 


The ratio of the number of remain- 
of the tota! phago particles added 
(log) 


Time in hours. 


Fig. 35. The progress of the inactivation of phage protein due to 
formaldehyde. Concentration of formaldehyde 0,05% (I) and 0,194 
(II). Concentration of phage protein: 0,05%: pH=7,5. 


presence of a factor or factors making the change leading to senescence 
proceed continuously. As already discussed, senescence is a pheno- 
menon occurring when the proteins or organisms continue to exist 
under the same environmental condition. Proteins having biological 
actions, such as toxins, antibodies, or enzymes, will achieve a gradual 
change, known as denaturation, when preserved 2” vitro after being 
separated from organisms. The change is. usually very gradual and 
frequently many years may elapse before complete inactivation occurs. 
Such a change is likewise attributable to the continued presence of 
some factors which cause the denaturation. Furthermore, the pro- 
duction of cancer from a somatic cell.as well as the development of 
adult from a. germ cell is considered to be brought about by a con- 
stant influence of a series of proper stimuli as already discussed. 
Here again, we have to consider the problem of reversibility. It 
is true that sometimes formalin-inactivated phage is. reactivated 
following the removal of formalin, but the reactivation occurs only 
seldom, usually being irreversible. The same can be said with phage 
inactivated by chemical or physical factors other than formalin. How- 
ever, the reduction of the virulence of phage, without inactivation, 
due to extremely small amounts of formalin is usually reversible (60). 
Namely, the virus reduced in its virulence by formalin may produce 
small plaques on agar plate, but after a period of incubation following 
the removal of formalin the original property to produce plaques of 
ordinary size will generally be recovered. Such a reversible change 
can be demonstrated not only with formalin, but also with such 
agents as antibody, mercuric chloride, and tannin (61) (62). In general 


378 V. THE NATURE OF EVOLUTION 


severe changes raised abruptly appear to be irreversible, while slight 
changes given rise to gradually may be reversible. 

The recovery of the original structure of protein, as previously 
considered, is dependent upon the memory of the path along which 
the change proceeded. If one goes without hurry tramping on the 
overgrown grass through a pathless field, he will be able to return 
easily along the beaten path, but if he goes hurriedly in long strides 
the return will be difficult. In like manner, if proteins are changed 
in their structure gradually, the return to the original structure will 
occur with ease, as the structural factor resisting the change may be 
removed by the gradual change, whereas in an extensive change raised 
abruptly the recovery of the original structure may be unable. This 
latter change must be mutation, while the former the reversible 
alteration of the gene. 

It will be expected from this concept that in mutation the re- 
covery of the original structure may be never impossible but only 
difficult. In fact, it has gradually become acknowledged that reversible 
mutation, commonly called back mutation, is by no means a rare occur- 
rence. As pointed out previously, even so great a change as that of 
somatic cell into cancer is reversible if the most favourable condition 
for the reversion is applied. It has been claimed that exposure to 
X-ray and ultraviolet-light irradiation increases the back mutation 
rate of microorganisms very substantially (63), indicating that appro- 
priate stimuli can also necessary for the promotion of the back 
mutation. 

The frequency of back mutation of bacteria from streptomycin- 
dependence to non-dependence is greatly increased by the treatment 
with ferrous compounds. According to Catlin (64) streptomycin-depen- 
dent colon bacilli show a considerably higher frequency of reversion 
to streptomycin-nondependence after exposure to ferrrous compounds. 
at 1°C. than after comparable treatment at 37°C. This suggests that 
in this case ferrous compounds may only contribute to initiate the 
return to the original structure which may be stable at lower tem- 
peratures. It should be remembered in this connection that besides 
ferrous compounds, ultraviolet and X-rays, and a number of chemicals 
such as manganeous compounds, also effective in the promotion of this 
back mutation. 

According to our observation, phage is frequently changed in its 
property by unknown causes to produce small plaque, which indicates 
the reduction of its virulence, and the changed property is sometimes 
inherited, showing that the change is a mutation. The maintenance 
of the changed property appears to be connected with the medium in 
which the phage affected the host bacteria. For example, as shown 


V. GRADUAL ALTERATION OF THE GENE ory 


in Table 10, the changed property is maintained in a usual broth, but 
in a broth prepared with Liebig’s meat extract it will soon return to 
the original property ; this may indicate that back mutation is induced 
by some factor present in the extract. When phage is developed ina 
medium containing neither calcium nor magnesium, the property to 
produce plaques of extremely small size is given rise to, and this 
property tends to be retained when the phage is incubated in an or- 


Table 10. 
Inheritance of Acquired Character in Phage 


Test tube 


(Phage and bact. are incubated) 
‘ 
Agar plate 
(Both large and small plaques exist) 
_—<——— EE TIE 


Large plaque Small plaque 
Ta. ae 
Extract-broth Ordinary broth Extract-broth Ordinary broth 
test tube test tube test tube test tube 
(Incubated (Incubated (Incubated (Incubated 

with bact.) with bact.) with bact.) with bact.) 
| | 
y y 
Agar plate Agar plate Agar plate Agar plate 
(Small plaques as (Large (Large plaques as (Small 
well as large plaques only) well as small plaques only) 
plaques appear) plaques appear) 


dinary medium, although frequently the original property is recovered 
(65). Thus it appears that there is no fundamental difference between 
the irreversible and reversible change in phage. 


3. The Production of Fitted Characters by Adaptation 


In addition to the reversibility, there may be another great differ- 
ence between mutation and the gradual, reversible alteration. The 
difference exists in that the gradual alteration will generally give rise 
to favourable effects upon the organisms, whereas the characters 
raised by mutation are mostly harmful. For example, although the 
possibility of using radiation to induce useful mutations has been 
extensively investigated, especially in crop plants, almost all of the 
experimentally produced mutations have proved to be deleterious in 
character (66). Further, it has been generally recognized that muta- 


380 V. THE NATURE OF EVOLUTION 


tions induced in the fruit fly, Drosophila, by X-ray are mostly asso- 
ciated with the production of lethal factors. 

Every existing organism must be the fittest or at least must have 
the characters of a survival value and accordingly its genes must have 
the structures well fitted for survival. Therefore, the probability for 
producing more fitted structures by a random change may be ex- 
tremely small and so it will naturally be expected that the changed 
Structures are mostly deleterious far from being useful. This may 
be the reason why mutation produces, as a rule, deleterious characters. 

However, strange to say, the gradual, reversible alteration of 
the genes, as pointed out above, seems commonly to produce characters 
favourable for the organism. This may depend upon that the revers- 
ible change is induced by naturally existing factors such as tempera- 
ture, light, and food, to the changes of which the organisms must 
always be exposed. If organisms could not endure the change of such 
common environmental factors, they would surely become extinct, and 
as a natural consequence only the organisms capable of enduring the 
change would be able to continue the existence. 

As discussed in the previous chapter, the proteins including the 
Organisms composed of the proteins are provided with the property in 
themselves to become adapted to the environmental factor under which 
they are placed for long periods; in other words, the protoplasm and 
accordingly the gene can acquire the structure which is most stable 
under an environment to which the organisms are exposed for prolonged 
periods, so that the environment may become the most suitable one 
to the organisms. Thus the changed environment which acted at the 
beginning as a stimulus upon an organism becomes normal having no 
influence upon the organism. If an organism achieves a change in 
its character in such a way according to the alteration of an en- 
vironmental factor, it may be said that an adaptation favourable for 
the organism has took place. 

Naturally existing environmental factors may usually change grad- 
ually, and so the protoplasm structure may also undergo the struc- 
tural change responding to this environmental change gradually, thus 
gradual, reversible change being established. If the structural pattern 
thus induced by the gradual environmental change was unfavourable 
for some organisms, the organisms would be perished; the organisms 
for which the newly produced pattern was favourable or at least unin- 
jurious would be left as the fitted. This must be the reason why 
reversible alteration of the gene always produces characters favourable 
or at least uninjurious to the organisms. 

On the other hand, the structural pattern induced by unusual 
stimuli such as X-ray or some synthetic chemical agents, even if the 


V. GRADUAL ALTERATION OF THE GENE 381 


pattern is induced gradually and accordingly is reversible, must be 
unfamiliar to the organism and the organisms are not accustomed to 
the pattern; and since, as above pointed out probability of a pattern 
produced at random of becoming favourable for the organism must be 
extremely small, patterns produced at random by unnatural factors 
must, as a rule, be injurious to the organisms. In addition, unnatural 
factors are usually applied suddenly in experimental researches, the 
changes are generally irreversible, resulting in mutation. 


CHAPTER VI 
SELECTION AND ISOLATION 


1. The Effect of the Selection 


As already pointed out, protein molecules appear to have the 
individuality. Every protein molecule is a replica of a template mole- 
cule, but it may be impossible to make a replica entirely equal to the 
template ; presumably every replica may differ slightly from the latter 
on some point or other. The degree and kind of such a difference may 
give rise to the individual character in protein molecules. 

It is well recognized that the molecules of even carefully prepared 
samples of a single protein from a single source may differ perceptibly 
in such diverse properties as molecular weight, charge density, com- 
position, molecular configuration, solubility, and biological activity. 
The conclusion is drawn that all protein preparations prepared so far 
represent populations of closely related members of a family, not 
collections of identical molecules (66a). 

Since genes consist of protein molecules, they should likewise 
possess the individuality, and again since organisms are determined 
in their character by the genes, they should likewise have the indivi- 
duality even when they belong to one and the same species. Indivi- 
dual variations may thus arise. 

Proteins will be changed in their structure by environmental effect 
but the degree and perhaps also the direction of the change may 
vary with the difference in the structure. In like manner, each indi- 
vidual may change the character in different ways even under the 
influence of the same environmental factor, and as a consequence varia- 
tions will disperse. 

As stated above, organic evolution is considered to be raised by 
the gradual change of genes. However, since even the individuals of 
the same species are thus not endowed with entirely the same series 
of genes, each may tend to undergo different changes, advancing into 
different directions. An individual exhibiting an extreme character, 
therefore, may have a gene tending to undergo an extreme change, 
and extreme character may be further advanced if the environmental 
effect, which has given rise to the character, continues to exist. 


VI. SELECTION AND ISOLATION 383 


Here, the well-known experiment of Castle carried out with hooded 
rats should be mentioned. He was able, through selection in succes- 
sive generations, to extend or diminish the black of the colour pattern 
of the rats. In his experiment it was clearly shown that the selected 
rats having the extreme colour-pattern tend to push the extremity in 
successive generations, the colour pattern becoming more and more 
conspicuous. His observation was spread over about 20 generations of 
the rats. Another well known example of this kind is Tower’s ex- 
periment with colorado beetles in which he likewise succeeded in in- 
creasing the peculiar colour pattern by selection. 

Many other evidences can be presented to show the effect of the 
selection, but it appears that they were not adequately interpreted, 
presumably because of the general acceptance of Johannsen’s pure- 
line theory, which, however, the writer is unable to accept as being 
legitimate. 

The beans with which Johannsen carried out the experiment and 
also the paramecia with which a similar result was obtained afterwards 
by another author might have been taking place a gradual change so 
slowly that the progress of the change was overlooked. It should be 
noted, however, that even during the experiments of Johannnsen, two 
mutations, one for higher and one for lower seed-weight, were de- 
tected ; this may show that his selection would produce high and low 
seed-weight respectively. Granting that such mutations were only rare, 
it may also conceivable that the peculiar condition under which the 
observations were made might remove the factor which was causing 
the gradual change. It should be remembered that the observations 
were made with the descendants from a single homozygous organisms 
exclusively propagating by self-fertilization. Whatever might be the 
reason why apparently unusual result were obtained for which the 
pure-line theory was derived, there seems little doubt that his findings 
were exceptional since as just stated there are many evidences showing 
the striking effect of the selection. 

However, most examples in which the selection appeared to be 
effective were commonly interpreted as being due to the accumula- 
tion of minute gene mutations occurring in a certain direction. Such 
an interpretation, on the other hand, may be proper as it may not be 
entirely impossible to consider the gradual alteration of the gene to 
be an accumulation of minute mutations. 

At any rate, the genes in each individual even under the same 
environment may perform a change peculiar to the individual according 
to the individual difference in the structure of the genes, giving rise 
to a peculiar character. When such a peculiar character becomes to: 
have a survival value, it will be selected by natural selection. Since 


384 V. THE NATURE OF EVOLUTION 


the individual thus selected must have the property to develop still 
further the character in question, the character will be advanced un- 
limitedly in so far as the environmental effect causing the change 
continues to exist, thus resulting as a natural result in overspeciali- 
zation or overgrowth, in which once useful parts are carried entirely 
beyond the point of utility until they become iujurious and possibly 
even instrumental in the extinction of the species. The tusks of the 
saber-toothed tiger, the antlers of the extinct Irish elk, the huge size 
of certain dinosaurs, and the tusks. of the Columbian elephant are 
often-cited examples of such disadvantageous development. 

As already discussed, although the genes seem to direct each 
particular restricted structure of protoplasm protein, the change at a 
certain structural portion of a protein molecule by a gene tends to 
exert influences upon other portions, and accordingly a development 
of a useful, selected character is inclined commonly to be associated 
with a development of certain other characters which may not be so 
useful or even utterly useless. In addition, as will be mentioned 
later in detail, hormones play a predominant part in the evolution in 
developing many characters by their peculiar effect upon genes. These 
may be the reasons why organs appearing to be of no use are also 
often overspecialized. 

In fact, useless characters, along with those that are useful, seem 
to be common: in organisms. This is particularly true for the struc- 
tural features of animals. In recent years a number of mass-selection 
experiments directed at increasing, as well as decreasing, the magni- 
tude of a given quantitative character have been reported. A constant 
feature of such experiments has been the occurrence of correlated 
changes in characters other than the one being selected (67). The 
difficulty of the independent occurrence of a change restricted to a 
portion of the protein molecule is shown also in the fact that the 
influence of genes is believed to be usually dependent upon their 
association with certain other genes. Evidences for this interaction 
of genes is clear-cut. At least twenty five pairs of allelomorphic 
genes are believed to be concerned with eye-colour. Conversely, a 
single pair of genes may influence more than one character. In many 
instances it is known that the three or more allelic states of the 
genes for a particular character exist. A single pair of genes may 
also influence the appearance of more than one character. It has 
also been known that genes have somewhat different actions accor- 
ding to what neighbours they possess. This is the so-called position 
effect. 

Anyhow, the individual having an extreme character shares the 
gene which will more and more extend the extreme character ; conse 


VI. SELECTION AND ISOLATION 385 


quently-the effect of the selection is enormous. Thus the wonderful 
variety of domestic animals:and cultivated plants were produced by 
artificial selection, whereby the individual that seems most disirable 
would be selected out of numerous individuals from generation to 
generation.. 


2. The Effect of Isolation and the Origin of Species 


As repeatedly described, newly acquired patterns of protein struc- 
ture are so unstable and liable as to return readily to the original 
patterns. This may hold true also for a selected character. A newly 
appearing character will be determined by a gene having a structural 
pattern newly acquired, and therefore, when an individual having an 
extreme character which has been advanced by selection is crossed 
with an unselected individual, the extreme character will soon be 
lost. Thus the new pattern of a gene of a selected individual is much 
weaker than that of the original. 

This fact was clearly shown in Castle’s experiment above cited. 
He found that the extreme color-pattern obtained by selection was 
lost when the selected individuals were crossed with the normal. 
Entirely similar phenomenon was likewise observed in Tower’s experi- 
ment with the beetles (68). 

Notwithstanding the fact that mutation is given rise to by irre- 
versible change of a gene, it is generally accepted that the overwhel- 
ming majority of characters resulting from mutations are recessive, 
indicating that newly acquired characters are weak even when they 
are irreversible, and so it should be expected naturally that characters. 
newly acquired by the reversible, gradual alteration of the genes are 
always weaker than the original. This must be the reason why iso- 
lation is regarded as the necessary frerequisite and the inevitable- 
cause of specialization. 

Isolation is considered thus to be indispensable for the development 
of new characters, but when a new pattern can deviate from the- 
original so far that the crossing between them becomes impossible, 
isolation is no more necessary and the new pattern is then a new 
species. There are thus commonly distinct discriminations between 
species, no intermediate types existing. 

As accepted generally, ecological factors may play the most im- 
portant role in the isolation, and when the deviation goes far enough 
the reduction in fertility will result, thus new species being establi- 
shed. The reduction in fertility involves interspecific sterility and 
hybrid sterility. Interspecific sterility may be based upon the produc- 


386 V. THE NATURE OF EVOLUTION 


tion of an inviable zygote. Hybrid sterility prevents the reproduction 
of hybrids that have reached the developmental stages at which the 
parents normally breed. Sterile hybrids produce either no functional 
gametes or gametes that give rise to inviable zygots. 

It should be noted, however, that while wild species, as a rule, 
thus cannot be crossed, domestic species can usually be bred among 
themselves. This indicates that wild species cannot exist as such 
unless the crossing with the original species becomes impossible, 
whereas domestic species can be preserved without the reduction in 
fertility owing to the isolation by artificial selection. 


CHAPTER VII 
THE MECHANISM OF ADAPTATION 


1. The Adaptation as a Reversible Change 


The close adaptations of animals and plants to their individual 
stations in life, fitting them to secure food and protect themselves, 
are the most striking features we find in'them. Since environment is 
usually not constant, it is plain that if living things are to continue 
to live, they must have the capacity for readjustment directly or in- 
directly to changed environment. 

It is a matter of common observation that plants of the same 
species, even if grown from seeds of the same fruit, may be quite unlike 
when grown in very different habitats. Thus, when plants of moist 
valleys are grown from seed or by transplantation in alpine regions, 
their internodes are generally Shorter, their leaves are smaller and 
thicker and their flowers are larger than when they grow in their 
natural habitat. If, however, the seeds of these plants are planted in 
the natural habitat again, it will be found that the modifications 
which the species displayed when grown in an alpine habitat have 
not been inherited. The reversibility of the change is thus definite. 

If we continue to use some of our muscles in excess, by and by 
the muscle will become hypertrophic so that we may be able to use 
it with facility. Such an adaptation is especially striking with bones 
and blood vessels as well as with muscles, but the change is reversible 
and the removal of the stimuli will soon be followed by the recovery 
of the normal state. 

