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Vou. XIX 


NOVEMBER, 1949 : Ne] 


THE CONTRIBUTIONS OF THE WOODS 
HOLE OCEANOGRAPHIC INSTITUTION 


C. O’D. IsELIN 
Director, Woods Hole Oceanographic Institution 

The dislocations of the war years have finally 
subsided at this Laboratory and it is hoped that 
the research program has become stabilized for 
a few years at least. The great majority of in- 
vestigations having direct practical applications 
have been terminated so that, of a total budget 
of about $750,000, which is now virtually assured 
through 1950, only about one-fifth comes under 
the heading of applied research or development. 
Thus, what we consider basic oceanography has 
grown at Woods Hole from a pre-war budget 
of about $110,000 to a post-war budget of about 
$600,000. 

This is not an exceptional increase. For the 
country as a whole, it has been estimated that 
the oceanographic budget has increased more 
nearly by a factor of ten and that it will double 
again during the next ten years. This is only to 
say that interest in the physies, chemistry, ge- 
ology and biology of the oceans is growing rap- 
idly. The Federal Government is especially 
aware of the need for increased knowledge of 

(Continued on page 3) 


THE MARINE BIOLOGICAL LABORATORY 
IN 1949 


Dr. CHARLES PACKARD 
Director, Marine Biological Laboratory 


The 62nd session of the Marine Biological Lab- 
oratory formally opened with the first Friday 
evening lecture by Dr. Gilbert Tyler. But long 
before this time investigators had been arriving, 
some in May and many more before the middle of 
June. Others will continue to come until late 
August. By the middle of September most of 
them will have left, but the workers in the Insti- 
tute of Muscle Research, under the direction of 
Dr. Szent-Gyorgyi will remain throughout the 
year. The laboratory welcomes these newcomers, 
and wishes that more investigators would find it 
possible to carry on their research here after the 
summer season. 

As usual, more people applied for places in 
the laboratories and in the courses than we could 
accommodate. A few more investigators could 
have been accepted if there were more rooms 
available in our residences and in the village 
houses. The housing situation will not material- 
ly improve until a number of investigators build 
their own homes in the Devil’s Lane Tract or 
elsewhere. There is no need to call attention to 


Marine Biclogical Laboratory, Dr. C. Packard... 1 
Contributions of the Woods Hole ccunoerep hie 
Institution, C. O’D. Iselin 
U. S. Fisheries Laboratory, D 5 
Serological Aspects of Fertilization, Dr. A. Tyler. 6 
Mechanism of Color Changes in Crustaceans, Dr. 
iraniaweAe SS TO WI) ise eee eee 
Labile P in Nueleie Acids, Abel Lajtha 


Structure of Fibrin Clots, Dr. E. Mihalyi 
Investigations on Muscle Fibers, Dr. A. G. Szent- 
Gyorgyi alhs) 


Evidence for Activity of DNAse i in Mitosis by Use 


of d-usnie Acid, Dr. Alfred Marshak... 16 
Cold as a Means of Combatting Asphyxia in New- 
born Guinea Pigs, James A. Miller, Jr. 17 


TABLE OF CONTENTS 


A Simple Method for Inducing Spawning of Sea 


Urchins and Sand-Dollars, Dr. A. Tyler 19 
Biological Specificity and Protein Structure, Dr. 

IDYONROH Me Seba CN 2 ee 20 
Reactions of Cells in Frog Tadpoles to Implants 

of Tantalum, Dr. Carl C. Speidel.__...___________ 21 
Growth and Metamorphosis of the Pluteus of 

Arbacia punctulata, Ethel B. Harvey_ 22 


Reversible Enzymie Reduction of Retinene to ‘Vita- 
min A, Dr, Alfred F. Bliss _ 2 
Effects of Ultra-violet Light on the Catalase Ac- 


tivity and on Photosystheses, Dr. A. Frankel _ 24 
Synthesis of Acetylcholine, Dr. D. Nachmansohn, 

S. Hestrin and H. Voripaieff : Pee), 
Directory of the Marine Biological Laboratory. pero 27 


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PSS 


November, 1949] 


THE COLLECTING NET 3 


the crowded condition of the Mess. 

Sixty years ago the laboratory acquired a 
small steam launch at a cost of $1,350. Later the 
classes were carried to the collecting areas in the 
Vigilant, a sailing vessel which was towed by the 
launch. Now we have a fleet of five power boats 
—Dolphin, Limulus, Nereis, Sagitta and Tern. 
The first two are new; the others have been in our 
service for many years. The Dolphin, purchased 
last year, and used by the classes and the collect- 
ing crew is now provided with a Diesel motor. 
Thus the danger of explosion or fire, has been 
reduced to a minimum. 

This summer there are five labor fellows and 
five Atomic Energy Commission fellows in resi- 
dence. The former are investigators who already 
have their doctorate and are competent to carry 
on independent research in the fields of biophys- 
ies, biochemistry or biological chemistry. The 
latter are in general a younger group. Each has 
a definite problem involving the use of radio- 
active isotopes. One purpose of these fellowships 
is to train investigators in the new and exacting 
techniques required in this type of research. 

On the third floor of the Brick Building is a 
‘‘hot laboratory’’ where transfers of highly ac- 
tive material can be made. Under Dr. Failla’s 
direction it has been equipped with the safe- 
guards necessary to protect the workers from 
radiations. Funds for the equipment were fur- 
nished by the American Cancer Society. 

The renovation of the Old Main Building, be- 
gun this Spring, is the first major change to be 
made in the oldest building of the Marine Bio- 
logical Laboratory. It was in 1888 that the 
south wing, now used by the Embryology class 
and some investigators, was erected. The next 


The Contributions of the Woods 


year the middle portion was added to serve as a 
lecture room and a library. Then in 1892 the 
north wing was built. Many notable biologists 
have occupied the little rooms on the first and 
second floors. Among these were Whitman, the 
first director; Loeb, physiologist; F. R. Lillie, 
embryologist; T. H. Morgan, geneticist; E. B. 
Wilson, cytologist ; and G. N. Calkins, protozoolo- 
sist. Some of the early pioneers—Conklin, R. S. 
Lillie, Mathews and Osterhout, have continued to 
return to the M.B.L., their places of summer 
work for more than fifty years. 

The renovation of the building was made pos- 
sible through the generosity of the Rockefeller 
Foundation, which has on previous occasions 
contributed to this laboratory. The basement, 
originally almost unexcavated, is now provided 
with well equipped laboratories used by the 
Physiology Class and by instructors in other 
courses. The upper floors will be remodeled 
next Fall. In the Embryology Laboratory the 
water table is to be rebuilt and moved toward 
the back of the room. On the Physiology side, 
the present stairway will be removed, and a new 
one will open from the street with stairs both 
to the basement and to the second floor. The 
present arrangement of rooms will be much al- 
tered. Upstairs the rooms will be enlarged, and 
some will be provided with salt water tables. It 
is hoped that insulation of the roof will keep 
room temperatures within reasonable limits. 

With all these changes, the building still re- 
tains much of its original character. It will al- 
ways be called ‘‘Old Main’’ to remind us of our 
debt to those who first worked in it and laid the 
foundations of American Biology. 


Hole Oceanographic Institution 


(Continued from page 1) 


the oceans and it is from this source that most 
of the money is coming to oceanography. How- 
ever, it may be significant to point out that our 
sister laboratory, the Scripps Institution of 
Oceanography, at present is receiving roughly 
$400,000 per year from the State of California. 
Many other coastal states also are making sub- 
stantial contributions for studies of the ecology 
of inshore forms, including the State of Massa- 
ehusetts which has given a contract to this In- 
stitution for the study of shellfish. 

There are, at present, 264 persons actively en- 
gaged in work at the Woods Hole Oceanographic 
Institution. These can be classified as follows: 


Full-time scientific and technical staff. 89 
Part-time scientific ; i.e., summers only — 44 
Fellowship holders So 
Visiting investigators 118 
Secretaries and clerks 19 


General maintenance and services ——_— 40 
Crews of vessels 
JA Ghoawbangyiee yan ee 


The total summer increase, including fellowship 
holders and visiting investigators, is 68 which is 
roughly the pre-war figure. 

Of what does this boom in oceanography con- 
sist? It will only be possible here to describe 
very briefly some of the main lines of investiga- 
tion in which especially rapid progress is being 
made. 

The most active group in oceanography today 
are those interested in the geology and_ geo- 
physies of the ocean basins. The recording echo 
sounder when combined with new, radio-naviga- 
tional techniques makes it practical to examine 
in detail the topography of the ocean bottom. 
During the last few years, the Atlantis has ac- 
eumulated about 70,000 miles of bottom records 


4 THE COLLECTING NET 


[Vol. XIX, No. 1 


and gradually it is being revealed that the bot- 
tom of the ocean is as varied and complex as the 
surface of the land. Recent soundings in the 
neighborhood of Bermuda show that the island 
is situated on a ridge of low hills, trending 
northeast-southwest. Several submerged sea 
mounts, nearly as high, have been located in the 
general area. The Hudson River Canyon has re- 
cently been traced nearly halfway to Bermuda 
and at present our newest research vessel Caryn 
is tracing the continuation of this remarkable 
feature towards Bermuda. 

It is evident that the course and character of 
the ocean currents may be very much influenced 
in passing through regions of pronounced bot- 
tom topography. The Atlantis left two weeks 
ago to study these relationships in the area east 
of the Grand Banks. 