Some animals like chameleons of the tropics can change color 
promptly according to the color of the background. Some of the most 
remarkable instances of variable concealment coloration are met with 
in the flounders and various other bottom-dwelling species of fish 
which change their colour pattern in conformity with the colour shade 
of the background. According to Kammerer (69) the salamander, 
Saramandara maculosa, could change their colour pattern in a similar 
way, but the change proceeded very gradually during the prolonged 
breeding periods with the background of certain colours. The obser- 
vation was continued for several years, but the change was not 
definitely proved to be heritable. As is generally recognized, the colour 


©88 V. THE NATURE OF EVOLUTION 


of some pupae can be altered by the colour of background on which 
the caterpillars are reared. 

Sumner (70) was able to change the tail length of rats by chang- 
ing the temperature at which they were breeded. High temperatures 
induced long tails and low temperatures short tails. In this connec- 
tion it should be remembered that, as is well known, cold climates 
reduce body surfaces such as ears, tail, and neck, resulting in the 
development of a generally more compact build of body. This tendency 
is particularly well exemplified by the size of ears in foxes as one 
passes from warmer to colder climate. This kind of adaptation may 
be valuable on the point of the thermal regulation of the body. 

It is said that the red primrose had red flowers if kept at a teém- 
perature ranging from 15° to 20° C. A plant with the same genes but 
reared at'a temperature of 30° to 35°C with other environmental con- 
-ditions unchanged, produced white flowers. If a plant with white 
flowers is brought into a room at 15° to 20°C the flowers that develop 
later will be red. This phenomenon may be likewise significant in 
the regulation of heat effect. 

Rabbits of a certain kind, when grow adult, have black hair on 
such heat-losing surfaces as ears, tail, and the ends of extremities, 
but the bandages to keep warm the part from where the black hair 
has been removed, will cause white hair to grow, whereas black hair 
will result-on the back or the abdominal region: when hair is cut to 
cool the region. 

A certain case of the fruit-Ay, Drosophila, is distinguished from 
the normal by the fact that these are very few black bands on the 
abdomen. When this race is reared ona rich supply of moist food, the 
abdominal bands are almost completely absent in all individuals. The 
same stock raised on scant, dry food exhibits normal banding of the 
abdomen. 

Thus organisms can readily change their characters according to 
the change of environment. Such a change is always reversible, and 
remarkably enough the change mostly appear to be favourable for the 
organism, at least. not deleterious. The last mentioned example of the 
fruit-fly may not be regarded as a favourable change, but at least 
harmless. 


2. Dauermodifikation 


The reversible, adaptive change observed in higher organisms can 
likewise be seen in unicellular organisms, such as bacteria, protozoa, 
and fungi. Especially with bacteria this phenomenon has been well 


VII. THE MECHANISM OF ADAPTATION 389 


investigated. Bacteria can usually acquire the resistance against a 
variety of chemical and physical agents, which exert some injurious 
effects upon them, when cultivated under their influences. The 
change is, as a rule, reversible, acquired characters being lost when 
the influence is removed. 

Attention should be paid, however, to the fact that usually some 
periods of time are required, after the removal of an injurious agent, 
for the complete disappearance of the resistance, that is, for the 
recovery of the former character, so that the change is often called 
“Dauermodifikation’’ or durable modification. This shows that the 
changed character is inherited through several generations although 
meanwhile the character will become less and less distinct until at 
last completely disappears. 

In higher organisms, acquired characters may appear not to be 
transmitted to progeny, but since in the majority of cases the charac- 
ters tend to continue for some periods after the romoval of the stimulus, 
it must be admitted that the character is transmitted from the somatic 
cells to other newly formed ones in the region. Accordingly, in this 
respect, the adaptation of higher multicellular organisms should like- 
wise be regarded as “‘Dauermodifikation’’ of somatic cells. On the other 
hand, as the structural pattern of a cell is to be determined by the 
genes contained in it, it should be considered that Dauermodifikation 
is caused by a temporary change of the genes in the somatic cell. 

As already described, the genes have the property to alter their 
structure responding to the environment or stimulus under which 
they are placed, but as the change is reversible the removal of the 
stimulus will lead to the recovery of the original structure. However, 
a considerable period of time is usually required for the complete 
recovery. Dauermodifikation is thus raised. 


3. Various Types of Adaptation 


As already mentioned, a new environment may act upon an 
organism as a stimulus but when the organisms is exposed for a pro- 
longed time to the same environment, the organism will complete the 
change induced by the environmental factor, that is, it will acquire the 
structure correponding to the new environment, which therefore comes 
to have no influence upon the organism. The adaptation thus depends 
upon the basic character of the protein itself, If the adapted struc- 
ture, that is, structure which had completed the change induced by the 
new environment was favourable for the organism, the organism would 
be more fitted to continue its existence ; therefore, the adapted struc- 


390 V. THE NATURE OF EVOLUTION 


tures are in the majority of cases favourable for the organism having 
been able to continue their existence up to the present. 

Now, if a factor causing such an adaptation is a chemical agent 
having a definite structure, the organism has to be changed to become 
complementary in their structural pattern to the chemical agent in 
order to become adapted to the latter, as the adapted structure of the 
organism must be the structure completely complementary to that of 
the agent. When such an adaptation is accomplished, the organism 
can readily combine with the agent through the complementary pattern 
but without being exerted any influences. As a good example of this 
type of adaptation will be mentioned the so-called adaptive enzymes. 

A certain strain of colon bacteria will gain the faculty to utilize 
lactose when cultivated in the medium containing this sugar. It is 
believed that in such a case an adaptive enzyme to ferment the sugar 
is produced. In order to utilize the sugar, the bacteria are to combine 
easily with it, and the combination will be achieved by the production, 
or rather reinforcement, of the combining group. The appearance in the 
bacteria of the structure complementary to that of the substrate asa 
result of the adaptation must account for the reinforcement of the 
combining group. The bacteria cannot utilize the sugar presumably 
because they are not provided with the strong combining group for 
the sugar. In like manner, antibody production is also regarded as 
this type of adaptation of certain cells against the antigen. Again 
virus is produced by a virus because the complementary structure is 
formed in the protoplasm. Thus it may be said that virus-formation 
is also involved in this type of adaptation. The production of such 
complementary structure in response to chemical structures of the 
stimulants appears to be based upon the general and essential charac- 
ter of the protoplasm. Viruses, antibodies, and adaptive enzymes are 
nothing but the structures freed from the protoplasm thus adapted ‘to 
respective stimulants. 

Anyhow, if a complementary structure given rise to in the proto- 
plasm of a cell or of an organism by a certain agent is transferred to 
the gene, or the structure is raised directly in the gene, then the 
cell or the organism can be regarded as having adapted itself to the 
agent with the acquisition of the property to utilize the agent. This 
type of adaptation may be designated as attractive adaptation, as 
attractive or combining structure is to be strengthened as a result 
of the adaptation. However, in addition to this, there seems to be 
another type which may be called rejective adaptation. Next a mention 
will be made of this type of adaptation. 

An agent, in order to act as a stimulus on an organism, must be 
received by the protoplasm of the organism. The reception may be 


VII. THE MECHANISM OF ADAPTATION 391 


established by some combining group present in the protoplasm or on 
the cell surface. In the case of the attractive adaptation, such a com- 
bining group is strengthened by the adaptation. A sugar or an antigen 
may combine with bacteria or antibody-producing cells, respectively, 
through such a combining group to act as a stimulus causing the 
adaptation ; and after the establishment of the adaptation the combin- 
ing group will be reinforced so much that the group, if liberated 
from the protoplasm, can act as the enzyme or the antibody, respecti- 
vely. Also a virus can affect a cell because a combining group directed 
to the virus may be present on the cell surface, and after the infection 
the protoplasm structure will become complementary to that of the 
virus; as a consequence the cell will combine readily with the virus 
but without being exerted any injurious effect by the latter. Thus 
the cell will become immune to the virus as a result of the attractive 
adaptation. 

However, such a complementary structure may fail sometimes to be 
produced following a virus infection as in the case of influenza virus 
affecting red-blood corpuscles, presumably because of the character of 
the cells unable to produce the pattern completely complementary to 
that of the virus. The failure in the production of complementary 
structure may give rise to only the disturbance in the protoplasm 
structure by the virus, leading to a type of coagulation of the protein, 
whereby various free polar forces including the combining group for 
the virus will be lost. As a natural result of this disappearance Of 
the combining group, the virus will be released from the cell and at 
the same time the cell will become immune to the virus, thus the 
rejective adaptation being established. 

For instance, when a virus such as that of influenza combines 
with a red cell, only a structural disturbance will result, causing the 
disapearance of the combining group, because the red cell is not sus- 
ceptible to the virus and so cannot reproduce the virus, that is, the 
cell cannot form the complementary pattern so as to establish the 
attractive adaptation. As a consequence the cell may acquire the pro- 
perty to reject the virus and the virus will be released from the cell. 
If such a change is given rise to by an agent other than a virus, 
it may be said that the cell is adapted itself to the agent not to be com- 
bined by it. The mechanism by which an enzyme can liberate froma 
molecule of a changed substrate to affect another unchanged substrate 
is elucidated in a similar way as discussed in chapter V, in Part IV. 

In the majority of cases, a number of combining groups directed 
respectively to each group of agents may be involved in some consti- 
tuents’ present on the cell surface. If such a constituent combines 
with an agent to be disturbed in its structure and undergoes denatu- 


392 V. THE NATURE OF EVOLUTION 


ration, the substance will be removed from the cell as it will become, 
as a result of the denaturation, a substance foreign to the cell. If the 
removal of such a substance is accompanied by the destruction of the 
enzyme systems concerning the synthesis of the substance, the sub- 
stance is not only lost but also its synthesis will come to cease, 
leading to the rejective adaptation of the cell against the agent. 

On the other hand, the inability of producing certain substances 
may be involved in the corresponding pattern of the protoplasm and 
accordingly also the gene itself may come to possess the pattern fol- 
lowing the establishment of a rejective adaptation. If so, the pattern 
may be transmitted to newly produced cells in so far as the gene 
retains the pattern thus induced. 

The mechanism of adaptive colour changes, such as those of sala- 
mander above cited, may be elucidated in this manner. If the struc- 
ture of a gene is changed so as to reject the visible ray of a certain 
wave length, under which the cell or the organism is always exposed, 
the cell or the organism will come to have the colour of the wave 
length. Thus organisms will become white, when they are exposed 
to the light of every wave length, since all the visible rays will 
come to be rejected, whereas they will become black when kept in a 
dark place as no light will be repulsed. Of course, such a change is 
valuable as a concealment colouration. In higher animals, hormone 
systems are amazingly advanced and protoplasm structure changed by 
environment may be liberated in the forms of hormones to effect the 
pattern of the protoplasm’and also even the genes of the other cells. 
Thus colour change is usually achieved by special pigment cells called 
chromatophores the function of which is under the control of hormones. 
In the case of temporary adaptation as in this case of concealment 
colouration, only the pattern of the protoplasm may be involved without 
being accompanied by the corresponding change of the gene. 

The rejective adaptation like the attractive one must be involved 
in one of the basic characters of the protein. Thus, on the one hand, 
physical of chemical agents which exert deleterious effect upon organ- 
isms may tend to cause, as a rule, coagulation of proteins. On the 
other hand, various polar groups of proteins are likely to disappear 
when they are coagulated. It appears, therefore, to be a natural result 
that organisms may adapt themselves to deleterious agents to reject 
them. In so far as an agent causes the coagulation whereby combining 
groups may disappear, an organism will achieve a rejecting adaptation 
to the agent, while if an agent has no action of coagulation, the pro- 
toplasm may produce the complementary structure in response to the 
structural influence of the agent with the establishment of the 
attractive adaptation. 


VII. THE MECHANISM OF ADAPTATION 393 


Organisms which can establish rejective adaptation to injurious 
agents are, of course, being fitted for existence and, will continue to 
exist, and therefore this adaptability has to be highly advanced. 

In higher animals including man various hormone systems are 
amazingly developed for their evolution, and adaptive evolution appears 
to be mainly achieved by hormones as will be described later. Hor- 
mones, aS mentioned above, can be regarded as protoplasm structures 
changed by environmental factors. Namely, changed structures of the 
protoplasm liberated as free forms may be looked upon as hormones, 
but as the influence of the environment may be greatly modified 
through hormones, and as the types of hormones are restricted in 
number in higher animals, the direction of evolution tends likewise to 
be restricted, not very specifically controlled by environment. 


CHAPTER VIII 


THE INHERITANCE OF ACQUIRED 
CHARACTERS 


1. The Forgetting of the Original Structure 


As pointed out above, adaptive change of genes, as a rule, can be 
regarded as ‘‘Dauermodifikation’”’ or durable modification. Since the 
gradual, reversible change of genes takes place gradually under the 
influence of environmental factor, the return to the original structure 
on the removal of the environmental factor, which induced the change, 
should take place also gradually, with the result that the changed 
character continues to exist for some periods of time after the removal 
of the factor. 

Durable modification, however, is found mainly with unicellular 
organisms. In the case of higher, multicellular organisms the modi- 
fication is, as a rule, restricted to one generation, not being inherited 
to progeny, although its transmission from soma to soma appears to 
be possible. This is apparently not to be attributed to the failure of 
the transmission of the change of the somatic cell to the germinal, 
since, aS will be discussed below, there are good reasons to consider 
that the transmission is quite possible. The general non-inheritance 
of acquired characters in higher organisms should be ascribed to the 
fertilization in which the changed pattern of the germ cell is to re- 
turn to its original state through the structural disturbance by the 
germ cell of the other sex. This return to the original pattern is 
nothing but the rejuvenation caused by the fertilization as already 
stated. 

This is also the case with unicellular organisms. It has several 
times been reported that the occurrence of conjugation or autogamy 
was particularly likely to result in rapid reversal of an adaptive 
change. In every case described the adaptive change can take place 
only during periods of asexual reproduction, in cells originally derived 
from single ancestral individual. 

For example, by culturing for long periods in the presence of 
sublethal amounts of arsenious acid, a strain of paramecium was 
obtained which was several times as resistant as its parent (72). This 


VI. SELECTION AND ISOLATION 395 


strain retained its resistance during hundreds of vegetative divisions 
in an arsenic-free medium before beginning to revert gradually to its 
former sensitivity. However, if conjugation of two resistant cells 
occurred, the progeny became completely sensitive. It should be noted 
that irregular fluctuations of temperature and other changing condi- 
tions hastened the de-adaptation, though not so conspicuous as conju- 
gation. As was already emphasized, the rejuvenation or the recovery 
of: the former structure is caused generally by the disturbance of the 
structure; the effect of the conjugation or the fertilization is based 
upon this disturbing action. 

A similar phenomenon is also kown with trypanosomes, rendered 
resistant against arsenophenylglycine. The resistance can be retained 
for considerably long periods, but a passage through a rat louse and 
then to a healthy rat the resistance is lost completely, thereby the 
trypanosomes become highly susceptible to the drug (73). As was 
previously discussed, in the respect of the rejuvenescence the host 
change is analogous to the fertilization. 

However, inspite of such rejuvenating action of fertilization, 
whereby the recovery of the original pattern will be facilitated, there 
are numerous evidences showing that acquired characters even of 
higher organisms are transmissible to progeny, though of a transient 
nature. The wheat of a south country can shorten the period required 
for reaching the maturity, when removed to the north and cultivated 
there for prolonged periods. Such an acquired character is likely to 
be retained for several generations even wheh the wheat is removed 
to the south. It is well acknowledged that various characters, based 
upon habitats, of cultivated plants tend to continue even when the 
plants are transplanted to entirely different places, although the char- 
acteristics will diminish gradually. The same has been reported 
with water-flea having the shape varying with the habitats. 

It has been confirmed that the acquired character to produce 
antibodies to some antigens can sometimes be transmitted to progeny. 
Thus, domestic doves and chickens immunized with some viruses 
transmit the character to produce the virus-neutralizing antibody 
through the egg to their offspring, although immunity conferred on 
the offspring is short, lasting a maximum of 12 weeks in doves and 
of 4 weeks in chickens (73a). This may be regarded as the transmis- 
sion to the progeny of the antigenic pattern impressed in the anti- 
body-producing cells by the virus. 

The transmission of the very pattern of bacteria or of the viruses 
to the offspring through germ cells of the host, a phenomenon which 
was repeatedly discussed, should be likewise regarded as a kind of 
a durable modification. The pattern is to be lost sooner or later even 


396 V. THE NATURE OF EVOLUTION 


if its transmission to the progeny is possible unless it is repeatedly 
engraved by infection. 

The Lamarckian theory of the inheritance of acquired characters 
is not generally accepted because of the failure to secure any definite 
evidence for the theory. However, since the change of the genes is 
reversible, the evidence demanded has never been, and will never be, 
obtained. The organism can develop a new character under a new 
environment, but, when it is returned to the original environment, the 
change is not lasting. For the continuance of a new character, the 
new environment inducing the new character is always needed, and 
in so far as the new environment continues to exist, the new character 
is not only lasting but also will gradually be stabilized. : 

Here we should remind the most remarkable character of proteins 
already discussed. Namely, proteins can recover their previous struc- 
ture, because the previous structure is remaining in their memory, 
but if it is forgotten the recovery will become impossible. This very 
forgetting is to occur when proteins exist for prolonged period away 
from the previous pattern.. Therefore, a new character produced under 
a new environment will be fixed when the new environment becomes 
constant, and as a result the character will not to be lost so soon 
even when the organisms are removed to the previous environment. 

Thus adapted characters of organisms to environmental factors, 
such as temperature, light, and nutrition, will not only remain in 
existence as long as the factor continues to exist, but will be 
gradually stabilized in being accumulated little by little in the genes, 
until the acquired pattern becomes the ordinary one with the oblivion 
of the previous pattern. 

If an environmental factor is again altered in a new direction 
after the former pattern has been forgotten or the return of the former 
pattern has become difficult, then adaptive change will begin anew on 
the basis of this acquired pattern; without returning to the previous 
one. In such a way the pattern of the genes will continuously ad- 
vance leading to the evolution of the organisms. Newly formed 
pattern is always unstable, and so the isolation is indispensable for 
the evolution. 

In the case of individual development or ontogeny, as previously 
stated, newly formed structures are likewise unstable, but they will 
gradually become stable and irreversible as a result of the oblivion 
of former structures. Also in this respect, ontogeny recapitulates 
phylogeny. ; 

In short, it may be concluded that the oblivion of the former 
structure is necessary for the evolution. However, for the function 
of the germ cell to recapitulate the phylogeny, an extremely strong 


VIII. THE INHERITANCE OF ACQUIRED CHARACTERS 397 


memory must be required. For this purpose higher animals may 
possess a certain group of cells, in which the memory is well kept 
by the repetition of the reversible change, to produce the germ cells. 