Another development in submarine geology 
has come about as the result of great improve- 
ments in coring tubes. The piston-type coring 
tube, first used by Swedish oceanographers, now 
permits cores thirty to fifty feet in length to be 
obtained from the deepest waters. Since the rate 
of sedimentation in deep water is relatively slow, 
there is great hope that much of the recent his- 
tory of the earth can be worked out rather quick- 
ly through studies of such cores. They form an 
undisturbed record of the changes in depth and 
climate extending over a period of several 1il- 
lion years. 

The seismic techniques developed in oil ge- 
ology have been adapted for use at sea and are 
allowing the examination of the rock structures 
underlying the ocean down to ten or fifteen miles 
below the bottom of the sea. Of special interest 
is the location of the edge of the granite on which 
the continents are built and to learn about the 
character of this edge. 

Turning to subjects more usually identified 
with oceanography, the study of the heat and 
water vapor exchange between the sea surface 
and the atmosphere is receiving special empha- 
sis. During the past twenty years it has been the 
fashion more or less to neglect heating and cool- 
ing as a cause of the general circulation, both in 
the atmosphere and in the hydrosphere. Recent 
studies of the Director of the Royal Netherlands 
Meteorological Institute are indicating that even 
on a day to day basis sea surface temperatures 
can exert a major effect on the development of 
the weather. The role of salt nuclei picked up 
by the air passing over the ocean also is proving 
to be a fascinating and important study connect- 
ing meteorology and oceanography. 

It will, of course, be a long time before the dis- 
tribution of temperature, salinity, oxygen and 
nutrient chemicals can be described in satisfac- 
tory detail. The broad seasonal and geographi- 


cal aspects of physical oceanography is a study 
requiring patience and a certain amount of sus- 
tained organization. If the collection of data 
was left entirely to the interests of individual 
investigators, it would proceed much more slow- 
ly than is desirable. In this sense, an oceano- 
graphic laboratory has somewhat the role of an 
astronomical observatory. Vast quantities of 
routine data must be collected and digested be- 
fore even the basic problems can be clearly de- 
fined. Improved instrumentation, both at sea 
and in the laboratory, is greatly accelerating the 
descriptive aspects of physical oceanography. 
It is not enough that the ships take in large quan- 
tities of, for example, routine temperature data. 
The process of correcting, sorting, averaging 
and digesting must, if possible, also be facilitated 
through machines of one kind or another. Al- 
though the instrumentation of oceanography 
has developed rapidly, it is clear that there is 
still much more to be done. The market for 
oceanographic instruments will probably always 
remain small. It is for these reasons that instru- 
ment design and construction remains an impor- 
tant activity at our laboratory. 

It would be nice to be able to report that bio- 
logical oceanography is going ahead with as 
much vigor as the physics and geology of the sea, 
but unfortunately in this case money is a serious 
limiting factor. Although it is clear that man 
will soon have to turn more and more to the sea 
as a source of protein, and this is already the 
case in several countries bordering rather bar- 
ren seas, there is little financial support in this 
country for basic studies of the produetivity of 
the oceans. We know about how many haddock 
are to be found on George’s Bank and about how 
much sustained yield can be expected from this 
area, but when it comes to the productivity of 
the oceans as a whole we know very little. Quan- 
titative studies of the smaller forms have been 
made, for they cannot easily escape a net, but 
as we advance up the food chain in the sea, the 
quantitative and geographical aspects beeome 
very vague indeed. 

Here again considerable instrumental develop- 
ment will be required. Two possible quantitative 
tools for marine biology are suggested by recent 
refinements in underwater acoustics and under- 
water photography. However, it also seems like- 
ly that marked improvements in the effective- 
ness of nets of various kinds can be made. Once 
really good sampling techniques have been de- 
vised the marine biologist will be face to face 
with the same problem that the physical ocean- 
ographer already has had to deal with. That is, 
he will soon be swamped with data, unless means 
are devised in advance to facilitate the analysis 
phase of a given investigation. 


November, 1949 | 


THE COLLECTING NET 


oO 


THE WORK OF THE UNITED STATES FISHERIES LABORATORY 


AT WOODS HOLE (fees 
= Lia e 
Dr. Paut 8S. GALTSOFP ce ; 
Director, United States Fisheries Laboratory \G la se 
ce a 


During the years following the end of the 
World War II, the United States Fisheries Lab- 
oratory at Woods Hole was gradually rehabili- 
tated and adapted for year-round operation. 

Investigators coming regularly to Woods Hole 
for the last ten years may recall that the build- 
ings and grounds of the Laboratory were seri- 
ously damaged by the hurricanes of 1938 and 
1942. Although the most serious defects have 
been repaired, the signs of the ravages caused by 
the wind and sea and are still noticeable; the 
sea wall along the southeast side of the small 
boat basin is still in ruins, and the pool in which 
the sharks and seals were formerly displayed 
has not been restored. The Laboratory was able, 
however, to rebuild the sea wall around the 
grounds and to rehabilitate the laboratory build- 
ing and the residence which have been made suit- 
able for all-year occupancy. The sea water ta- 
bles, chemical benches and other laboratory 
equipment which were removed when the sta- 
tion was occupied by the U. 8. Navy during the 
war have been completely restored. The water 
pipes were repaired and the buildings rewired 
and reconditioned. 