2. The Transmission of Acquired Pattern to Germ Cells 


In the case of unicellular organisms, the transmission of the 
change, given rise to in the protoplasm, to the gene may readily 
take place, but in multicellular organisms, its transmission to the 
genes of the germ cell appears at first sight to be impossible, although 
for the establishment of the inheritance of acquired characters the 
transmission of the pattern from soma to germinal seems indispen- 
sable. The writer holds the opinion, however, that this is actually 
possible, and factors involved in this process are both hormones and 
virus-like agent as will be mentioned below. 

For a multicellular organism, to exhibit various functions as a 
single living organism, that is, to behave as an individual, a variety 
of cells or cell systems involved in the organism must be coordinated, 
or must work in harmony. This coordination is established usually 
by two ways. One is by means of substances that circulate in the 
blood, namely hormones, and the other is by means of impulses that 
pass along the nerves. As the result of secretions which enter the 
blood from endocrine glands, what is known as chemical coordination 
is possible. 

It has been known for a long time that various external influences, 
especially deleterious agents, applied to higher animals lead to a striking 
change in the function of endocrine glands. At present, it is generally 
believed that the endocrine system plays a prominent part in the adap- 
tive reactions which occur irrespective of the specific nature of the 
damaging agents. The sum of all these non-specific, systemic reactions 
of the» body which ensue upon long-continued..exposure to stress has 
been termed the ‘“‘general-adaptation-syndrome”’ (74). It is charac- 
terized by a number of morphological and functional change. Among 
the most prominent of these is the enlargement of the adrenal cortex 
with increased corticoid-hormone secretion, involution of the thymus 
and of other lymphatic organs. 

A number of deleterious effects such as reduced atmospheric 
pressure, extreme heat or cold, severe muscular work, hemorrhage, 
burns, traumatic shock, and a large variety of drugs, cause histologi- 
cal signs of increased adrenaline and corticoid hormone secretion. All 
these phenomena run closely parallel with the general damaging 
effects of agents and must be regarded as part of the general-adaptation- 


398 V.. THE NATURE OF EVOLUTION 


syndrome which they elicit. Their influence upon the cortex is me- 
diated by the anterior lobe of hypophysis and prevented by hypophy- 
sectomy. 

It is considered that the effect of such damaging agents or the 
stress is first received by the anterior lobe of hypophysis with the 
increased secretion of adrenocorticotrophic hormone which induces the 
adrenal cortex to produce corticoid-hormone; the production of gonad- 
trophin and thyrotrophin, on the contrary, may be diminished by the 
Same Stress. 

Of the various environmental factors,-climatic ones seem to be 
most closely connected with animal and vegetable life, whereas it is 
supposed that also the climatic effects are received mainly by hypo- 
physis or pituitary gland, and that seasonal cycles of pituitary activity 
that are reflected by cytologic change or bioassay are known to occur 
throughout animals especially in vertebrates. It is reported that a 
combination of environmental factors, including amount of rainfall, 
mean temperature, and a food supply, affects the pituitary cycle of 
some animals (75). Moreover, it is stated that the breeding season 
rhythm of all amphibia is under the anterior pituitary control. which 
is in turn influenced by nervous factors, and the final release of pitui- 
tary hormones is thought to occur in response to complex climatic 
changes. Internal sexual rhythms are usually adjusted to externa. 
seasonal change. 


It is generally daltepeledeed that water metabolism of man is 
extensively effected by climatic factors, wheras it is known that the 
water metabolism is subjected to the function of adrenocorticotrophic 
hormone of hypophysis. On the other hand, the features of piant 
activities such as flowering and fruit bearing are, as is well known, 
governed by climatic factors, such as temperature and light, whilst it 
is believed thatin this connection hormones or related substances play 
an important part. Some animals such as the chameleon can promptly 
change the colour-pattern in response to the colour of back ground. 
Such a colour change in several vertebrate species depends upon 
stimulation of the eyes which is transmitted to the endocrine glands, 
which in turn produce hormones acting directly upon the pigment 
cells, chromatophores. 

The interfering action of hormones upon genes is remarkable as 
already emphasized, and consequently changes in the feature of 
hormone secretion induced by environmental factors may be able to 
have influences peculiar to the changed feature upon the genes of 
germ cells leading to the formation of peculiar characters of the orga- 
nisms. However, the effect of hormones cannot be so specific as can 
produce the amazingly specific adaptation of organisms. The specific 


VIII. THE INHERITANCE OF ACQUIRED CHARACTER 399 


change corresponding to a given environmental factor will be produced. 
in the cells or tissues upon which the factor is directly supplied ; and 
such a specific change may be unable to be transmitted to the germ 
cells by hormones. However, if the specifically changed structure of 
the protoplasm is liberated from the cell in the form of a “‘virus’’ the 
structure will be easily transmitted to the germ cells. 

In ontogeny, as was already considered, the development of proto- 
plasm structures inducing the advancement of various characters are 
apparently achieved by both hormones and virus-like agents. It may, 
therefore, be reasonable to suppose that in phylogeny also virus-like 
agents, in addition to hormones, are involved, since the miniature of 
phylogeny is seen in ontogeny. 

It is most interesting that this concept is well in accord with 
Darwin’s pangenesis theory. Darwin considered that minute particles 
termed gemmules may be distributed throughout the body of the 
organism in order to accept external effects and subsequently to 
transmit them to germ cells. Of course, gemmules are no more than 
“‘viruses’’ in the writer’s concept. 

It was already pointed out that primitive structures of the proto- 
plasm are unstable and weak, as the primitive structures are the 
newly produced as compared with the differentiated ones. Chick em- 
bryos have primitive structures and so can readily affected by various 
viruses. In a similar way, since germ cells or mother germ cell are 
of primitive nature, their structural pattern will be readily changed 
by the ‘‘viruses’’ or gemmules. Consequently, environmental effect 
may be easily transmitted to germ cells or mother germ cells through 
virus-like agents. 

The structural correlation between soma and germ cells or mother 
germ cells, suggested in Chapter I, 3, in this Part, can be thus 
achieved by ‘‘viruses.’’ The genes in germ cells are considered, there- 
fore, to be always accepting the structural pattern of every part of the 
body through the ‘‘viruses’’; and accordingly the genes in the germ 
cell must be the representatives of all patterns of somatic cells. The 
patterns engraved in the genes will be developed during ontogeny 
when the conditions become fitted for their development. 

To sum up, if certain somatic cells are changed in their protoplasm. 
structure by a certain environmental factor, the changed structural 
pattern will be transmitted by the ‘“‘virus’’ to the germ cell; and the 
character corresponding to the pattern will be developed in the pro- 
geny, if the pattern is strong enough not to be returned to its original 
during the development. 

Hormones may be able to induce striking changes in the structure 
of the genes, but their action is rather non-specific as compared to the 


400 V. THE NATURE OF EVOLUTION 


highly specific effect of the ‘‘virus.’’ Presumably hormones may act 
as helpmates of viruses, assisting the development of the specific 
structure determined by the latter. As was already pointed out, 
mammary cancer virus or leucosis virus can act as such only when 
sexual hormones or related factors are present. At an early stage of 
the individual development, hormone-like substances may assist the 
function of organizer, which can be looked upon as a strong structure 
comparable to a virus. 

During pregnancy, striking changes take place in the feature of 
hormone secretions, especially leading to hypertrophy of the hypophysis 
associated’ with its increased function, indicating the requirement of 
unusually increased activity of hormones for the development of the 
foetus. 


3. The Effect of Use and Disuse 


It is a familiar fact that during the life of an individual, new 
characters can be acquired by use, and that disuse leads to the 
disappearance of the characters. For example, the use of muscles 
increases their development, while disuse results in the deficiency or 
even complete loss of the function. According to the Lamarckian 
theory the characters thus acquired in an individual are inherited by 
its offspring. Although the majority of authors claim that this theory 
has no support from modern biology, the writer is able to provide 
the theoretical basis to this doctrine. 

The development of a structure by use can be regarded as a sort 
of the general phenomenon that the cells are increased in their acti- 
vity upon the reception of stimuli, a phenomenon which in turn may 
be based upon that the protein molecules may be activated by a proper 
physical or chemical factor as a result of temporary liberation of free 
polar groups as: previously stated. The change in certain somatic 
cells thus produced by use may be transmitted to the germ cell 
through the ‘‘virus’’, as in the case of other structures changed by 
environmental factors. There is, of course, no reason to suppose that 
the change brought about by use is different from that induced by 
usual environmental factors. The change induced by use must likewise 
be reversible, and so disuse will result in its disappearance. 

Also in the development of the pattern induced by use, just as in 
that caused by usual environmental factors, hormones appear to be 
involved. As above stated a severe muscular work leads to the 
increased corticoid-hormone secretion. It is a well-known observation 
that cows may be brought into full lactation some time before partu- 


* 


“VII. THE INHERITANCE OF ACQUIRED CHARACTERS A401 


rition, if they are milked regulary, and lactation may be induced in 
virgins, or women many years past the menopause, by repeated stimu- 
lation of the nipples through sucking. In this connection, many workers 
believe that nervous impulses from the nipple affect pituitary func- 
tion. Further, it is stated that the acquisition of new modes of 
behaviour as ,a result of experience cannot occur in the absence of 
hormonal influences (76). 

Thus the pattern developed by use in the local somatic cells can 
be transmitted to the germ cells by the “‘virus’’ whose fenction may 
be strengthened by the cooperation of hormones. If the pattern trans- 
ferred thus to a certain gene in the germ cell is strong enough to be 
preserved, even if partially, until the individual developed from the 
germ cell begins to use the organ in question, the pattern will be 
accumulated in successive generations resulting in a striking develop- 
ment of the organ. However, since such a development may be also 
reversible, disuse will lead to the disappearance of the character, 
although the disappearance will become more and more difficult if the 
use extends over generations. 


CHAPTER IX 
EVOLUTION AND MUTATION 


1. The Significance of Mutation 


As discussed in Chapter V, environmental effects may give rise in 
organisms to adaptive changes, which are usually advantageous, as a 
result of the gradual, structural changes of the genes. The environ- 
mental effect is gradually transferred to genes through the cytoplasm 
or hormones. Such a change of genes is reversible, so that the change 
will disappear when the environmental condition is returned to the 
original state. 

On the other hand, occasionally, strong physical or chemical agents 
can act directly upon the genes, producing sudden, irreversible changes. 
A change belonging to this category is called mutation. It is usually 
deleterious to the organisms in contrast to the gradual, reversible 
change. Up to the present, this latter change has been disregarded 
presumably because of its reversible nature, whereas the former, irre- 
versible change, namely mutation, has attracted universal attentions 
and is generally regarded as one of the main causes of evolution. 

However, such a random change can by no means be considered to 
be significant for the evolution, as was already emphasized by Osborn. 
In fact, aS just mentioned, characters raised by mutation are mostly 
deleterious to the organism far from being useful. 

The mechanism, by which the abrupt change of the gene is not 
only prevented but on the coritrary favourable gradual change is induced 
in the gene, must be of the utmost importance for the establishment 
of the evolution, and consequently such a mechanism might have been 
considerably developed even when the organisms were in rather 
primitive stages. It should be remembered that the adaptive, rever- 
sible changes are common even in the primitive organisms, such as 
bacteria and protozoa, whilst mutation is rather unusual. In higher 
animals, environmental effects may be modified and unspecialized by 
hormones, thus abrupt changes being completely prevented. But, at 
the same time, the reaction modus of higher organisms against com- 
mon environmental factors may become restricted, and the direction of 
the evolution is accordingly confined in a narrow scope. 

It is believed that Australia and New Zealand have been separated 


XI. EVOLUTION AND MUTATION 403 


from the Asiatic continent probably ever since the Cretaceous period, 
and nowhere else in the world do we find so many old-fashioned relics 
of earlier mammalian forms. At the time of the Australian region 
was cut off from the continent of Asia, marsupials were probably the 
highest mammals in the region. In their isolation they were not 
brought into competition with the higher types of mammals which were 
evolving on the other continents; hence they have lingered along the 
path of development at the lower levels of mammalianism. Evolution, 
however, has not been at a standstill among them, for we find that 
special types have arisen to occupy many of the available physical and 
nutritional stations of animal existence. Thus, there are carnivorous 
forms such as the so-called Tasmanian wolf, and herbivorous forms 
like the kangaroos; or in terms of dwelling place there are land forms 
{Tasmanian wolf, kangaroo, wallaby), burrowing forms (bandicoots, 
marsupial mole), arboreal forms (phalangers, Koala, Dasyurus) and vol- 
planing forms (flying phalangers). 

It should be noted that these forms of divergency, commonly called 
adaptive radiation, are very similar to those of other continents where 
various higher types of mammals have been developed, indicating that 
the direction of the evolution of mammals is strikingly restricted. 
Presumably this is due to the highly developed mechanism by which 
environmental influences are reduced to limited forms, and the mech- 
anism may consist of the hormones. 

Organisms especially higher animals appear to be able to avoid 
abrupt mutations by such elaborate means. It may be said, therefore; 
that organisms liable to undergo mutations are only defective specimens, 
never fitted to continue their existence. 


2. Genie Changes due to Chemica! Agents 


Typical, gradual changes in higher animals are considered to be 
induced by physical environmental factors, such as climatic ones, which 
are transferred to the genes after greatly modified by hormones, 
whereas chemical agents appear to affect the genes directly and ab- 
ruptly ; accordingly, the change due to chemical agents is rather a 
mutation, occurring rapidly and tending to be irreversible. 

Viruses afford a good example of the mutation of this category. 
The specific structure of viruses is transmitted directly to the genes, 
and the change occurs rather rapidly, tending to be irreversible and 
usually even injurious to the organism. The action of pneumococcus- 
transforming principle, as discussed repeatedly, may belong to this 
category. This principle causes no injurious effect unlike usual viruses. 


404 V. THE NATURE OF EVOLUTION 


On this point the change raised by this principle is different from 
mutation more than is the change raised by usual viruses. 

This tendency is more manifest with the production of the so-called 
adaptive enzymes. Adaptive enzymes may be produced as a result of 
the adaptation of an organism to chemical substances whose specific 
structure is transmitted to the genes of the organism. The production 
of antibodies can be likewise regarded as a type of such adaptation 
(71). In these cases, although the specific structure may be taransmit- 
ted to the genes rather abruptly, the change can no more be called 
mutation, since it is not only completely reversible but very useful to 
the organism. 

Such a change is called adaptation because it is useful to the 
organism, and such a usefulness has been undoubtedly established be- 
cause the organisms capable of performing such a change could con- 
tinue to exist, and therefore the character to adapt themselves to 
chemical agents have been advanced amazingly in the organisms. In 
this respect, it bears a striking resemblance to the gradual change due 
to physical agents, both being purposeful and have been developed by 
organisms for their existence. It should be realized that local changes 
in somatic cells would never be transferred to the germ cells without 
virus-like agents. This must be a very useful function of ‘‘virus’’, a 
mutagenic agent. 

This seems the case not only with a single individual, but also with 
an organized colony of gregarious animals such as termites and honey- 
bees, aS various individuals in such a colony may be regarded as so 
many cells; the colony may be equivalent to an individual of multicel- 
lular organisms, and so the kings and queens may correspond to germ 
cells, and workers and soldiers to somatic cells. 

The soldiers of termites have greatly enlarged mandibles, guard 
the king and queen and defend the nest against the attacks of intruders. 
Again, the specialized structures and habits of the workers of the 
honeybees is remarkable, food is collected and stored, the young are 
tended and fed. Each individual groups of these insects exhibit such 
amazingly instinctive reactions, which must be the result of inherited 
reflexes. How can have arisen and then inherited these characteristics 
of each individual group, since the workers and soldiers leave no des- 
cendent, unless the characteristics are transferred by virus-like agents 
to the kings or queens? 

Such a transmission of characteristics by virus-like agents appears 
to occur even among fruit flies which are kept in laboratory. In an 
observation with a certain strain of the fruit fly characterized by a 
high mutation rate, Mampell (77) found that the mutability is increased 
not only in this species but also in the individuals of another species 


XI. EVOLUTION AND MUTATION 405 


raised.in the same cultures. He has suggested that the characteristics 
may be transmissible through a virus-like agent. Again, L’Heritier 
(78) has published a full account of the behaviour of the ‘‘viroid’’ or 
cytoplasmic particle responsible for CO, sensibility in Drosophila. 
The agent is capable of transmitting the sensibility from an sensitive 
individual to other normals as do usual viruses. 

As already stated, besides virus-like agents hormones are involved 
in the communication of characters among different cells or organs in 
a multicellular organsism. Similarly, the balance of castes in a termite 
society is believed by some workers to be maintained by means of 
certain special hormones (79). 

The castes of termites develop according to the needs of the colony, 
just as the embryonic cells of a single animal differentiate into blood 
cells, bone cells, muscle cells and so on. Thus a colony of a termite 
is a kind of superorganism. The caste of a termite is not fixed; any 
nymph in a colony of termites can develop into a soldier or a king or 
queen of the supplementary type, developing on what the situation 
demanded. Such a development is controlled by means of some kind 
of the so-called social hormone, produced by the differentiated adults 
and acted upon the undifferentiated nymphs. When the colonies were 
separated by a screen which prevented any contact, the orphanized 
colony was produced and supplemented its own king and queen in the 
normal way. An active principle transmitted by contact, that is, a 
social hormone, is responsible for the development of differentiation. 

As fully discussed in Chapter II, at an early stage of the develop- 
ment embryonic cells are endowed with unstable structures and so can 
change in every direction according to the change of environmental 
factors, but as the development advances, the structures will become 
gradually fixed and unchangeable. Entirely the same phenomenon is 
recognized in the development of the castes of a termite society. Thus, 
probability of change in a nymph decreases with time after molt. With 
the proper stimuli all nymphs that have just molted will change, but 
of the nymphs that molted 19 days earlier, only 50 per cent will change 
and 50 days earlier less than 20 per cent. 

These facts clearly show that the castes of termites correspond to 
cells or organs in an ordinary multicellular organism, which the latter 
in turn is equivalent to a colony, and that the development of the 
castes is achieved by the same principle as that of the cells of an 
embryo. Therefore, the above concept that the transmission of the 
characters of soldiers and workers to the king and queen is achieved 
by ‘‘virus’’ seems to be only natural. 