The hatching of marine fish (cod, flounder 
and mackerel), which for many years had been 
carried out by the old Bureau of Fisheries, has 
been discontinued and the hatchery equipment 
adapted for biological research. In 1947, ar- 
rangements were made to transfer the headquar- 
ters of the section of the North Atlantic Fish- 
eries Investigations from Cambridge, Mass., to 
the Woods Hole Laboratory. The necessary re- 
arrangements to provide additional office and 
laboratory space for investigators and docking 
facilities for the research vessel Albatross III 
were completed that year. A comprehensive 
program of fishery research in the North Atlan- 
tic and its progress will be discussed in a sepa- 
rate article by Dr. William F. Royce, in charge 
of the project. 

Besides the studies carried on by the North 
Atlantic Section, the Woods Hole fisheries labo- 
ratory is engaged in shellfishery research con- 
ducted by Dr. Paul 8. Galtsoff and serves as a 
temporary headquarters for the clam investiga- 
tions carried on by John B. Glud. 


Following a well-established old tradition of 
working together with other scientific institutions 
at Woods Hole, the Service made a cooperative 
agreement in 1947 with the Marine Biological 
Laboratory for an exchange of services and facil- 
ities. The plan has proved mutually pleasant 
and profitable. 

Full cooperation with the Woods Hole Ocean- 
ographic Institute is likewise a very important 
factor in carrying on the research program of 
the Laboratory. Close association with the offi- 
cers and personnel of both institutions, mutual 
assistance in case of emergencies and free ex- 
change of ideas, creates a favorable environment 
which stimulates researeh work. 

The Aquarium of the Laboratory was re- 
opened to the public in 1947. Thanks to the 
cooperation of the Supply Department of the 
Marine Biological Laboratory, it was possible to 
assemble and display to the public from 55 to 65 
different species of fish and invertebrates which 
occur in local waters. As an innovation, part of 
the former hatchery room on the first floor was 
set aside for special exhibits showing the various 
techniques used in marine biology. Of special 
interest are the exhibits arranged by the Woods 
Hole Oceanographic Institute, which show ocean- 
ographic instruments, automatic recording de- 
vices, underwater photography, sounds recorded 
in the depths of the sea, and various methods em- 
ployed by modern science in the study of the 
ocean. 

The Aquarium is open to the public every 
day, including holidays, from 8:00 A.M. to 8:00 
P.M. The number of visitors, particularly on 
Sundays and holidays is surprisingly large, fre- 
quently exceeding 1,000 persons a day. 

Thus far, the Service has not been able to ob- 
tain sufficient appropriation for the complete re- 
habilitation of buildings and grounds and for 
the modernizing of scientific equipment and the 
Aquarium. Every year, however, the work of 
reconstruction and rehabilitation continues with 
the limited funds available for this purpose. The 
investigators of the Laboratory are confident 
that with this increased scope of scientific activi- 
ties the Laboratory will become an important 
center of research and training in fishery biology. 


6 THE COLLECTING NET 


[Vol. XIX, No. 1 


SEROLOGICAL ASPECTS OF FERTILIZATION 


Dr. ALBERT TYLER 


California Institute of Technology, Pasadena 


It seems quite appropriate that a talk on ferti- 
lizin and related substances should be presented 
here at Woods Hole since it was here that the 
subject was first developed by the late Professor 
Frank R. Lillie, who was director of this labora- 
tory for many years. It was here, too, that most 
of the early work in this field was done by Jac- 
ques Loeb, Otto Glaser, Alvalyn Woodward, C. 
R. Moore, E. E. Just, Myra Sampson and G. H. 
A. Clowes. After the highly interesting early 
work investigations along this line practically 
ceased from alout 1922 until 1939 when Max 
Hartmann and his colleagues, working at Naples, 
and we, in Pasadena, undertook a series of inves- 
tigations which have continued, with some inter- 
ruption, during the war years. More recently 
John Runnstrém and his co-workers in Stock- 
holm have entered this field. In general the re- 
sults of the latter investigators agree very well 
with our own while those of Hartmann and his 
co-workers differ in several points. Recent de- 
tailed reviews of the subject have been written by 
Bielig and Medem (1949) and by Tyler (1948, 
1949). 