It is well known that numerous varieties of flowering plants are 
attributed to virus infection. The most usual effect of a virus infection 


406 V. THE NATURE OF EVOLUTINN 


on the flower is to cause a characteristic change in the colour. The 
oldest known example is tulip-break due to an aphis-transmitted mosaic 
virus. Another common virus variegation occurs in the flowers of 
wallflowers and stocks and other cruciferous plants (80). These ex- 
amples should be regarded as mutations due to viruses rather than as 
virus-diseases. 

Many breeders believe that domestic animals will sometimes breed 
the young resembling on some point to the male animal which is not 
the true father of the young but with which the mother animal was 
once crossed, a phenomenon called telegony. This phenomenon is, of 
course, never consistent with the modern theory of genetics, and ac- 
cordingly it has been dismissed as a mere bigotry. Nevertheless, this 
appears possible according to the writer’s concept. 

It has been well established that males of cancer strains of mice 
may transfer the virus-like agent capable of producing the cancer at 
the time of copulation to infect females of either susceptible or rela- 
tively resistant strains and that these infected females may propagate 
and pass the agent to their progeny (81). If this is caused by ‘‘infec- 
tion’’, not by the transmission of the pattern through the germ cell to 
the progeny, such a female mouse may be able to transfer the character 
of the male to the young of another male. In case where the sperm 
of a male after the fertilization developed in the body of a female into 
the foetus, the influence of a male pattern upon the female would 
probably be far more striking. Moreover, it may not be quite im- 
possible that every married couple tends to become Jack and Jill after 
a prolonged cohabitation not only through the mental connection but 
through virus-like agents. 


CHAPTER X 
THE MECHANISM OF EVOLUTION 


1. The Evolution without Natural Selection 


An alteration of the environment results in a change in the char- 
acter of the organism. The change thus raised is mostly favourable 
for the organism and will be advanced as long as the environmental 
factor to induce the change continues to exist, until at last the changed 
character becomes fixed and inheritable. For example, if a mammal 
is kept under the environment of a high temperature, its tail and ears 
will become long and expanded as shown by the Sumner’s experiment 
with rats as above cited. Such a change is indeed significant for the 
temperature regulation, and hence the mammal is said to have adapted 
itself to the high temperature. If the mammal is kept under such a 
high temperature for successive generations, the acquired character 
will become hardly reversible and fixed so as to be inheritable. 

According to the experiment of Sumner, some of the rats exposed 
to high temperatures for only one generation tended to transmit the 
character to the progeny. The phenomenon, known as Allen’s rule, 
that mammals of the warmer climates expand body surfaces by the 
development of parts, such as ears and tails, may be the result of this 
adaptation fixed by Nature. Here, it should be realized that such 
adaptive evolution is possible to occur without any natural selection, 
that is, organisms can evolve without natural selection in adapting 
themselves to environments. 

The evolution must be far more rapid and easier if the effect of 
use is added to the environmental factor. Thus, if a tail, lengthened 
by a high temperature, can be used to grasp objects and if its use is 
required, the animal will use it repeatedly and this effect of use will 
be added to the influence of the temperature to develop the tail ex- 
tensively. In such a way, tails of certain animals such as monkeys 
and rats have probably developed. ; 

The stimulus resulting from the use would become greater, the 
higher the organ was advanced, because of the more unusual and 
incessant use, and therefore the development of the organ would be 
advanced unlimitedly unless it ceased to be used. Orthogenesis may 
be thus established. Natural selection may help more or less the 


408 V. THE NATURE OF EVOLUTION 


development, but there is no doubt that the evolution is quite possible 
without selection. 

Every character of organisms is wonderfully fitted for the purpose 
of their existence. Since the character to achieve the evolution must 
be of the most importance for the organism, extensive advancement 
should be expected also in this character. In addition, since the char- 
acter must be advanced with the progress of the evolution, it must be 
the more advanced, the higher the organimsms are evolved. 

Nevertheless, at present the majority of authors seem to believe 
that the organic evolution was advanced mainly by virtue of random 
mutation and natural selection, a wonderfully clumsy means which 
might only be adopted by extremely primitive organisms as mentioned 
in the next section. 


2. The Evolution of Evolution Mechanism 


New protein molecules are considered to be syntheszied as replicas 
of the template protein constituting the protoplasm or assimilase, but 
the newly formed replicas may not be precisely identical to the tem- 
plate because of partial failure of the replication, thus arising the 
individual differences in the structure of protein molecules. Such an 
individual difference of the structure may in turn give rise to the 
difference in the behaviour of each protein molecule or each polymerized 
product of molecules towards the environmental factor, and accordinly 
each may tend to undergo each peculiar change or mutation. Vari- 
ations of primitive organisms might be thus raised and become distinct. 
An individual having a structure tending to be changed in a definite 
direction under the effect of a certain environmental factor might 
continue to be changed in its structure in the direction under the 
influence of the factor. 

Among the individauls thus coming to have different structures, 
only those most fitted to exist would be able to survive, that is, the 
fittest would be selected by natural selection. As a result of this 
selection, the faculty to evolve spontaneously into a well adapted 
direction might be gradually advanced. 

Although the protein itself possesses the basic character to adapt 
itself to environments as already stated, the character might be insignifi-~ 
cant when it was not yet developed enough, and presumably random 
mutation and selection might play a predominant part in the evolution 
of protein molecules. 

The evolution brought about thus mainly by mutation and selection 
might continue until the proteins were polymerized into complete form 


IX. EVOLUTION AND MUTATION 409 


of assimilase, or until the assimilases were endowed with characters 
as primitive organisms. The progress of the evolution in such a man- 
ner, however, was most probably highly tedious, and hence a dreadfully 
long span of time might pass between the appearance of assimilase-like 
protein aggregates and the production of the organism-like assimilases. 
The very remarkable evolution mechanism belonging to the present 
organisms was presumably advanced thereafter, and since the mecha- 
nism might be advanced the more, the more evolved the organisms, the 
rate of the evolution of the modern organisms may be extremely great 
as compared with that of primeval organisms. 

It seems reasonable to regard the acquisition of resistance of bacteria 
to several antibacterial drugs as a type of the adaptation seen in higher 
organism, but a number of authors claim that the resistance is at- 
tributed to spontaneous mutation and natural selection, that is, the 
emergence of a population resistant to a given drug results from the 
selective effect of that agent upon mutants arising spontaneously from 
the original population (82). Since in primitive organisms such as 
bacteria a highly advanced mechanism of the evolution cannot be ex- 
pected, adaptation may be not so distinct as in higher organisms. But 
it should be borne in mind that adaptability is one of the basic char- 
acters of protein itself and so the protein molecule itself can be adapted 
to environments though in an undeveloped way. 

Mutation may be looked upon, in a sense, as a generation of a 
virus as discussed already. Hence, the primary organisms in extremely 
primitive stages may be said to have been evolved by the successive 
production of viruses. Accordingly the primary organisms may be consi- 
dered as being originated from viruses like the secondary. In this 
respect, there appears no fundamental difference between the primary 
and the secondary organisms. 

In fact, it may be difficult to draw a distinct line of demarkation 
between these two main groups of organisms. It may be said, however, 
that the secondary organisms were generated on the basis of preexisted 
protoplasm or organisms, whilst the basis on which the primary org- 
anisms were developed was the incomplete protoplasm or the aggrega- 
tion of protein molecules which had been produced without organisms. 
Therefore, the secondary organisms unlike the primary have their host 
at least when they are in their primitive stages. Nevertheless, in the 
case of the secondary organisms that developed on the basis of the 
primary organisms existing at thé most primitive stage, there might 
be no distinct difference between the parasite and the host, and so the 
parasitic nature would be insignificant in such secondary organisms. 

In this respect it may be suggested that the Mollusca may be the 
oldest secondary organisms, because their parasitic nature seems only 


410 V. THE NATURE OF EVOLUTION 


trifling, if exists, and the Echinodermata which may be the oldest pri- 
mary organisms are being adopted by them as the host (83). 


3. -; The Survival of the Fittest 


As we have seen above, natural selection appears not so important 
for the evolution of the higher organisms as customarily believed, but 
the selection may play sometimes a prominent role in the determination 
of the direction of evolution. 

The genes are provided with the property to achieve the gradual 
change in a certain direction. However, as the genes have the indivi- 
duality, even under the same environmental effect. every gene will 
behave differently, changing in different rates and in different directions. 
Nature will select the-gene which has been changed in the most fitted 
way to the environment. As the gene thus selected may have the 
property to continue the change favourable for the organisms, the or- 
ganisms having the gene will continue the favourable change as long 
as the environmental effect in question remains in existence. 

For example, if a colour change brought about in an insect under 
a certain environment becomes significant as a mimicry, the insect will 
be selected and separated from other individuals, and since the insect 
thus selected has the property to advance the colour change and, in 
addition, since the selection will continue without resting, the mimicry 
will sooner or later be completed, although it may be possible that the 
change may go so far as becomes: useless as mimicry. 

If herbivorous animals need fleetness in order to escape from their 
enemies, by which the slower-footed individuals are to be devoured up, 
the swifter-footed individuals will be selected:and separated from the 
original type. Since the selected individuals may have the tendency 
to develop the character, the fleetness will gradually increase even 
without further selection, but the continual enforcement to the use and 
the successive selection in each generation will no doubt accelerate the 
development.. Thus, swift-footed animals like horses have presumably 
been created. 

The phenomenon known as vernalization can be regarded as result- 
ing from the natural selection by which a character fitted to a peculiar 
environment has been adopted and developed. As is well recognized, 
spring rye, in common with other spring cereals, ‘requires no low tem- 
peratures during germination for normal flowering, whereas winter 
rye, unless exposed to low temperatures during germination, as is 
known as vernalization, shows a great delay in ear formation and 
emergence. ‘ 


X. THE MECHANISM OF EVOLUTION 411 


As was already discussed in detail, germ cells in general, whether 
they are of animals or vegetables, may develop various characters in 
response to the environments to which they are exposed. The seed of 
the winter rye is to be always exposed to low temperatures during 
germination and so if in some individuals a character favourable for 
the subsequent development was produced by this stimulus, the indivi- 
duals capable of acquiring such a character under the effect of low 
temperatures would be selected as the fittest, and as a result the in- 
dividuals thus selected would fail to show usual development without 
preliminary exposure to the low temperature. 


4. The Degeneration of Organs and Atavism 


Organic evolution is induced by the structural evolution of the 
genes, whereas the structural change of the genes is reversible. There- 
fore, organic evolution itself must likewise be reversible. 

Our eyes are no doubt one of the most advanced organs. The 
development of the eyes is probably mainly brought about by continuous 
and exceeding uses, so that the cessation of the use will lead to the 
return to the original, undeveloped state, that is, the eyes will become 
atrophied by disuse. Thus, many species of fishes living in deep seas 
or in caves are known to be blind. Horses kept in mines and man kept 
in dungeons are said to have had their eyes so impaired that they could 
be restored to sight only by gradual exposure to light, showing the 
wonderful swiftness by which the eyes would degenerate. This sug- 
gests that their evolution has occurred rapidly and comparatively 
recently. 

The theory of orthogenesis holds true in so far as the environmental 
effects including the stimulus from use are constant and unchanged. 
Any change in the effects, of course, would lead to a change in the 
direction of the evolution. In an extreme case, the direction may be- 
come entirely reverse. For example, when human ancestors might live 
on trees like apes, their feet might have been developed so as to be 
able to grasp objects, but their leaving from the trees followed by the 
cessation of the use of the feet to grasp, might result in the return to 
the original form. Again, whales might be able to walk when they 
lived on land, but their return to water was followed by the degenera- 
tion of the legs. 

The reversibility of genes is attributed to the memory of the original 
structure. Hence, the diminution in the memory will lead to the dif- 
ficulty in the manifestation of the reversibility. Since newly acquired 
characters may have strong memories, their disappearance may occur 


412 V. THE NATURE OF EVOLUTION 


readily. Thus a character acquired during one generation may disap- 
pear rapidly on the removal of the causative effect. However, when 
the effect continues to exist for generations to stabilize the acquired 
character, the memory of the former structure will become faint, while 
the new one being fixed. It may be supposed, therefore, that organs 
like eyes having the tendency to degenerate rapidly are newly produced 
organs retaining still the fresh memory of the original pattern. 

Attention has been paid for a long time to the fact that old organs 
which were useful in the old environment but which have ceased to 
have a function in the new, linger on in a reduced form with aston- 
ishing persistence, the grip of ancestry still upon them. Various land 
birds, particularly among those of oceanic island, have lost the power 
of flight. Externally no trace of wing is visible, but close inspection 
reveals a minute rudiment of wings still persisting under the long 
hairlike plumages, although wholly without significance as an organ 
of flight. Familiar examples of vestigial organs are presented.by the 
degenerative eyes of many cave animals. 

The writer believes that such vestigial structures were produced 
as a result of a random change of the genes, or, more precisely, were 
produced involuntarily being associated with the development of a 
peculiar structure of a gene causing a certain useful character, and 
when a certain structure thus produced involuntarily came to have the 
value of use, the use would be commenced with the rapid development. 
Therefore, the portion which is to be degenerated by the disuse must 
be the structure that was developed rapidly by the use, vestigial 
structure thus being left unchanged. 

Many useless structures are more fully developed in the embryo 
than in the adult, thus presenting an instance of the recapitulation of 
phylogeny by ontogeny. Some of the most striking cases are those of 
functionless organs which develop in the embryo but which disappear 
again before birth. For example, in whales both anterior and posterior 
limbs are formed, though the latter subsequently atrophy and in some 
species wholly disappear. Likewise the embryo is densely covered with 
hair, although the adult whale is devoid of hair. Furthermore, it was 
found in general that blind cave fishes develop what appear to be 
normal eyes in their early life history, but later the eyes are lost again 
through atrophy. 

If the individual develpoment is interrupted halfway, some char- 
acter may remain in an embryonal state, a phenomenon called atavism. 
The well known salamander, axolotl, shows atavism and remains for 
life in an immature form, performing respiration by gills instead of 
by lungs as in the larvae. This atavism is known as neoteny. Under 
a peculiar condition associated with a food change or water deficiency, 


X. THE MECHANISM OF EVOLUTION 413 


however, this salamander may develop into the adult form of lung- 
breathing. The administration of thyroid gland substance seems most 
effective to lead the salamander to the adult form, suggesting that the 
inability to recover the adult form may be involved in the deficiency 
of thyroid hormone. 

Environmental effects, as previously stated, appear to exert their 
influences upon genes mainly through hormones. Accordingly, hormones 
only, without accompanying any environmental effects, may naturally 
be able to induce certain changes in genes. As discussed previously, 
hormones play an important role in the establishment of individual 
development. The metamorphosis of ,tadpoles is accelerated by the 
administration of thyroid-gland substance and also, as is well known, 
that of insects is subjected to the function of endocrine glands. The 
pattern of individual development of the stag antler may show the 
pattern of its phylogenical development, but its development is inter- 
rupted by the castration, showing also the involvement of hormones. 
Thus certain environmental alterations having influences upon hormones 
may cause atavism as in axolotl. 

The primary organisms were generated and evolved in water, so 
that their parting from water might be achieved only with unusual 
difficulties. In like manner, for the secondary organisms the departure 
from the parasitism may also be not an easy task. However, the re- 
turn of land animals to water or of free-living secondary organisms 
to parasitism may occur comparatively easily, because the return must 
only be atavism or the recovery of the former structure. To be para- 
sitic at the larval stage and free-living at the adult may be a natural 
form of the individual development recapitulating the phylogeny. The 
reverse is, however, unnatural, and so may be a type of atavism. 

The advancement of parasitic animals into free living may be thus 
possible. On the other hand, the entry of free-living into parasitism 
appears to be impossible, because any spontaneous production of char- 
acters which are suited to parasitism, a most particular and extremely 
restricted environment, cannot be considered. From this point of view, 
the writer regards animals which are parasitic at any stage of life as 
the secondary organisms. 


CHAPTER XI 


THE CHANGE OF HUMAN CHARACTER 
BY ENVIRONMENT 


1. Climate and manicind® 


Sérensen (84) has concluded from his experimental results’ that 
proteins in biological fluids are composed of a variety of components in 
a state of equilibrium depending upon the environmental conditions, 
and that this equilibrium is shifted reversibly and readily through 
changes in the composition of the solution. According: to the writer’s 
concept, organisms by themselves are such systems of protein com- 
ponents, their state of equilibrium changing reversibly through the 
alteration of the environment. On the other hand, as the state of 
protein comlexes in organisms is governed by the’ genes, it can be 
considered that changes of organisms are induced by the genes chang- 
ing reversibly in response to the varying state of environment. 

Of such environmental factors, climatic ones appear to be of the 
most importance for us humans, for we are on ail occasions exposed 
to the climatic factors which are always changing, and accordingly our 
genes are expected to be always changing with them. In the extensive 
investigation into the relation between civilization. and climate, Hunt- 
ington concluded that. a stimulating. climate is the main condition 
which promotes civilization. 

His contention, as set out in “Civilization and: Climate’’ is that 
a certain type of climate, now found mainly in’ Britain, France and 
neighbouring parts of Europe, and in the Eastern United States, is 
favourable toa high level of civilization. This climate is characterized 
by a moderate temperature, and by the passage of frequent barometric 
depressions, which give a sufficient rainfall and changeable stimulating 
weather. Now it is well known that the great centers of civilization 
in the past lay in more southernly latitudes than those of to-day, begin- 
ning in Egypt, Mesopotamia, and the Eastern Mediterranean and then 
passing to Greece and Roma. Huntington attributes these changes of the 
centre of civilization to climate change associated with the northward 
shifting of the belt of cyclonic activity. In this connection, he stated 
as follows in ‘‘Principles of Human Geography”: 


THE CHANGE OF HUMAN CHARACTER BY ENVIRONMENT 415 


“To understand the relation of climate to civilization, let us com- 
pare the province of Ontario, where the climate is one of the best in 
the world, and the Bahama Islands, which have a warm, monotonous, 
’ tropical climate. The original white settlers in both places were of the 
same stock. They were English colonists, many of whom left the U. 
S. at the time of the Revolution because of their loyalty to England. 
Today the descendants of the Loyalists in Canada are one of the strong- 
est elements in causing that country to be conspicuously well governed 
and progressive. In the Bahama the descendants of similar Loyalists 
are also one of the finest elements, but many of them are relatively 
inefficient. Among the Canadians practically every one has a fairly 
good education. Among the Bahamans a large number have never been 
to school, and many who learned to read and write in their childhood 
have forgotten these arts because they do not practice them. 

““The main cause of these differences is the climate. As the Baha- 
mans themselves say “This climate is very’ healthful and pleasant. 
The only trouble is that it doesn’t make one feel like work. In winter 
it’s all right, although even‘then we can’t fly all very well for you 
Americans to think we're lazy, but try living here a year or two 
yourselves, and you'll be as lazy as we are’.. The regular routine of 
daily life can be carried on without much difficulty, but when a new 
kind of work is to be done, he says, ‘Wait till tomorrow’. It must 
not be forgotten that a stimulating climate is only one of the conditions 
which promotes civilization’’. 

We, the writer and Ohashi, observed the same tendency in Shang- 
hai, while staying there for several years, and after the inquiry was 
made of Japanese students of the Dobunshoin University, a conclusion 
similar to that of Huntington was reached, although we were ignorant 
at that time of his splendid work. The climatic conditions in Shanghai 
were found to be comparable to those in Bahama in the example cited 
above. According to our investigation into the difference of climatic 
factors between Shanghai and Nagasaki, a Japanese city situated at 
the opposite side of the sea about in the same latitude, there was no 
significant difference in the average temperature or humidity, but it 
was noted that the extent of deviation of the factors including atmos- 
pheric pressure were far greater in Nagasaki than in Shanghai. 

We expressed the opinion that in China, including Shanghai, as 
well as in tropical countries there is some deficiency in climatic factors, 
a deficiency causing a disorder in the function of endocrine glands 
especially adrenal contex (85). The lack of mental and physical activi- 
ties and abnormal pigmentation common in the inhabitants in these 
countries may be accounted for by the hormonal disorder. The symp- 
toms appear to be similar to those of Addisonism, a diseased state 


416 V. THE NATURE OF EVOLUTION 


‘due to the disfunction of adrenal cortex. As we have seen above, 
climatic factors exert influences upon hypophysis, which in turn governs 
adrenal cortex. We beleive still at present that our conclusion was 
reasonable. 

The lack of stimulating climatic factors proposed by Huntington 
‘may induce a disturbance of the pituitary function followed by a change 
in the feature of hormonal secretion, especially by a disorder of adrenal 
cortex resulting in the change of human characters. The continued 
lack of the factors for successive generations may lead to the fixation 
of the characters peculiar to the climate. The increase in the strength 
of the factors may, on the other hand, change the characters in the 
opposite direction. 

There has long been a strong conviction among anthropologists 
that head form is the most reliable element in the classification of 
race, but Boas (86) in measuring with many immigrants and their 
children in New York City proved that even the cephalic indexes 
which were formerly considered permanent racial traits are changeable 
by environment. Thus, the children of broad-headed Jew from Poland, 
born in New York, proved to be less broad-headed than the parents. 
Moreover, the reduction in breadth of head of the children increased 
in accordance with the duration of the residence of the mothers in New 
‘York. Another group of immigrants consisted of long-headed people 
from southern Italy. In their cases, also, there was a slight but 
significant difference between the American-born children and their 
parents or brothers and sisters born in Europe. This difference, too, 
increased as the time elapsed since the immigration of the parents. 
In this case, however, the change was towards greater instead of less 
‘breadth of head. In other words, each type of children tended somewhat 
away from the standard physique of the parent, and towards a less 
extreme middle type. 

This middle type might be, therefore, the standard physique in 
New York, determined by the environmental factors, probably chiefly 
those of climate, prevailing there. It should be noted that the change 
in the children was increased with the duration of residence of the 
mothers. This fact indicates that the influence of the prolonged resi- 
dence exerted on the mother was transmitted to the children, proving 
clearly the inheritance of the acquired character. 


2. The Inheritance of Habitude 


The environmental effects which change the human characters may 
not be involved in climate only as will be considered later, but the fate 


THE CHANGE OF HUMAN CHARACTER BY ENVIRONMENT 417 


of civilization can be easily explained even by taking only climate into: 
consideration. It is true that the people living in the regions of the 
climate lacking in the stimulating factors, especially in tropical regions, 
seem to be born tired, whether or not it may be due to a disorder in. 
endocrine glands, and seem to dislike to use their brains. On the 
other hand, disuse of the brain will result in its atrophy. Thus,. 
disuse of the brain for successive generations may lead to the production 
of mentally inactive people, whilst people live in climate full of sti- 
mulating factors may become more and more mentally active by the 
constant use of the brain. 

The change in the brain pattern thus produced may be transmitted 
to germ cells by virus-like agents. In addition, hormones may be 
concerned with the transmission as in the pattern of other organs. 
The majority of authors are inclined to believe that hormones exert 
their most direct and pronounced effects upon behaviour through the 
induction of changes in the central nervous system. As pointed out 
already, the acquisition of new modes of behaviour as a result of 
experience cannot occur in the absence of hormonal influences, although. 
all evidences point to the conclusion that capacity to learn is not 
directly and immediately dependent upon the secretion of a single 
endocrine product. 

In addition, certain hormones appear to exhibit pronounced effects. 
upon emotion in human (87). The conspicuous difference in human 
emotions due to the difference in sex can, of course, be attributed to 
the sexual hormones, indicating how great are the hormonal influences 
upon the emotion. On the other hand, certain endocrine functions. 
appear to be strongly influenced by psychic factors. Observing that 
repeated gentling of rats changes their reactions towards man from 
flight or pugnacity to relaxation and docility, Hammett (88) reports 
that 79 per cent of ‘‘non-gentled’’ rats die within forty-eight hours. 
after parathyroidectomy, while among ‘“‘gentled’’ individuals mortality 
within the same period amounts to only 13 per cent. There is a 
widespread impression that thyroid secretion may in certain instances. 
increase general irritability and contribute to the intensity of emotional 
reactions. Administration of thyroid to Plymouth Rock hens is said 
to result in a change from a gentle and phlegmatic to a highly neryous 
temperament (89). In this connection, Rickey (90) claimed that the 
offspring of rats which have been fed thyroid gland substance are 
more nervous and erratic than the progeny of normal parents. On 
the other hand, as will be mentioned later, adrenal extract exhibits. 
the function to release the nervous temperament in contrast to thyroid. 
Thus it may be concluded that a change in the function of hormonal 
glands due to the effect of environment, including climate, can induce 


Ber V. THE NATURE OF EVOLUTION 


the change in the brain pattern even if the effect of use or disuse is 

‘not'involved, Therefore, the addition of the effect of use and disuse 
to that of the hormones may result ina striking change in the mental 
‘pattern, and when the effects continue to have their influences in 
successive generations, the mental characteristics will become strong 
and fixed and accordingly inheritable. 

As to the inheritance of memory, a detailed consideration was 
made in the beginning of this Part. The.memory in our brain is 
considered to be a structural change in the protoplasm protein of 
brain cells. Such a structural change can be transmitted to the newly 
formed proteins; thus the memory can be transferred successively to 
newly formed proteins or protoplasm. If this change is transmitted 
to germ cells by protoplasm particles, namely by virus-like agents, 
and if the changed structure does not return to the original during 
the development of the germ cell tothe young individual, the memory 
can be inherited by the young. The memory of phylogeny has been 
inherited from generation to generation for a dreadful long span of 
time as the ontogeny. Moreover, every instinct of creatures must 
depend upon the memory thus inherited for generations. 

One may say that not only do English children have to learn their 
own language, but they learn it no quicker than they would learn 
French if brought up from the first in a French-speaking household, 
and that this fact demonstrates evidently the failure of the inherit- 
ance of memory. However, if this is true, it can be considered that 
minute structural differences corresponding to the memory of difference 
between French and English cannot be transmitted, but that the faculty 
to learn words as a whole must be inherited and strengthened when 
man continues to speak a language. 

A number of experimental evidences suggesting the possibility 
of the inheritance of habit has been presented by several workers, but 
mostly have been regarded as very doubtful like other experimental 
data concerning the inheritance of acquired character. Since the struc- 
tural change in the protoplasm in central nervous system must also 
be reversible, it should naturally follow that the clear demonstration 
of the inheritance of habitude likewise failed when observations were 
made only for a few generations. From the theory of the writer thus 
far described the inheritance of habitude seems to be a natural con- 
sequence. The splendid advancement of animals, especially of mankind 
as seen in the present day, might not have occurred without the 
inheritance of habitude. ’ 

Domestic animals such as dogs and horses are very faithful as 
well as obedient to their masters. This may probably not only due 
to the artificial selection, but also to the inheritance of habitude. 
Thus, habit does become a nature. 


CHARTER XII 


THE EVOLUTION OF MANKIND 
AND ITS FUTURE 


1. The Orthogenesis of Protein Molecules 


The denaturation or the structural change of proteins appears to 
proceed without stopping in so far as the change-inducing agents are 
present as seen in the change of phage by formalin. The process is 
compared to the gradual alteration of the genes by environmental 
factors. It is worthy of note, however, that the velocity of the process 
or the velocity constant, if calculated as a monomolecular reaction. 
tends to become smaller as the reaction proceeds. It is reasonable to 
regard the process as a monomolecular reaction, but if calculated as 
a bimolecular reaction the decreasing rate of the velocity constant is 
lessened, and therefore the writer attributed this phenomenon to the 
infection of denaturation, that is, he considered that the denaturation 
was autocatalytically accelerated by the transmission of denaturation 
from the protein undergoing denaturation to other intact molecules 
(61). Frequently, however, the decrease in the progress is so manifest 
that it cannot be explained even by this assumption and there seems 
to exist still another cause to lessen the velocity. 

At present the writer believes that this must be based upon the ten- 
dency of the protein to recover its original structure, that is, upon the 
reversibility. As previously discussed, protein denaturation sometimes 
proceeds in oscillation like a physical phenomenon. This is interpreted 
as resulting from the competition between the effort of the protein 
to recover its original structure and that of denaturating agent to 
promote the change. If so, the velocity of the change would be the 
more lessened as the change progresses the more, since the repulsive 
force or reversibility should become stronger with the progresseof the 
change. This appears actually the case. As every one knows an 
elastic substance can be bent easily at first, but further bending may 
become difficult on account of the increase in the force to spring back 
to the original shape. Protein molecules can be looked upon as such 
an elastic substance. 

In Figs. 36 and 37 the progress of inactivation of rennin respec- 
tively by its antiserum and tannin is illustrated (91). In both cases 


420 V. THE NATURE OF EVOLUTION 


100 


WI 


XC) 
SN 
GD) 
iS 
eer) 
= 40 
a 
2 
30 
ee 
20 


NAG ee SER) 
UG ee es | 
See Se ee 


— time (hours) 
Fig. 26 Decrease in progress in the action of rennet due to anti-rennet serum. 
Antiserum: anti-rennet rabbit serum. 
Rennet solution (0.0194), 1 cc+antiserum 0.996 NaCl solution, 
lec+milk (196 CaCl,) Icc 
Dilution of antiserum: I= 1:160, I1=1:80, III=1:40, IV=1:20, 
en SI} 


100 g= 


— Rennet quantity remaining, * 


—— time (minutes) 
Fig. 37 Derease in progress in the action of rennet due to tannic acid. 
Tannic acid: Kahlbaum, used about a month after its dissolving. 
Rennet solution (0.019), 1 cc+tannin 0.996 NaCl solution, 1 cc+millc 
(196 CaCl,), lcc. 
Concentration of tannic acid: I: 0.0031259, II: 0.0062596. III: 
0.012596, IV: 0.02594 


XII. THE EVOLUTION OF MANKIND AND ITS FUTURE 421 


the inactivation progresses rapidly at first, but later distinctly slows 
down presumably because of the increase in the intensity of the repul- 
sive force. 

The bending force of the inactivating agents, on the other hand, 
should be determined by the kind of the agents, and in one and the 
same agent, the higher the concentration the more rapid and the more 
extensive may be the change. The change of rennin due to antiserum 
or tannin in low concentrations appears usually to cease to proceed 
after some periods of progress, whereas the inactivation due to heat 
proceeds without diminishing the velocity until the inactivation be- 
comes completed. Thus heat is regarded as an agent strong enough to 
be able to ignore the repulsive force. 

In the case of phage, formalin acts as a strong agent, so that 
almost no diminution in the velocity is observed as already shown in 
Fig. 35, whereas some diminution is seen with HgCl: or with heat, 
and in both cases of antiserum and tannin diminution is usually mani- 
fest (62). It may be said, therefore, that, if the force to push forward 
the change is much greater than the repulsive force, the change will 
proceed without decreasing, whilst if the former force is weak, the 
repulsive force will be revealed to diminish the changing velocity. 

Presumably this holds true for the change of genes. The effect 
of environment including climate seems to exert its action comparatively 
rapidly on human genes as shown in the above cited observations of 
Boas, but a considerable span of time may be necessary for the immi- 
grants from various parts to become an entirely similar type, if possible, 
which is to be determined by the environment of the settlement. 

Here a great question may arise. If the repulsive force of the 
genes becomes greater as the change proceeds, the change is expected 
to be suspended sooner or later. If so, evolution is impossible unless 
the environmental factor is strong enough to disregard the force. Is 
this ever the case? 

Here again we should remember the nature of the reversibility of 
protein structure. The reversibility depends upon the memory of the 
former structure. Therefore, the loss of the memory may result in 
the disappearance of the repulsive force, and consequently if a gene is 
left for a long time in the same changed state under the same en- 
vironment causing the change, its former structure will be forgotten 
with the disappearance of the repulsive force, the change thus being 
gradually pushed forward. This seems not only the case with genes, 
but also with elastic substances in general. The organic evolution as 
already stated cannot take place unless the memory of the former 
Structure is lessened or lost. 

Proteins can thus be looked upon as an elastic substance and the 


A22 V. THE NATURE OF EVOLUTION 


elasticity is revealed most strikingly in the genes. As previously men- 
tioned, there are actually many other evidences that protein molecules 
are elastic in themselves. The organisms having genes provided with 
only weak rigidity or feeble elasticity must be very sensitive to en- 
vironmental changes, by which will be induced profound and extensive 
alteration in their characters. Therefore, if the rigidity in the structure 
of the genes is reduced by some or other causes, the organisms will 
commence an orthogenetic changes advancing rapidly with the extensive 
specialization until they become extinct due to the overspecialization. 
The progress of phage inactivation by formalin does not decline 
as stated above, showing the relative weakness of the reversibility, but 
in a weakly acid solution the diminution of the velocity is manifest, 
and the progress frequently oscillates. Thus it can be said that phage 
aS a gene-model had a strong reversibility in a weakly acid solution, 
but that in a neutral or an alkaline solution the reversibility is 
lessened and accordingly formalin will cause an rapidly-advancing 
orthogenetic change leading to the extinction of the phage. 


2. The Orthogenesis of Mankind 


As we have already seen, environmental factors including climate 
appear to exert the influence mainly through the endocrine glands 
especially hypophysis.. Shapiro’s work (92) on Japanese immigrants 
to Hawaii has led to the same conclusion as that of Boas on migrants 
from various Eeuropean countries to New York City. The Japanese 
migrants are taller than their stay-at-home relatives, and the children 
of the migrants depart further than do their parents. The children 
of Japanese in Los Angeles are also known to be taller than their 
kindred in Japan, the increase in size being especially distinct in legs 
and arms as compared to that in the trunk. Such an increase in 
stature may be readily explained by the assumption that the climate 
or other environmental factors in: Los Angeles and Hawaii stimulate 
the function of hypophysis as will. be detailed below. A similar 
change is reported to occur also in New York in the immigrants of 
Central European people, such as Bohemians, Slovaks, Hungarians, 
Poles, and Hebrews, suggesting the presence of the same environmental 
factor or factors also in New York. 

It has long been known that pathological hyperfunctions of 
hypophysis result, on the one hand, in the gigantism in man, while, 
on the other hand, the experimental gigantism in rats or dogs was 
actually demonstated by injection of an extract of hypophysis. The 
final proof for the existence of the growth hormone comes from the 


XII. THE EVOLUTION OF MANKIND AND ITS FUTURE 423 


isolation of the hormone in a crystalline form from anterior pituitary 
glands (93) (94). The administration of this hormone leads to weight 
gain, muscle hypertrophy, increased skeletal dimensions, and skin thick- 
ening in hypophysectomized rats. Climatic factors in general appear 
to stimulate the function of pituitary gland, and hence people living 
under a stimulating climate will increase in stature owing to the 
increasd secretion of this growth hormone. 

On the contrary, people live in the climate lacking in the stimulating 
factor may become smaller because of the want of the growth hormone. 
In addition, it has been. believed that adrenocorticotrophic hormone 
(ACTH) of hypophysis whose production is considered to be increased 
by the stimulating agent is antagonistic to both gonadotrophic and 
thyrotrophic hormones which are also produced by the anterior lobe of 
pituitary gland (95) (96), so that the absence of stimulating climate 
may increase the production of these latter principles. 

In man, hyperthyroidism is often followed by degeneration and atro- 
phy of adrenal cortex. There is an evidence of a decrease in cortical 
function after long-continued increase in thyroid activity. Moreover, 
adrenal extract counteracts the effect of thyroxin administration 
even in normal animals, reducing the increased loss of nitrogen and 
lowering the increased pulse rate.. In addition, it is observed that 
corticosterone and desoxycorticosterone counteract the effects of mild 
hyperthyroidism of the liver (97). Corticosterone is known to be able 
to modify or correct the symptoms of hyperthyroidism. 

There are also many experimental evidences that administration of 
cortisone or ACTH will suppress thyroid function (98). Similar findings 
have been obtained under experimental conditions which are known 
to be accompanied by increased secretion of cortical hormone including 
injection of epinephrine or formalin and exposure to abnormal temper- 
ature (99). 

On the other hand, it is well recognized that animal metamorphosis 
fails to occur after thyroidectomy. Further, gonadotrophic principles 
are known to lead to the development of precocious sexual maturity. 
In this connection, it should also be mentioned that metamorphosis of 
a South African frog is retarded by the administration of adrenal 
extract or desoxycorticosterone acetate (100). Thus, if metamorphosis 
is promoted by thyroid hormones small precocious animals may result, 
whilst this may be prevented by adrenal cortex. Stockard, (101) in 
his extensive studies of dogs and their hybrids, showed that dwarfs, 
such as the dwarf bulldogs and other dwarf breeds, have a larger 
thyroid per weight of dog than such normal breeds as the fox-hound 
while the giant breeds have a relatively small thyroid. During preg- 
nancy, a remarkable hypertrophy of pituitary gland is forthcoming, 


424 V. THE NATURE OF EVOLUTION 


followed by the demonstrable acromagaly or gigantism, that is, enlarge- 
ment of hands and feet, and the thickening of the lips and nose, 
probably due to the hypersecretion of growth hormone, whereas the 
gonadotrophic content of the pituitary is found to be reduced (102). 