The present summary is based primarily on 
the work of the author’s laboratory. This work 
has been concerned with four kinds of substances 
that have been isolated from eges and sperm of 
marine animals; namely, fertilizins from eggs, 
antifertilizins from sperm, antifertilizins from 
egos and egg-membranes lysins from sperm. 
Early in this work it was shown that the ferti- 
lizin of eges of the sea-urchin and other animals 
is the macromolecular material of the gelatinous 
coat, and this has been confirmed by Hartmann, 
Runnstr6m and others. The gelatinous coat 
slowly dissolves as the eges stand in sea-water, 
yielding the so-called ege-water that has the 
property of ageglutinating homologous sperm. 
The gelatinous coat can be rapidly dissolved in 
slightly acidified sea-water, without injury to the 
rest of the egg, and concentrated solutions of 
fertilizin thereby obtained. Various tests, in- 
eluding the action of purified proteinases, 
showed the fertilizins of the sea-urchin and the 
keyhole limpet (as defined by their agelutinating 
action) to be of protein nature. By relatively 
simple extractions and precipitation procedures 
we have been able to prepare sea-urchin ferti- 
lizin in electrophoretically and ultracentrifu- 
gally homogeneous form. The purified material 
contains both amino-acids and sugars and may, 


therefore, be termed a glycoprotein or mucopoly- 


saccharide (depending upon the terminology 
adopted). It is of highly acidie character, show- 


ing little change in electrophoretic mobility be- 
tween pH 8.6 and 2.0 This is evidently due to 
the fact, discovered by Vasseur (1947) and con- 
firmed by us, that it contains over 25 per cent 
sulphate, most probably linked in the manner of 
a sulphuric ester. Some of the analytical data 
obtained on purified fertilizin is given in Table 
I. The values for the amino-acid and reducing 
sugar content are minimum due to the fact that 
a fair amount of not readily analyzable humin 
residue forms upon acid-hydrolysis. Galactose 
has been identified as the osazone. Paper chroma- 
tography has shown the presence of at least seven 
different amino-acids which are, most probably, 
aspartic acid, elutamic acid, threonine, lysine, 
arginine, phenylalanine and isoleucine in addi- 
tion to tryptophane in the humin residue. 


TABLE I 


Analysis of electrophoretically homogeneous preparations 
of fertilizin of Strongylocentrotus purpuratus 


IND trogen! eae - 9.6-5.8% 
Carbon 33.3% - 
Hydrogen cual eee 5.5% 
Sulphate asa omen 23% 
Phosphate 0.06% 


Reducing sugar >25% 
Amino acids —__ >20% 
Glucosamine (?) 1.6% 
Galactose pos. 
Glucuronie acid neg. 

neg. 
Molecular weight (eale. as sphere) — 82,000 


On the basis of the results of attempts to sepa- 
rate protein and polysaccharide fractions of fer- 
tilizin it is concluded that these do not exist as 
loosely bound distinct entities in the molecule but 
rather that amino-acid and sugar residues are 
firmly inter-linked. In this connection it may 
be noted that other glycoproteins of somewhat 
similar composition, in particular those exhibit- 
ing the human ABO blood-group activity, have 
likewise proven refractory to attempts to disso- 
ciate protein and polysaccharide constituents 
(see Morgan, 1947). 

The antifertilizin from sea-urchin sperm has 
also been prepared in electrophoretically homo- 
geneous form. It is an acidic protein, isoelectric 
at pH 3. Investigations of its amino-acid com- 
position are in progress. One of the workers in 


November, 1949] 


Runnstrém’s laboratory (Hultin, 1947) has sue- 
gested that it may be a basic protein, but this is 
refuted by results published recently by Metz 
(1949) as well as by Runnstrém’s (1942) and 
our own data concerning its electrophoretic mo- 
bility. Evidence from electron microscopy of 
extracted sperm shows the antifertilizin to be 
located on the surface of the nuclear region of 
the head. 

The antifertilizin from eges and the egg-mem- 
brane lysin have likewise been shown to be of 
protein nature. Dr. Max Krauss, of our labora- 
tory, has obtained e@ood evidence showing that 
the action of the lysin of keyhole hmpet sperm is 


dependent upon the presence of sulfhydryl 
eroups. Electron microscopy of extracted sperm 


indicates that the acrosome may be the source of 
the lysin. 

Lillie considered the interaction of fertilizin 
and sperm to be analogous to that of serological 
agelutination and evidence has since accumu- 
lated that the kind of interaction exhibited by 
the various substances extracted from the eggs 
and sperm is essentially that of antigen and an- 
tibody. The finding of an antifertilizin within 
the ege alone with fertilizin in the coat, means, 
then, that in one and the same cell there are a 
pair of substances that are capable of interacting 
in antigen-antibody manner. This along with 
consideration of certain information from the 
literature of immunology has led to the develop- 
ment of a so-called auto-antibody concept of cell 
structure, growth and differentiation that has 
been presented recently in some detail (Tyler, 
1947). Briefly this view states that the macro- 
molecular substances of which cells are con- 
structed bear the same relationship to one an- 
other as do antigen and antibody and that they 
are formed in essentially the same manner as are 
antibodies. In addition to various experiments 
of others that can be interpreted on the basis of 
the occurrence of such natural auto-antibodies 
the author has been able to demonstrate the pres- 
ence of an auto-antivenin in a venomous reptile, 
the Gila monster. The view also offers interpre- 
tation for certain serological anomalies, such as 
the Wassermann reaction, auto-agelutination 
phenomena, specific interaction of virus with cell 
surface, ete., and it offers the possibility of ob- 
taining protective agents against pathogenic or- 
vanisms by extraction of the organisms them- 
selves. 