It may be concluded, therefore, that the absence of stimulating 
climate makes a precocious, small man tending to Addisonism, owing 
to the hyperfunction of both gonadotrophic and thyrotrophic hormones 
together with the insufficiency of growth hormone and ACTH, accompa- 
nying the dysfunction of adrenal cortex. This is the feature of man, 
common in tropical regions. A similar phenomenon may be observable 
also in animals. Thus, each group of garter snakes is found to show 
independently dwarfing as the distance increases from the center of 
dispersal in the Mexican plateau, no matter whether the group is 
pushing into the tropics or temperate regions (30). This may be 
ascribed to the degree of deficiency in climatic or other factors, which 
can stimulate the secretion of growth hormone in the snake, resulting 
in the hyperfunction of thyroid gland. 

As we have seen above, our genes possess rigid structure with 
strong elasticity, so that they can show a resistance to environmental 
factors which are able to induce a change in them. If any change 
is induced, usually repulsive force will become stronger as the change 
proceeds, so that the progress of the change may come to be very 
slow, if not stopped, a consequence which may not issue when the 
genes have weak structures with feeble elasticity. A loss of the 
rigidity in genes, therefore, may lead to a rapid, unceasing change 
of the characters of the organism having the genes. In such a case 
the organism under a climate or other environments that stimulate 
the hypophysis will become continuously bigger to attain a gigantic 
figure which may result in its extinction. As is known as Cope’s 
law, overspecialized animals usually would become extinct with gigan- 
tism. The extensive specialization results from a rapid change of 
a gene, which would be in turn a result of a loss of the structural 
rigidity in a gene. 

Here a very remarkable fact should be pointed out that a fearful 
tendency to this gigantism is most manifestly being revealed in 
mankind. The statistics indicating the increase in stature of man go 
back a century and a half in Switzerland and fifty years or so in 
many other places. Among European countries data covering one or 
two generations are available for Norway, Sweden, Denmark, Germany, 
the Netherlands, Italy, and Spain. People in the United States also 
share this trend. Japanese date from 1878 onwards are partcularly 
significant because they show that an Asiatic people is undergoing 
the same lengthening process as those of European extraction. The 


XII. THE EVOLUTION OF MANKIND AND ITS FUTURE 425 


stature of army recruits or others is known to have increased almost 
steadily since 1792 in Switzerland, 1836 among Harvard students, 1841 
in Sweden, 1852 in Denmark, 1855 in Norway, 1863 in Holland, and 
1885 in Japan (103). From measurements of about half a million 
males of twenty years from 1892 to 1926, the mean height of Japanese 
was found to have increased by 3.23 cm, the increase being steady 
and uniform from year to year (104). 

In this connection, another most remarkable fact should be cited. 
During almost the same period of time the resistance to tuberculosis 
has been increased steadily in man. The disease has shown since 
1849 a steady decline, along with approximate mortality rate, for the 
United States and for England and Wales, during the same period (105). 

The dramatic change in the relation of the tubercle bacillus to 
man took place over most of the Western World during the same 
100-year period. In other words, mortality rates began to decrease in 
Europe and North America around 1850 and they have continued to 
decrease steadily eversince, in conformity with the increase of the 
stature, except for short and local interruptions in the downward 
curve during and after the first and the second World Wars as shown 
in Fig. 38. It should be noted that the rate of decrease in tubercu- 
losis mortality had already reached its maximum slope around 1990, 


Comparative mortality indices 


1939—45 War 


1904 1945 


Fig <8. Tuberculosis all forms, under 15 years, England and Wales. 
1904-1945. Reproduced from Dubos, R. J. : Science in 
Progress, Sixth Series, 1949. 


426 V. THE NATURE OF EVOLUTION 


long before any specific immunization or therapeutic measures were 
available. 

The writer claims that these two amazing phenomena are closely 
related with each other. As mentioned above, adrenal cortex is 
controlled by hypophysis, and environmental factor stimulating the 
anterior pituitary gland causes the secretion of adrenocorticotrophic 
hormone, which in turn induces the secretion of cortex hormone, 
whereas the same factor leads to the involution of thymus and other 
lymphatic organs. On the other hand, as is well known the so-called 
“status thymicolymphaticus’’ is a status liable to be affected by pulmo- 
nary tuberculosis. Its characteristics are an excessive development of 
the thymus, tonsils and other lymphatic organs, with a concomitant 
hypoplasia of the adrenal cortex. Moreover, intimate relation between 
tuberculosis and Addisonism, known as a morbid state associated 
with a dysfunction of adrenal cortex, has also been generally accepted. 
It can be concluded, therefore, that the hyperfunction of hypophysis 
caused by hypophysis-stimulating environment may result in the in- 
crease in the growth-hormone secretion, thereby man obtains a 
gigantic figure, and at the same time, it may remove the status 
thymicolymphaticus and Addisonism, thus rendering man insensitive 
to tuberculosis. 

The average span of man’s life is said to have been lengthened 
steadily during the same time in civilized nations. This must be 
a natural result of the increase in man’s stature, since in general 
the bigger the animal, the longer its life. On the contrary, the 
hyperfunction of thyroid gland, which seems to be antagonistic to 
the action of adrenocorticotrophic hormone and which may be brought 
about by the want of the stimulating factor may result in the 
shortening of life span because of the premature growth due to the 
hormone. 

It is little doubt that the remarkable morphological change in 
man, above mentioned, has begun to occur comparatively recently. 
According to Schwidetzky (106) as illustrated in Table 11, the head 
form of man has shown a great change at least after the year 1200; 
it should be noticed that almost no significant change occurred during 
the period ranging from 1200 B.C. to 1200 A.D. Moreover, it is 
reported that the actual increase in the stature in a generation among 
Yale University students has been about 17/. inches (103). If man 
had continued in the past to become bigger at this rate, only 1,000 
years ago he would have been a dwarf of only about 2 feet; such 
was of course never the case. It is evident, therefore, that the 
change began recently and is continuing to proceed with a frightlful 
speed. Of course, the period of time extending over only a few 


XII. THE EVOLUTION OF MANKIND AND ITS FUTURE 427 
Table 11. 
Change in Human Head Form. 
After Krogman, W. M.: The Science of Man in the World Crisis, 
Columbia Univ. Press. 1946. 
Nordic East European Type 
Date Cranial | Cranial | Cranial | Cranial | Cranial | Cranial 
index | length breadth index length breadth 
1200, B.C. 69.2 | 76.1 
B00} Aw: 69.6 | | ial 
| 
1200, A. D. Migr) 189.0 137.6 fies. |) dley.s} 144.8 
1985; A: D: ca 81.0 | 183.3 7a ca 86.0 176.5 527 


generations of man must be solely one moment as compared with the 
awfully long history of organic evolution. 

Mankind seems to have begun to make a dash at the fate of 
extermination with a dreadful speed like many other animals that 
have already become extinct because of rapidly developing orthogenesis. 


3. The Cause of the Orthogenesis of Man 


What is the cause of this dreadful orthogenetic change of man ? 
Since any gain in height seems neither to have occurred nor to be 
occurring in tropical countries or in polar regions such as Greenland 
and Alaska, where tuberculosis is still increasing far from showing 
any decline, the main cause of the change must be attributed to 
climate. It was believed that a gradual lowering of temperature 
was the primary factor for the gain in height (107), but the writer 
claims that what has changed and is now changing is the character 
of genes of mankind rather than climate. 

It may be reasonable to consider that the genes of man have 
changed by some causes so as to be rendered sensitive to climatic 
factor and that still at present are being rendered more sensitive to 
the factor, that is, the genes are changing to lose the rigidity or the 
elasticity in their structure. 

Many causes are considered by which genes may lose the rigidity, 
but the most important one may be found in that man becomes 
well nourished with the advancement of civilization. The writer 
believes that the effect of deficiency in food counteracts the stimulating 
action of climate on hypophysis. There are many evidences that 
inanition injures the pituitary function. Chronically underfed rats 


428 V. THE NATURE OF EVOLUTION 


shows a decrease in weight of the adrenals, and it has been sug- 
gested that this is a result of insufficient adrenocorticotophic hormone, 
since implantation of pituitaries increases the weight of the adrenalis 
in such animals (108). Such adrenal atrophy is by no means attributed 
to the general deficiency of calory, for the administration of carbo- 
hydrate cannot alleviate the symptom. Presumably, protein deficiency 
plays a predominant part, since it is said that a deficiency of certain 
amino acids in the diet causes a severe atrophy of cortex, although 
it is also reported that a fat-free diets produce a relative atrophy of 
both cortex and medulla (97). . 

A series of symptoms following hunger can be well interpreted 
as the hypofunction of adrenocorticotrophic hormone of hypophysis. 
The so-called ‘‘famine oedema”’ or “‘hunger swelling’’ can be regarded 
as the manifestation of the pituitary hypofunction. War oedema has 
appeared in epidemic form in many of the recorded wars of history 
among both the troops and the civil populations. A similar oedema 
occurring in a malnourished infants or in those fed principally on 
a high carbohydrate diet has long been recognized. A considerable 
nocturia is seen in mild chronic cases, as we ourselves miserably 
experienced during and after the War, whereas as generally accepted 
water metabolism is severely affected by adrenal insufficiency, thereby, 
despite the negative water balance, water diuresis is extremely limited. 
In addition, albumin-globulin ratio of the blood is greatly altered in 
hunger as in Addisonian syndrome, all globulin fractions usually 
showing some increase, while albumin decreasing (109). It is well 
established that such a change in albumin-globulin ratio in the blood, 
which also characterises some cases of rheumatoid arthritis and other 
diseases of mesenchymal tissue, is frequently returned to normal 
during administration of cortisone or adrenocorticotrophic hormone 
(110). Again, a number of investigators have reported that, as a 
result of hypophysectomy in animals there is a decrease in the 
albumin content of the blood (111). It has actually been confirmed 
with rats that inanition has a similar effect (112). According to a 
recent studies by Ulrich e al. (113), treatment of hypophysectomized 
animal with growth hormone results in a great stimulation of albumin 
synthesis, while ACTH has no such effect. Moreover, it should be 
remembered that fatigability and muscular weakness peculiar to 
Addisonian syndrome are also manifest in inanition, as we had 
likewise rich experience of it thanks to the War. 

In addition to the want of food, an irritable, uneasy life associ- 
ated with nervous tenseness of animality, peculiar to a barbarous 
life, may contribute to the prevention of the loss of the rigidity in 
gene structure. As already described, there appears to be a closely 


XII. THE EVOLUTION OF MANKIND AND ITS FUTURE 429 


related connection between hormones and temperament; hormones 
effect the latter, while the latter effects the development of endocrine 
glands. Such a relation is especially obvious in the relation between 
thyroid gland and temperament, and general irritability goes parallel 
to the increased function of the gland. Therefore, an uncivilized life 
requesting beastly tenseness and nervousness may lead to the develop- 
ment of thyroid gland, or hyperfunction of thyrotrophic hormone of 
the pituitary gland, which in turn exerts antagonistic influence upon 
the function of adrenocorticotrophic hormone and probably also on 
growth hormone, thus reducing the stimulating action of climate. On 
the other hand, civilized life makes man feel at ease resulting in the 
loss of the beastly strain, contributing to the effect of climate, and 
may thus promote the loss of the rigidity. Environmental factors 
are considered to affect hormonal glands mainly through autonomous 
nervous system, and hence it may be a matter of course that psychic 
conditions such as irritability and nervous tenseness are capable of 
exerting a similar effect through the nervous system. 

In this connection, it shoud be mentioned that adrenal cortex ex- 
hibits the function of releasing the nervous irritability in contrast to 
thyroid. Also in this respect these two hormonal glands act antago- 
nistically with each other. Thus, it has been reported that adrenal 
extract produced improvement in some cases of pathological irritabi- 


+0.20 
+0.15 
+0.10 


+0.05 


Excess of centimeters 


1 2 3 4 5 6 7 8 
Number of children 


Fig. 39. Excess of stature over averge stature for families of various 
sizes. Reproduced from Boas: Race, Language and Cul- 
tures, New York, 1949. 


430 V. THE NATURE OF EVOLUTION 


lity, and a sense of well-being, even euphoria, replaced depression, 
and abnormal irritability disappeared (114). Even normal individuals 
at times respond, the immediate effect being drowsiness and release of 
nervous tension, if present. These effects are not due to suggestion, 
because sheep, in a state of neurosis due to overtaxing the nervous 
system in conditioned reflex experiments, showed marked improve- 
ment under adrenal extract therapy (115). 

In corformity with this concept, according to Boas (86) the increase 
in the stature of children is greatly influenced by their number in 
the family, and the increase is the more manifest the fewer the 
number, as shown in Fig. 39. It can be expected that nervous tenseness 
of animality may be necessary the more and the chance of hunger 
may be the more frequent, the larger the size of the family. 

This is most clearly demonstrated in the investigation of the 
Department of Education of Japan into the bodily change of Japanese 
children during and after the Second War. It is reported that the 
stature of the children was remarkably reduced during and after the 
War and that the reduction was distinct in the length of legs as 
compared with the almost unchanged trunk (116). It should be 
realized in this connection that the increase in Japanese stature in 
Los Angeles occurs chiefly in legs not in trunk. Such a reduction of 
stature is, of course, mainly ascribed to the want of food which 
might not only lead to the insufficiency of ACTH. and growth hor- 
mone, but also might stimulate the production of thyrotrophic hormone. 
However, in addition to this, irritable, uneasy life during the war 
might play a prominent part in this reduction in stature. 

On the other hand, the removal of uneasiness will result in the 
increase in stature, as evidenced actually by an experiment with 
animals. Thus, according to Weininger (117), albino rats, gentled for 
three weeks for ten min. a day following weaning, would show 
significantly greater mean weight. The difference in weight between 
experimental and control groups was related to a significantly greater 
proportion of adipose tissue and to a significantly Sieh skeletal 
length for the former. 

Porter (86) found that students with high scholastic standing are 
generally above the average in height. Entirely similar phenomenon 
was afterwards confirmed by Sargent (103), They considered that 
the growth of bright boys was more rapid than that of dull boys, 
but it may be more reasonable to suppose that the children able 
to be endowed with a higher education are rather civilized and better 
nourished, and perhaps need less nervous tenseness of animality. 
The Provident Mutual Life Insurance Co. of Philadelphia surveyed © 
270,000 men to see if there was any relationship between height and 


XII. FHE EVOLUTION OF MANKIND AND ITS FUTURE 431 


success. The company chose as its barometer the size of the policy 
each man held, long considered an excellent key to earning power. As 
the result it was found that an amazing parallel was present between 
the average policy and height, indicating clearly that the richer a man 
the taller his stature (118). It is well known that the increase in the 
stature of Japanese is more striking in towns than in the country. 
Dwarf horses have arisen on various islands where a limited food 
supply was probably the most effective cause; the pygmy elephant of 
Malta and some other cases in animals may have had a similar origin. 

Another probable cause of lessening in the rigidity of the genes 
may be the growth hormone itself. Overspecialization of animals 
appears to be usually connected with the increase in the bodily size, 
suggesting that growth hormone is required for the specialization. 
The specialization, on the other hand, may not be achieved without 
the decrease in the rigidity or the elasticity of a gene. Growth hormone, 
therefore, may be essential for the lessening in the rigidity. The 
effect of the hormone revealed in the establishment of unusual growth 
may be a manifestation of the reduction of the genic rigidity itself, 
since unusual growth may be unable without the loss of the rigidity. 

A stress causing a rapid change in animal characters appears to 
exert Stimulating effect upon hypophysis. Growth hormone may be 
yielded in response to such a stress in order to achieve with ease an 
adaptive change. If this conception is true, when a hypophysis-stimulat- 
ing factor continues to exist with the continuous production of growth 
hormone, the decrease in the rigidity will be unlimitedly pushed 
forwards, thus the speed of a change directed to the destruction being 
accelerated. 


4. Viruses, the Fatal Enemy of Man 


The loss of the rigidity in the genes will be revealed in the 
protoplasm as the acquisition of an easily changeable character. Such 
a character of the protoplasm, on the other hand, may be nothing but 
a high susceptibility to various viruses, since the structural change 
of protoplasm by a virus is no more than the occurrence of the infec- 
tion with the virus. If the protoplasm is provided with a strong 
reversibility, virus structure will soon be expelled and the protoplasm 
will recover its normal state even when the protoplasm is once affected 
by the virus. 

Cancer is a disease presumably resulting from the loss or the reduc- 
tion of the rigidity in the protoplasm including genes, a disease in 
which genes are coming out of order leading to the abnormal reduc- 


432 V. THE NATURE OF EVOLUTION 


tibility to their primitive structure. Sometimes it is regarded as a 
type of virus diseases. Now, it is a noteworthy fact that cancer is 
prevailing extensively among civilized nations. For example, in the 
United States present cancer mortality is over 200,000 per year and it 
is increasing at the rate of approximately 3 per cent or about 5,000 per 
year. Specifically, 207,721 deaths from cancer and leucaemia were 
reported in 1948. If cancer will continue to increase at this rate, in 
only a few hundred years it will so happen that all the people in 
United States are doomed to die of cancer. This fact strongly sug- 
gests that the decreasing process of the structural rigidity in human 
genes is rapidly proceeding in civilized people, presumably because of 
hypohysis increasing in its function. Moon ef af. (119) have actually 
confirmed that hypophysectomy does exhibit inhibitory effect upon 
cancer of the rat induced by methylcholanthrene. Moreover, the 
significance of pituitary function in neoplastic diseases including cancer 
is indicated by the many and diverse tumours that develop in the rat 
following the prolonged administration of growth hormone (120). 

During pregnancy, as was already pointed out, a remarkable hyper- 
trophy is observed in hypophysis; at the same time, attention is paid 
on the increased incidence of poliomyelitis during pregnancy. Domestic 
animals and cultivated plants have been highly specialized probably 
by the lessening in the rigidity of the genes. Their character highly 
susceptible to viruses must be based upon this; as is well known the 
more specialized, the more susceptible they are to viruses. Civilized 
people showing the tendency to increase in stature and becoming more 
and more resistant to tuberculosis may be regarded.as highly specialized 
species. At the same time, they are very susceptible to some virus 
diseases including cancer as seen above, whereas among small uncivilized 
people inclined to be severely afflicted by tuberculosis, the incidence of 
virus diseases including cancer appears to be much fewer than among 
civilized. 