Experiments; relating to the spontaneous re- 
versal of sperm agelutination by fertilizin in the 
sea-urchin (a phenomenon now known to occur 
also in the so-called Hirst reaction of hemagelut- 
ination by viruses) led to the finding that ferti- 
lizin could be converted. by simple treatments. 
into a non-agelutinating, ‘‘univalent’’ form. Evi- 


THE COLLECTING NET 7 


id 


dence was also accumulated that fertilizin occurs 
normally in such ‘‘univalent’’? form in many 
species of animals and C. B. Metz (1945) dis- 
covered that specific agglutination of sperm 
could be obtained with such non-agelutinating 
fertilizins by the addition of a non-specific ad- 
juvant obtained from hen’s ege-white, serum- 
albumin or other sources. This latter result is 
paralleled by recent experiments of Wiener 
(1948) on ‘‘univalent’’ Rh antibodies which like- 
wise can cause specific agglutination in the pres- 
ence of certain non-specific proteins. Metz’s re- 
sults provide strong support for Lillie’s view 
that the fertilizins are of general distribution 
throughout the animal kingdom. 

As a side-line of some practical, as well as 
theoretical, interest a series of experiments were 
undertaken in which immune antibodies against 
manunalian blood cells, pathogenic bacteria and 
toxins were converted into the ‘“‘univalent’’ form 
by photo-oxidation and various properties of 
such antisera examined. The anaphylactic prop- 
erties of such treated sera were found to be 
greatly reduced. The evidence also showed that 
the ‘‘univalent’’ antidiptherial antibodies were 
capable of neutralizing the toxin but that ‘‘uni- 
valent’’ antipneumococcal antibodies were in- 
capable of acting as protective antibodies. With 
respect to serum-sickness factors there is, then, 
considerable improvement in the antisera in the 
former case but not in the latter. It was found, 
too, that the ‘‘univalent’’ anti-blood cell anti- 
bodies were incapable of acting as hemolytic 
sensitizer, or of fixing complement, and this offers 
some support of Heidelberger’s views concerning 
the mechanism of complement fixation. 

Investigations of the role of fertilizin and anti- 
fertilizin in fertilization have shown that, when 
present on the surface of the respective gametes, 
they facilitate the process. When present in solu- 
tion. however, they block fertilization, presuma- 
bly because the interaction of the sperm with 
fertilizin, or of the eges with antifertilizin, is 
completed before contact is made between the 
effective surfaces of the gametes. It has not, as 
yet, been possible to determine with any cer- 
tainty whether or not fertilizin-antifertilizin in- 
teraction is also essential for fertilization since, 
in the experiments on removal of fertilizin by 
methods that do not injure the rest of the eee, 
a minute laver of this substance evidently re- 
mains firmly bound to the surface. However, 
results of experiments employing immune anti- 
bodies against antifertilizin favor the view that 
the interaction is essential for fertilization. The 
role of the ege-membrane lysin, in species in 
which this agent has been demonstrated 1s mani- 
festly to enable the sperm to penetrate the mem- 
brane barriers that surround the unfertilved 


8 THE COLLECTING NET 


[Vol. XIX, No. 1 


ege. For the antifertilizin within the egg, Lillie 
had proposed a role in activation and establish- 
ment of the block to polyspermy but evidence 
concerning this is still lacking. Lillie’s demon- 
stration that fertilizin is obtainable from no 
other tissue than the gametes has been amply 
confirmed and this serves as a basis for under- 
standing the tissue-specificity of fertilization. An 
extensive investigation has been made, and is in 
progress, concerning the problem of species-speci- 
ficity. In general, the results show that the 
degree of cross-reaction of fertilizin and anti- 
fertilizin of various species is greater than the 
degree of cross-fertilization. Thus the specificity 
of fertilizin-antifertilizin interaction is not in 
itself sufficient to account for that of fertiliza- 
tion. Similar results are obtained with the lytic 
agent of sperm. Also, the specificity of these sub- 
stances as antigens in rabbits is not as great as 
that of fertilization. It appears then that other 
specific factors must be involved and this is not 
too surprising since it is quite likely that many 
other substances besides those discussed here are 
concerned in various steps in the process of ferti- 
lization. On the other hand, it should be noted 
that where cross-reaction between fertilizin and 
antifertilizin is lacking fertilization also fails to 
occur. 