Poliomyelitis is extensively prevalent among civilized nations es- 
pecially in United States and Canada, whereas low incidence of the 
paralytic disease, even among children, of the tropics and subtropics, 
such as Africa or Far East, has been reported (121). In Japan, recent- 
ly poliomyelitis is becoming not a rare disease, while the decrease in 
tuberculosis has become conspicuous. In 1916 there was an explosive 
outbreak of poliomyelites in America, and in was then impressed by 
the fact that patients so often were of the same physical type, usually 
large, overgrown children (122). 

On the other hand, many experimental results suggesting that the 
hyperfunction of hypophysis is disavantageous to virus infection have 
been recently accumulated. Thus, adrenal cortex hormone (cortisone) 


XII. THE EVOLUTION OF MANKIND AND ITS FUTURE 433 


or adrenocorticotrophic hormone (ACTH) has been found to affect the 
course of virus infection to the disadvantage of the host. This has 
been demonstrated for influenza and mumps virus in chick embryo and 
in mice, for poliomyelitis and Coxackie virus in mice and hamsters, 
vaccinia in guinea pigs (123) (124). 

The administration of these hormones, however, is known to be 
inhibitory to inflammatory or allergic reactions. For example, it has 
been reported that ACTH and cortisone reduce the febrile response of 
rabbits to typhoid vaccine and that cortisone markedly reduces the 
temperature of patients with typhoid fever (125) (126). Moreover, it 
has been shown that these hormones can suppress the febrile response 
of rabbits to bacterial endotoxin (127). Cortisone and ACTH are known 
also to inhibit cellular inflammatory reactions to a wide variety of 
other irritants including burns, trauma, and various chemical agents 
(128). 

Inflammatory reactions against bacterial infection may thus be pre- 
vented by the administration of cortical hormones. However, replica- 
tion of the bacterial pattern in the host, like that of the viral pattern, 
seems to be accelerated by these hormones. Thus, cortical hormones 
have been shown to increase the susceptibility in a number of hosts 
of many unrelated bacterial diseases (129). Effective doses of ACTH 
or cortisone are usually associated with evidence of increased multi- 
plication of the pathogens and more wide spread dissemination, al- 
though adrenal cortical hormones are not stimulatory to bacterial 
growth 7m vitro (128). This seems the case even with tuberculosis. 
Clinical experience suggests that administration of cortisone may 
reactivate an apparently healed tuberculosis infection in man (130). 
Laboratory studies with various animal infected with tubercle bacilli 
have shown that the administration of cortisone causes a more 
extensive diseases than that exhibited by controls. 

The favourable effect of the heightened function of hypophysis upon 
tuberculosis may, therefore, be not dependent upon the hypersecretion 
of cortisone, but upon the involution of thymus and other lymphatic 
organs which favour tuberculosis as already pointed out. It should 
be mentioned, however, that although cortisone exerted its deleterious 
effect upon tuberculous animals, corticotrophins. of hypophysis could 
be administrated in excess of human dose levels without harmful effect 
and was deleterious only at levels so high as 20 times human doses 
(131). 

Not only tuberculosis but the infectious diseases of bacterial origin 
in general appear rather rare in civilized people, in contrast to the 
high prevalence of some virus diseases among them, despite the above 
mentioned fact that the susceptibility of hosts to bacterial diseases is 


434 V. THE NATURE OF EVOLUTION 


enhanced by the cortical hormones. This may be dependent on the 
prevention of the infection of the diseases presumably owing to the 
hygienic life modus of the civilized people. Civilized life may thus 
prevent the infection of not only bacterial but also of some viral 
diseases. 

However, in general, adrenalectomy has been found to diminish the 
capacity of the body to withstand such stresses as infection, and a 
more or less complete return to previous resistance is achieved by the 
administration of sufficient amounts of adrenocortical hormones. This 
is the case also with Addison’s disease. The value of adrenocortical 
hormones to the patients with Addison’s disease hardly requires no 
comment (128). In view of this fact, it may safely be concluded that 
if disfunction of adrenal cortex is prevailed in some barbarous life, 
infections by various. pathogens including tuberculosis should occur 
strikingly arnong the uncivilized people. 

Whooping-cough is similar in many respects to an ordinary virus 
disease, and the incidence of the disease is much higher in United 
States and European countries than in Japan. Even in Japan it is more 
frequent in towns than in the country. According to the investigation 
of Nukada (132) in our laboratory, the incidence of Whooping-cough in 
Japanese children is the lesser, the greater their number in a family, 
provided that the number is greater than three, as shown in Fig. 40. 


Morbidity (2%) 


(oof te 


1 2 3 4 5 6 7 


- Number of children 


Fig. 40. Mobidity of whooping-cough in families of various sizes. 


XII. THE EVOLUTION OF MANKIND AND ITS FUTURE 435 


On the view point of easy infection, the increase in the number of 
children must result in the increase in the incidence; this is shown 
when the number is less than three, but the reverse is the case 
when the number is greater than three, showing the overwhelming 
influence of another factor acting in the reverse direction. This re- 
minds us of the fact, already shown in Fig. 39, which indicates that the 
stature of the children is the taller, the fewer their number in a family. 
Thus there is little doubt that the easy going life of children of small- 
sized family increases in them the sensitivity to whooping-cough as in 
more civilized nation. 

It has been confirmed by us (133) that the incidence of measles and 
whooping-cough in Kanagawa Prefecture in Japan was reduced remark- 
ably at the end of, or immediately after, the War; this fact indicates 
that the uneasy life of the children at the end of the War bears a 
striking resemblance to the usual but irritable, and food-insufficient 
life of the children of a large family. In addition, it is generally 
accepted that also intestinal toxic disorders of children and appendicitis 
almost entirely ceased to occur in Japan during the War. Even com- 
mon bacterial infectious diseases such as dysentery and typhoid fever 
were Said to have been likewise remarkably diminished. The develop- 
ment of the pattern of causative agents of these diseases, whether 
they were viruses or bacteria appeared thus to have been inhibited by 
the same factors. As jis generally accepted, tuberculosis, on the 
contrary, increases strikingly during wars, a phenomenon most probably 
due to the want of nutrition which causes a hormonal unbalance 
including the dysfunction of adrenal cortex favouring the disease. 

It is needless to mention that inanition is generally accepted to have 
an intimate connection with tuberculosis. However, if one considers 
that inanition causes a general decrease in the resistance to infectious 
diseases he commits a great mistake, because it is well established 
that an ill-fed animal is less susceptible to virus diseases including 
cancer than a well-fed one, as is readily expected from the theory 
above stated. 

One of the earliest demonstrations of this phenomenon was that of 
Rous (134), who in his experiments on fowl sarcoma virus, emphasized 
that healthy, well nourished fowls were more susceptible to the virus 
than the thin and ill. Olitzky, ef al. (135) found that guinea pigs 
suffering from malnutrition were resistant to infection upon inocula- 
tion with the virus of foot-and-mouth disease than were normal, 
healthy animals. Recent reports dealing with nutrition. and poliomye- 
litis have contribute further data on the way in which nutritional 
deficiency can decrease susceptibility. It is a note-worthy fact that 
cancers are very difficult to transplant in otherwise susceptible animals, 


436 V. THE NATURE OF EVOLUTION 


if the food intake of the animals is restricted severely. Even with 
the tumours growing well, the animals survive longer on restricted 
amounts of food (110). 

Thus it may be concluded that the nutritional defficiency and 
perhaps uneasy life requesting nervous tension of animality during 
the war were the cause of the reduction both in stature and in the 
incidence of some infectious diseases such as whooping-cough and 
measles. The same cause may increase tuberculosis. 

However, attention should be paid to the fact that, as shown in 
Fig. 38, although the decline in mortality index of tuberculosis was 
interrupted during the war, compensative, extensive decline occurring 
after the war. Again, as already stated, Japanese children were re- 
duced miserably in their stature during the War, but now they have 
already not only recovered their former status but have become even 
bigger, showing also the compensatory alteration. These facts may 
suggest that the factor pushing forwards the orthogenetic change was 
exerting its influence without stopping during the war, and that the 
mode of social life may have only a superficial influence. However, 
the more reasonable explanation is that the genic pattern having gra- 
dually been attained by the orthogenetic change is so strong that it 
cannot be altered permanently by the transient uncivilized life modus 
with both nutrient deficiency and nervous uneasiness which may only 
temporarily alter the pattern in the opposite direction, and that there- 
fore on the removal of the factors involved in the uncivilized life 
modus the former pattern can be readily recovered. Of course, if the 
War lasted much longer, the former pattern would have been for- 
gotten and as a consequence the compensatory alteration would have 
never or hardly occurred. 

Huntington claimed that the only factor which promotes civiliza- 
tion is a stimulating climate. Indeed, under a stimulating climate 
man may become active both mentally and physically owing to the 
stimulating effect of the climate upon hypophysis, and may tend to use 
extensively the brain and related organs, which may subsequently be 
developed, contributing to the promotion of civilization. The advanced 
civilization will enable man to live an easy life with sufficient food, 
thereby the factor arresting the stimulating action of climate exert- 
ing on hypophysis is removed. Consequently, stimulating climate can 
exert its full action, thus growth hormone being produced abundantly ; 
at the same time, for the development of the brain or other organs 
growth hormone may be required, and unusual use of the brain or 
other organs may stimulate hypophysis. Thus man may increase 
in his stature followed by the rapid promotion of civilization, which 
may further lead to the more sufficient supply of nutrition with the 


XII. THE EVOLUTION OF MANKIND AND ITS FUTURE 437 


diminution in the requirement of nervous strain of animality, con- 
tributing to the more extensive production of growth hormone. 

In.this way, causes becoming results, results becoming causes, the 
speed of the orthogenetic progress of man towards gigantism will be 
increased acceleratively. 

As described above, the average increase in height in a generation 
of Yale University students has been reported to be about 17/: inches. 
Swedish stature has increased 9cm in the last hundred years (104). 
If man will continue to become bigger at this rate, after 10,000 years 
he will be a giant over 40 feet. On the other hand, as the body 
weight is directly proportional to the square of height, animals becom- 
ing bigger over a certain extent would fail to resist the terrestrial 
gravitation, thus there being no choice but to become extinct. The 
whale can exist notwithstanding its gigantic size owing to its aquatic 
life as water alleviates the gravitation. 

However, more dreadful foresight may arise from the point that 
man is losing rapidly the structural rigidity in the genes. The fright- 
ful speed of specialization may be dependent upon this point, which 
seems to be accelerated by the growth hormone; at the same time, 
this loss of rigidity in genes may give rise to an extremely changeable 
character of protoplasm with profound and incessant production of 
various malevolent viruses including cancers. Thus mankind is bound 
to become extinct in a near future as did the gigantic reptiles. 

As is well known, a great many reptilian orders, including those 
containing the dinosaurs and pterydactyls, are entirely extinct, but 
still at present a number of small reptiles are existing. In like man- 
ner, some of the mankind may be left behind on the earth even when 
the majority of them will be extinct, and presumably those who are 
to be left are the people who are living in a climate lacking in the 
stimulating factor and who are compelled to live in‘a nutritious 
deficiency with a nervous tenseness of animality on account of their 
uncivilized life, which must always be associated with the climate. 

Irritability and uneasiness as well as the want of food peculiar to 
uncivilized life may result in the precocious sexual maturity through 
the hormonal pattern induced by the situation, presumably involving 
the hyperfunction of thyroid and gonadal glands, and this may cause 
the uncivilized, small people to have many children, in contrast to 
civilized, specialized people. This seems to be a phenomenon common 
to the animal kingdom in general. The smaller the animal, the more 
numerous enemies it must have, and accordingly the more nervous 
tenseness must be required, and again therefore the peculiar hormonal 
pattern mainly concerned with thyroid and gonadal function should be 
developed the more. Thus the smaller the animal, generally the more 


438 V. THE NATURE OF EVOLUTION 


numerous itS youngs, a consequence which must be most purposeful 
for the survival of small animals, since numerous victims may always 
be demanded by their enemies. This may likewise contribute greatly 
to the natural selection of the fittest, so that the character will 
bocome more and more fitted for survival. The reverse may be true 
when the animal increases in its size, there being no room for the 
natural selection on account of its poor procreative power, and also in 
this respect there is a strong reason for the extinction of large-sized 
animals. 

Since there may be no natural selection in such animals, their 
evolution should be orthogenetic and animals bound to be extinct by 
their overspecialization are always to advance on an _ orthogenetic 
course. It appears, however, that Nature has prepared an excellent 
mechanism to check this terrible orthogenesis. The mechanism may 
be based upon the want of food, which is expected to occur when 
animals greatly increase their size. Inanition may not only inhibit 
the active function of hypophysis, but may stimulate thyroid or related 
glands to make the evolutionary course entirely reverse. Reptilians 
might accomplish the orthogenesis until they became extinct presumably 
because there were too plentiful food to make them hungry. 

Orthogenetic, unlimited increase in size, therefore, may occur 
rather rarely. In general, an animal having increased in its size toa 
certain extent may cease to become bigger through the want of food, 
as the bigger an animal, the food may be required the more. On the 
contrary, an animal having become smaller and smaller may sometimes 
begin to increase in size owing to the sufficiency of food-supply be- 
cause small animals need only a small quantity of food. In this way, 
the quantity of food may always control the size, accordingly, the 
specialization rate of animals. 

However, civilized man is able to escape this want of food, however 
big he becomes, owing to the high civilization, and therefore, for the 
future, the character difference between civilized and uncivilized man 
will become amazingly great, and when the overspecialized, civilized 
man is extinct, the latter will represent the mankind, but those who 
escape the extinction will follow the fate of the former after migrating 
into the stimulating climate. 

When one surveys the entire field of animal evolution, the 
significant fact becomes apparent, not only from the geological but 
from the facts of comparative anatomy, that each great distinctive 
new group in animals has always sprung from the more primitive 
member of the preceding group, fnot from those highly specialized. 
Mankind may not provide an exception. 

The extinct Cro-Magnon people are known to have a Stately stature 


XII. THE EVOLUTION OF MANKIND AND ITS FUTURE 439 


comparable to the present and live a considerably high civilized life 
in the old stone age nearly 20,000 years ago. Their cranial capacity 
appears to have been even somewhat greater than that of modern 
Europeans. Their extinction might be caused by the loss of structural 
rigidity in the genes, followed by the prevalence of some severe virus 
diseases. In their days, the ancestors of the present Europeans must 
have been only a barbarous, insignificant existence, but now they are 
the representative of mankind and apparently are going to follow in 
the steps of Cro-Magnon. 

How fatal are some viruses to overspecialized people may be 
understood if one remembers the dreadful prevalence of influenza which 
outbroke during the Ist War; the victims of the disease were far 
greater than those of the War. Human being have presumably achieved 
since then a great advance in their specialization by losing remarkably 
the rigidity of genes; who can warrant you there is no chance of out- 
break of an influenza or other virus diseases so severe that almost all 
the civilized people will be sacrificed to it ? 

However, the writer’s forecast concerning the future of man thus 
far described will be failed if his theories are accepted generally and 
counter-measures are taken against the human orthogenesis. Not only 
the prevention of human extinction as a race but also as an individual 
may be not utterly impossible. Since the individual devolopment is 
raised by hormones, administration of a proper hormone or hormones 
may be able to stop or retard the advancement; even to accomplish 
the rejuvenescence may be not imppossible as seen in the production of 
germ cells. As already stated, thyroid hormones will promote matura- 
tion leading to premature decay, while certain pituitary hormones 
appear to be involved in prolonging the life span. Carlson and Hoelzel 
(136) compared the effects of omnivorous and herbivorous diets upon 
the life of rats, and confirmed a favourable influence of the diet rich 
in meat. This may be accounted for by the positive effect of the 
protein rich diet upon the pituitary function. Moreover, in most ex- 
periments recorded in the literature the female of a species such as 
the rat lives longer than the male, a phenomenon which has long been 
recognized also in the case of man. This must be attributed to the 
difference of hormones due to the difference of sex. 


CHAPTER XIII 
THE SUMMARY OF PART V 


Protein having A-structure may be converted into B-structure 
under the effect of b-factor, whereas the protein thus converted to have 
B-structure will return to A-structure under a-factor. The writer 
designates such a phenomenon as reversibility of protein structure, 
and regards it as the ‘‘memory”’ of the former structure. This pheno- 
menon appears to be common in usual proteins but especially manifest 
in the protoplasm, and may be indicated as follows: 


b-factor 
A-structure ame B-structure, 


a-factor 


wherein both a- and b-factor are various chemical or physical agents, 
such as temperature, light, or hormones. A-structure may be stable 
under the effect of a-factor, and B-structure under b-factor. 

However, protein having B-structure frequently fails to be conver- 
ted into A-structure by the mere change of the environmental factor. 
In such a case, some ‘‘stimulus’’ is needed to induce the change, the 
change being prompted by a disturbance in the protein structure in 


duced by the ‘‘stimulus’’. This may be compared to the conversion 
of supercooled water into ice by a certain ‘‘stimulus”’ like a mechanical 
agitation. 


If unfolding and refolding of polypeptide chains are required for 
the structural conversion of a protein, ‘‘stimulus’’ may favour the 
unfolding, that will be followed by the refolding, which the latter is 
specific to the factor or environment under which the protein is 
placed. 

The change from A to B structure and from B to A structure 
appears to take place easily when the change is repeated; in other 
words, the repetition of the reversible change may result in the fa- 
cility of the change, the memory being thus strengthened. 

The intensification of the memory is presumably raised by the 
removal of structural factors which prevent the reversible change. 
The removal of such preventing factors may be achieved the more 


THE SUMMARY OF PART V 441 


completely, the more frequently occurs the repetition of the change. 
The memory is, therefore, a structural pattern of proteins, given rise 
to by the removal of the structural factors which prevent the rever- 
sible change. 

Proteins produced as replicas of memory-carrying proteins, which 
constitute the protoplasm acting as a template, must consequently have 
the memory. The memory in our brain may thus be established. As 
the structural pattern specific to a memory is transferred to newly 
synthesized proteins, the memory can continue for life. 

On the other hand, the structural pattern involving a memory also 
has the tendency to recover its original pattern to produce again the 
preventing structural factors, and therefore the persistence to the 
original pattern render the reversible change difficult leading to the 
oblivion of the memory. Hence, for the maintenance of memory is 
needed the repetition of the reversible change, namely continuous 
training, thereby the production of preventing structural factors is 
inhibited. 

In addition, if a protein remains under the same environmental 
condition in taking the structure determined by the environment, the 
structure will be gradually altered so as to be more fitted to the 
environment, so that the structure is fixed and its change in any 
directions becomes difficult, thus the original structure being forgotten. 

The gradual alteration of protein structure to be fitted to the 
environment is the adaptation of the protein to the environment, and if 
the adaptation goes so far as the structure becomes too fixed to lose 
its elasticity it may be called senescence. 


2 


Individual development may be based-upon the reversible character 
of proteins constituting the genes of germ cells. It is considered that 
a germ cell can act as an independent, unicellular organism because 
of the primitive structure of its protoplasm and that this primitiveness 
is brought about by the structural return of a certain somatic cell to 
its primitive stage, from which the organism producing the germ cell 
has been developed. Sucha return to the primitive structure must be 
brought about by the reversible character of protoplams protein. Since 
the structure of the protoplasm is governed by the genes, the return 
of the protoplasm to the primitive structure must be raised by the 
structural change of the genes. The structure of the genes may be 
reduced to the primitiveness under the influence of a factor or factors 
that make the primitive structure more stable than the developed. 


442 V. THE NATURE OF EVOLUTION 


The gene of a germ cell thus reduced to the primitive stage can 
again recover its previous, advanced structure owing to the reversible 
character, when factors or environmental conditions are provided which 
favour the development of the advanced structure. The recovering 
change is generally initiated by fertilization, but the fertilization can 
be substituted by a proper physical or a chemical stimulus, which may 
act like a mechanical stir in the case of the transformation of super- 
cooled water into ice. 

Various characters are revealed in a developing individual with 
the progress of the recovering change of the genes, because the proto- 
plasm is looked upon as a kind of liquid crystal, whose crystal shape 
is determined by the structure of protein components, which is in turn 
controlled by the genes. The change is continued until the complete 
recovery of the advanced structure is established, whereby various 
morphological and functional characters are successively revealed with 
the progress of the change. 

Regeneration, which is common to plants and some lower animals, 
can be explained in the same way. The somatic cells in the site of 
cutting are reduced in their structure to a primitive, undifferentiated 
state, from which subsequently the differentiated structure is anew 
recovered. Regeneration, therefore, is dependent upon the reversible 
character of protoplasm protein just as ontogeny. 

The formation of organs or tissues in the various body portions 
during the ontogeny is apparently due to the environmental effects 
varying with the portion. Since, as above cited, protein structures are 
determined by environmental factors, different environmental effects 
produce different structures which in turn iuduce different organs or 
tissues. 

Different structures thus produced in the different places of develop- 
ing body are unstable at the start, and tend to change to other 
structures if the environments are changed, but by a prolonged ex- 
posure to the same environment the structures are gradually fixed and 
become so stable that they can retain their specific structure even 
when transplanted to another part. The so-called organizer is an 
embryonal part whose structure has been thus fixed. 

The protein of organizer, if liberated as protoplasm particles, may 
be able to transfer its specific structure to other unfixed parts of the 
embryo as a ‘“‘virus’’, but the protein molecules, which fail to form 
virus-like particles, or other components, such as steroid, liberated 
from the organizer may behave as hormones by exerting on the sur- 
rounding embryonal parts their structural effect which is peculiar to 
the organizer, though they are not so powerful as being capable of 
transmitting the specific structure itself as a “‘virus’. Thus, if a 


THE SUMMARY OF PART V 448 


local structure is so fixed as to be called an organizer, a series of 
peculiar structures are formed around it, with the formation of com- 
plicated shapes and functions. 

The structures thus varying with the portion of an individual are 
produced because the local genes have been changed in their structure 
by the environmental effects which vary with the portion. Therefore, 
every organ and tissue must have peculiar genes. This conclusion 
seems natural, since, as is generally accepted, every organ is provided 
with protein antigenically specific to the organ, while it is also be- 
lieved that what determine the protein structure are genes. 


3 


The so-called biogenetic law that ontogeny recapitulates phylogeny 
can also be readily explained by the assumption that the germinal 
cells are the reductive form of the somatic cells, and the change 
giving rise to reduction is reversible. Since the structural reversibility 
of a protein is considered to be based upon the memory of the course 
along which the protein has attained to the present structure, the 
somatic cells, in the course of reduction to the germinal, have to 
pursue reversely the course of phylogeny, whilst, in the course of 
development of the germ cell, the course of phylogeny itself is to be 
followed. This reversible change must be repeated in each generation, 
and hence the memory is correct, and ontogeny recapitulates phylogeny 
without fail. 

In higher animals, it seems that the memory is retained only in 
the particular cells involved in the production of germ cells, and in 
ordinary somatic cells there appears to be some mechanism by which 
the reduction of the structure is made difficult. Ordinary cells are, 
however, also inclined to be reduced to the primitive structure when 
the preventing mechanism is rendered weak, and in such a case cancer 
cells, the structure of which is primitive like germ cells but different 
in many respects from the latter presumably on account of this 
abnormal reduction, are produced. Sexual hormones play an important 
role in the production of germ cells, whilst the same hormones or 
related substances appear to be involved in the production of cancer 
cells. The reduction of somatic cells to cancerous is an abnormal 
process and so the return of the cancerous to somatic cells appears 
to be not an easy occurrence as in germ cells, but it does sometimes 
occur. 

he structural reversibility of genes is extremely strong. The 
genes have the property to alter their structure according to the 


Adit V. THE NATURE OF EVOLUTION 


environment under which they are exposed, as in general do proteins, 
the main component of the genes. Owing to this strong reversibility 
a gene can return to its original structure when the environmental 
effect which has caused the change is removed. 

The recovery of the original structure, however, is difficult, if a 
profound change arises abruptly ina gene. The genes can recover their 
original structure because the course leading to the original structure 
is remembered, but the memory of the course may not be retained 
clearly, if the change occurs too rapidly and extensively to remove the 
structural factors which obstruct the progress of the change. In such 
a case the change is inclined to.be irreversible and called mutation. 

Mutation is uncommon, and usually the genes undergo extremely 
gradual changes, which continue to proceed by degrees in directions 
which are determined by the environmental factors. Such a change is 
completely reversible, and can be regarded as the normal change of 
the genes. 

The factors, which induce directly or indirectly the gradual, rever- 
sible change, involve the stimulus coming from organs which are 
continuously used excessively, as well as ordinary environmental factors 
such as temperature, light, and diet. 

The change of a gene leads necessarily to a change of character 
of the cell or of the organism which is governed by the gene. Organ- 
isms may alter their characters when brought under a new environ- 
mental factor. Since such a change is reversible, the acquired character 
is lost when the environment returns to the original state. 

However, if organisms are exposed always to a new environmental 
factor without returning to the original environment, the previous 
structure will be gradually forgotten with the fixation of the new 
character responding to the new environmental factor. Thus acquired 
characters will become fixed, not easily to be lost by the change of 
the environment. 

Fossils forming an evolutionary series suggest that evolution 
proceeds in definite directions, from the directing tendency of internal 
structures, and this phenomenon is called orthogenesis. If a certain 
environmental effect continues to exert its influence upon a gene or 
genes, in producing a gradual change in the gene in a definite direc- 
tion, the organism having the gene is to undergo orthogenesis. 

Bacteriophage can be looked upon as a free gene, and when acted 
on by an inactivating agent such as formalin or antiserum, it tends to 
change gradually in a definite direction until completely inactivated. 
This change is usually reversible if the change does not proceed too 
far and too rapid, the original character of phage being recovered on 
the removal of inactivating agent, which can be compared to the 


THE SUMMARY OF PART V 445 


stimulus coming from the use of organs or from a changed environ- 
ment. In this case, phage is of course a gene-model. 

The gradual change of genes is completely reversible, but some 
periods of time are necessary for the reversion to the original 
structure after the removal of the changing factor, as the reversion 
occurs also gradually. Consequently acquired characters, which have 
not yet been fixed enough, usually remain for some periods after the 
removal of the factor, a phenomenon known as ‘‘Dauermodifikation.”’ 


4 


Characters given rise to by mutation are generally injurious to the 
organisms, whereas those given rise to by the gradual, reversible 
change of genes are usually favourable. 

This may depend on that the reversible change, unlike mutation, 
is induced by naturally existing environmental factors to which the 
organisms have to be always exposed. If organisms could not endure 
the change of usual environmental factors, they would inevitably 
become extinct; as a natural result only the organisms capable of 
bearing the change would be able to continue the existence. In order 
to become the fittest, the organisms would have to acquire the faculty 
not only to endure the change but to obtain a favourable character 
through the change, so that the present-day organisms, which must 
be the fittest, can attain characters adapted to the changed environ- 
ments. 

The change of genes thus raised by the environmental change is 
gradual and reversible, and leads to the production of favourable 
character, whereas the abrupt action of some unnatural chemical or 
physical agents upon genes may cause random changes; thus muta- 
tion may arise. The genes of an organism existing in the present day 
must have structures most fitted to the existence of the organism, so 
that the majority of random changes taking place in the genic struc- 
tures would naturally result in the production of less fitted or 
unfavourable characters. Thus, mutation is usually injurious. 

The adaptability of organisms to a new environment is based upon 
the gradual change of genes producing the character suitable for the 
new environment. The adaptability must have been developed because 
it is an indispensable character for the organisms. The organisms 
without this character would surely fail to continue their existence. 

Such a favourable character, however, is presumably not yet suf- 
ficiently developed in extremely primitive organisms, but as they were 
evolved higher, the mechanism of the gradual change of genes might 


446 V. THE NATURE OF EVOLUTION 


be developed so elaborately that evolution is sometimes possible even 
without any natural selection. 


5 


The principle of adaptation may exist in the nature of protein 
itself. Protein molecules are usually inclined to be coagulated by 
agents which appear to be injurious to organisms, in the coagulation 
polar groups being folded and lost, while the injurious agents presu- 
mably can act upon the protein by combining with it through certain 
polar groups of the protein, and therefore the disappearance of the 
polar groups can be regarded as the adaptation of the protein not to 
be further combined by the agents or to reject the agents. 

On the other hand, when the environmental factor is not injurious, 
the protein adapts itself to the new environment in rearranging its 
structure to become stable under the environment. And, if affected by 
some chemical agents possessing certain structures which are not in- 
jurious, the polar groups of the protein may be rearranged instead of 
being folded in response to the structural arrangement of the agent, 
thus the protein becoming able to combine more easily with the latter 
with the establishment of adaptation to utilize the agent. The trans- 
mission to genes of such structural changes occurring in the protoplsm 
protein may result in the adaptation of cells to environmental factors. 

The adaptive change thus raised in local somatic cells can be 
transferred to the germinal by both virus-like agents and hormones. 
If the changed structure is liberated in the form of protoplasm par- 
ticles or virus-like forms, the structure can be precisely transferred to 
the geminal cells. On the other hand, if the changed structure is 
released in the form of a certain hormone or a hormone-like agent 
corresponding to the structure, some influence related to the structure 
will be exerted on the germ cells through the agent. In higher organ- 
isms, the environment appears to exert usually its effect on hormonal 
systems, whereas it is known that hormones have great influences 
upon the character of organisms. 

In the transmission of a precise structure of somatic cells to ger- 
minal, a virus-like agent is involved whilst hormones may act as 
carriers of inexact structures. 

The continued effect of a certain environmental factor on germ 
cells may cause a fixed change in the genes of the germ cells, with 
the result that the original structure of the genes is forgotten, thereby 
the acquired character becoming inheritable. 

In like manner, structure corresponding to the character resulting 


THE SUMMARY. OF PART .V 447 


from use or disuse of an organ can be transmitted to the genes of 
germ cells and will gradually be fixed in them to become inheritable. 
The influence of the use of an organ upon genes appears to be so 
intensive that it leads to a swift and extensive development of the 
organ. 

The atrophy of an organ following disuse is similarly rapid, which 
should, however, be naturally expected, since the memory of the pre- 
vious structure must be strong and fresh if the development has 
occurred swiftly by the use. Vestigial organs, remaining persistent 
after a prolonged disuse, may therefore involve the structures whose 
development was never raised by the use. Probably, vestigial organs 
were produced aimlessly by an orthogenetic change of a gene not 
directly involved in any use, and when they came to have a useful 
value, the use was commenced leading to a rapid development. 

A gene is usually concerned with a restricted, peculiar structure 
of protoplasm protein, but a change in a restricted portion of a protein 
molecule cannot, as a rule, occur without exerting any influence upon 
other portions of the protein. Hence, an extensive development of a 
certain character by the change of the corresponding gene is to be 
associated more or less with developments of other related characters, 
thus overspecialization of useless organs being sometimes established. 
The production of primitive organs, which may be left as vestigial 
organs, might also occur in this way. 


6 


A gene can undergo a gradual change in a certain direction under 
a given environmental factor, because it has the property to achieve 
the change, that is, because it has the predisposition to undergo the 
change. Sucha predisposition may somewhat vary with the difference 
in individuality of the genes. Individuals with genes having each parti- 
cular predisposition may accordingly change the character in different 
ways or at different rates even under the same environment, thus in- 
dividual variation becoming more manifest. Out of them the fittest 
will be selected by natural selection. Since the fittest thus selected 
has the predisposition to proceed further in the change which will 
produce the fitted character, the individual will continue to evolve in 
an orthogenetic direction in so far as environmental factor persists. 

Newly acquired structure of a gene is unstable and may readily 
return to the previous structure as the memory of the previous struc- 
ture is fresh, and accordingly. a new structure of a gene, produced by 
a certain gradual change, may be put back readily to the previous 


~ 


448 V. THE NATURE OF EVOLUTION 


state when the gene falls in with some other genes having the original 
structure. Therefore, in order to establish a peculiar, gradual change 
the gene must be isolated from other genes having the original 
structure. This may be the reason why isolation is needed for the 
evolution. f 

When a gene has achieved a gradual change to such an extent 
that the genes having the original structure can no more exert their 
influence upon it, the organism having the gene may be called new 
species. Since changes of a gene, not great enough to reject the in- 
terference of the unchanged, original gene, are always to be returned 
to the previous structure by the latter, there should usually be a dis- 
tinct line of demarkation between any two species, no gradual trans- 
mission from a species to another being found among individuals exist- 
ing without separation. 


ri 


It seems probable that the fate of civilization is subjected to 
climate as Huntington believed. The want of stimulating effect in 
climate may cause the loss of mental and physical activity in man, 
leading to the disuse of brain and related organs, resulting in the 
degeneration of the organs. 

Stimulating climates exert their influence on hypophysis, probably 
in the main through autonomic nervous systems, and as a result the 
secretion of corticotrophic and growth hormones may be enhanced at 
the expense of thyrotrophic and gonadotrophic principles. Consequently, 
under a stimulating climate man may tend to be increased in his 
stature owing to the growth hormone, and at the same time his sus- 
ceptibility to tuberculosis may be reduced, since status thymico-lym- 
phaticus and cortical insufficiency, both of which raise the suscepti- 
bility, are removed by the pituitary hyperfunction. 

It is known that civilized people have been showing the tendency 
to be increased in their stature since about a hundred years, while on 
the other hand, during the same period the resistance to tuberculosis 
has been increasing steadily in them. ‘These two amazing facts can 
be readily explained by: the assumption that the stimulating effect of 
climate on hypophysis was heightened rapidly. 

This heightening of the effect, however, is probably not attributed 
to the increase in the stimulating action of climate itself, but to the 
increase in the susceptibility of man to the climate. The promotion of 
civilization may enable man to live an easy life with a plenty of 
nutrition. The cause of the increase in the susceptibility to climate 


THE SUMMARY OF PART V 449 


may lie in this, because the deficiency of food and also the irritable, 
uneasy life peculiar to uncivilized life presumably exert upon hy- 
pophysis counteraction against climatic factor. 

The want of food may result in adrenal insufficience by counter- 
acting against stimulating climate, and in addition nervous tension and 
irritability attending on uncivilized, barbarous life may also counteract 
against climate by stimulating some hormonal systems mainly  thy- 
roid. Because of the presence of such preventing factors, uncivilized 
people may neither increase their stature nor become insusceptible 
to tuberculosis even under the stimulating climate. However, with 
the development of civilization these preventing factors are removed to 
enable the stimulating climate to exert its full influence. 

During and immediately after the Second World War, Japanese 
children were reduced in their height remarkably, suggesting strongly 
that the want of food and nervous tension inhibited the production of 
growth hormone. Even in peace time, country man is smaller than 
town man, and students with high scholastic standing are generally 
above the average in height. Moreover, children of a large family are 
remarkably below in height than those of a small family. 

Excessive use of certain organs may lead to the enhanced secretion 
of growth hormone, which may presumably contribute to the rapid 
development of organs continuously used. The speedy development of 
an organ would be achieved by a swift change of the genic pattern, 
and this swift change may be favoured by growth hormone. This 
hormone, on the other hand, would promote the growth by causing a 
swift change in genic pattern. Therefore, the vigorous production of 
growth hormone resulting from the pituitary hyperfunction may 
cause the lability of the structural pattern of genes, rendering the 
protoplasm very unstable so that the susceptibility of the organism 
to some viruses including cancer may be strikingly increased. 

It has actually been established that adrenocorticotrophic and 
cortical hormones affect the course of some infectious diseases mainly 
those caused by viruses including cancer to the disadvantage of the host, 
whereas nutritional deficiency which inhibits the production of these 
hormones is known to increase the resistance to viruses and cancer. 
During a war, the incidence of tuberculosis may be strikingly increased, 
but that of some virus diseases is declined. Children of a large family 
show a great resistance to some viruses, and children in the country 
are also less susceptible to the viruses than those in towns. The 
same is the case with uncivilized and civilized people. 

The specialization of civilized people as revealed in the increase 
in the stature may be ascribed to the structure of genes becoming 
unstable as shown in the fact that their susceptibility to some viruses 


450 V. THE NATURE OF EVOLUTION 


and cancer is also increasing strikingly. 

Overspecialized animals, in general, appear to have become extinct 
with the unusual development of bodily size. Likewise mankind 
seems to be bound to become extinct in a near future as did the rep- 
tiles. However, uncivilized: people who can remain small in stature 
with a high resistance to virus diseases owing to the absence of stimu- 
lating climate in their abode, which inhibits the promotion of civiliza- 
tion, may escape the extinction and will represent the mankind when 
the civilized are extinct. 


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