A scheme has been proposed (Tyler, 1948) for 
the manner in which fertilizin-antifertilizin in- 
teraction may account for the approach and 
specific attachment of sperm to egg surface. As 


noted above Lillie also suggested that activation 
of the egg might involve these substances. At 
present the best available hypothesis concerning 
activation is that proposed by Heilbrunn (1943), 
which involves a protoplasmic gelation or clotting 
reaction initiated by a release of calcium. It is 
of interest to note, then, that the fertilizin-anti- 
fertilizin reaction is largely dependent upon the 
presence of calcium, as Loeb first showed and as 
Vasseur (1949) has recently demonstrated in 
some detail and, that fertilizin shows (see Im- 
mers, 1949) some heparin-like activity. 


References 


Bielig, H. J. and Medem, F. 1949 Experientia, 5: 11. 

Heilbrunn, L. V. 1943 An Outline of General Physi- 
ology, 2nd ed. Saunders, Philadelphia. 

Hultin, T. 1947 Arkiv Kemi, Mineral., Geol., 24B: 
No. 12. 

Immers, J. 1949 Arkiv Zool., 42A: No. 6. 

Metz, C. B. 1949 Proc. Soc. Exper. Biol. Med., 70: 

Morgan, W. T. J. 1947 Experientia, 3: 257. 

Runnstrom, J., Tiselius, A. and Vasseur, E. 1942 Arkiv 
Kemi, Mineral., Geol., 15A: No. 16. 

Tyler, A. 1947 Growth, 10 (suppl.) : 7. 

Tyler, A. 1948 Physiol. Revs., 28: 180. 

Tyler, A. 1948 Anat. Record, 101: 658. 

Tyler, A. 1949 Amer. Nat. (in press). 

Vasseur, E. 1947 Arkiv Kemi, Mineral., Geol., 25B: 
No. 6. 

Vasseur, E. 1949 Arkiv Kemi, 1: 105. 

Wiener, A. and Gordon, E. B. 1948 J. Lab. Clin. Med., 
33: 181. 


422. 


Nore: An abstract of a Friday Evening Lecture de- 
livered July 1, 1949, at the Marine Biological Laboratory. 


THE MECHANISM OF COLOR CHANGES IN CRUSTACEANS 


Dr. FRANK A. Brown, Jr. 
Professor and Chairman of the Department of Biological Sciences, Northwestern University 


The capacity for exhibiting changes in body 
coloration is widely distributed among higher 
animals. It is usually found only in species which 
possess well-developed eyes and ‘central nervous 
systems and the color changes are ordinarily 
complexly controlled through these organs. Of 
all the organisms showing color changes no group 
shows more striking examples of this capacity 
than the decapod crustaceans. Within this group 
the character of the changes spread through a 
wide range. Some species, as for example the 
fiddler crab, Uca, has as its major natural re- 
sponse simply a darkening of the body by day 
and a blanching by night. The sand shrimp, 
Crago, and the common prawn, Palaemonetes, on 
the other hand, are able to darken upon dark- 
colored backgrounds and lighten upon light-col- 
ored ones. In addition, both of these latter ani- 
mals are able to a good extent to become even 
the color of colored backgrounds upon which 
they come to rest. This is especially true of Palae- 


monetes. Still other crustaceans as, for example, 
the gulf-weed crab, Planes, and the shrimps, 
Latreutes and Hippolyte, appear able to imitate 
not only the color but also the pattern of the 
coloration of the algae upon which they live. 
These color changes in the crustaceans are ac- 
complished by means of special effector organs, 
the chromatophores, which are widely distributed 
over the surface of the body. These chromato- 
phores are highly branched, multinucleate cells, 
each containing one (monochromatic), two (di- 
chromatic) or more (polychromatic) pigments. 
The chromatophores normally function in both 
of two ways in the production of color changes. 
One of these two activities is referred to as physi- 
ological color change and involves the mechanical 
movement of the pigment within the chromato- 
phore. Any given pigment may concentrate into 
a spherical mass in the chromatophore center and 
thus not contribute to the gross coloration of the 
animal, or it may disperse out into the chromato- 


November, 1949] 


THE COLLECTING NET 


9 


phore branches and contribute to the coloration. 
By an appropriate differential activity of the 
chromatophoral pigments in physiological color 
changes, a prawn like Palaemonetes, possessing 
red, yellow, blue, and white pigments can readily 
assume the color of any background upon which 
it is placed. The rates at which physiological 
color changes are accomplished are relatively 
rapid, the time required for a pigment to pass 
from a maximally concentrated to a maximally 
dispersed state, or vice-versa, ranging from 
about five minutes to a few hours depending 
upon the chromatophore type. As would be ex- 
pected, the accuracy with which the background 
color can be matched by physiological color 
changes alone, depends upon the amounts of the 
required pigments present within the chromato- 
phores. 

The second of the two primary activities of 
chromatophores is termed morphological color 
change. This activity involves a differential syn- 
thesis or destruction of the pigments within the 
chromatophore. In Palaemonetes, for example, 
the red and blue pigments gradually disappear 
from the chromatophores of specimens kept upon 
a white background. Animals kept upon a black 
background show, on the contrary, a gradual 
gain in the amount of these dark pigments in the 
chromatophores. These morphological color 
changes are much slower than the physiological 
ones and require days or even weeks before the 
maximum adaptive change is completed. Extrac- 
tion ofsthe total pigment in the bodies of animals 
undergoing extensive morphological changes 
shows clearly that these changes involve chemi- 
eal alteration in the pigments and not simply a 
translocation of the pigments within the body. 

It was for many years assumed that the chro- 
matophores of crustaceans were directly under 
the control of nerves. Numerous attempts to dem- 
onstrate histologically the presence of nerves 
passing to the chromatophores all resulted, how- 
ever, in failure. Furthermore, when all the 
known nerve supply to a region of the body was 
transected, there seemed to be no disturbance, 
whatsoever, of the capacity of that region for 
color change. Even the accurately detailed tint- 
adaptation of a shrimp like Palaemonetes occurs 
in such denervated regions of the body. 

About twenty years ago, two investigators, 
working independently, demonstrated an action 
of blood-borne principles in the control of crus- 
tacean chromatophores. Koller, in Germany, 
utilizing the technique of blood transfusion, 
showed that blood from a black donor darkened 
a pale recipient and that blood from a yellow 
donor caused a pale recipient to become yellow. 
Perkins, in this country, observed that occlusion 
of a blood vessel resulted in an immediate cessa- 


tion of the color response of that region of the 
body supplied by that vessel, and that the abil- 
ity to respond reappeared at once following res- 
toration of the normal blood supply. Perkins 
demonstrated, in addition, that an aqueous ex- 
tract of the eyestalks of the shrimp, Palaemon- 
etes, hghtened dark specimens into which it was 
injected, and, conversely, removal of the eye- 
stalks left them permanently darkened. On the 
basis of these observations Perkins postulated the 
eyestalks to contain sources of a lightening hor- 
mone. At about the same time Koller reported 
that aqueous extracts of the rostral region of the 
sand-shrimp, Crago, darkened pale specimens 
and destruction of the region resulted in their 
remaining permanently pale. Koller therefore 
postulated a source of a darkening hormone to 
lie in the rostral region. These last observations 
of Koller have not yet been confirmed despite a 
number of attempts to do so. 

It appears clear at the present time that hor- 
mones conveyed by the blood constitute the only 
chromatophore- activating agents for the crusta- 
eceans. The great complexity of the endocrine 
mechanism which must be involved becomes ap- 
parent when, one recalls the independent activi- 
ties of the several pigments in single individuals 
in the.gourse of adaptation to different colored 
backgrounds. Palaemonetes possesses four kinds 
of pigments each of which is capable of activity 
independent of each of the other three. It is 
clearly evident that a minimum»of three hor- 
mones must be present in this shrimp to account 
for this complex differential activity of the pig- 
ments. 

Investigations during the past few years have 
provided us with considerable information as to 
sources, numbers and the activities of hormones 
influencing the various chromatophore types. The 
two major sources appear to be the sinus glands 
in the eyestalks and various regions of the cen- 
tral vervous system. 

The sinus glands are minute organs which are 
richly charged with secretory granules; these 
granules render the gland readily visible in the 
freshly dissected eyestalk. The glands are com- 
plexly innervated from the brain and optic gang- 
lia. Sinus glands may be easily dissected free of 
the remaining eyestalk tissue, extracted, and 
tested for activity by injection into other speci- 
mens. It seems quite clear at present that sinus 
glands of crustaceans possess at least three chro- 
matophorotropic principles. Evidence for this 
conclusion has come from two types of experi- 
ments: comparative physiological studies and 
chemical fractionation. Physiological studies of 
the actions of extracts of sinus glands of various 
species of crustaceans as tested by simultaneous 
assay of their actions upon two widely different 


10 THE COLLECTING NET 


[Vol. XIX, No. 1 


responding chromatophore types, Uca black pig- 
ment and Palaemonetes red pigment, have dem- 
onstrated that the activities of the extracts with 
respect to these two types do not vary in any 
correlated manner. This is what would be ex- 
pected were the activity upon one chromatophore 
type the result of one substance and the action 
upon the other, of another. Compelling the same 
conclusion are experiments upon chemical frac- 
tionation. An alcohol-soluble fraction of sinus 
glands from a wide variety of species of crusta- 
ceans exhibits a strong action upon Palaemonetes 
red pigment and little upon Uca black; the aleo- 
hol-insoluble fraction, on the contrary, has a 
strong action upon Uea black pigment and rela- 
tively little upon the red of Palaemonetes. There 
seem, therefore, definitely to be at least two prin- 
ciples in all sinus glands studied. 

Further comparative studies of the effects of 
sinus gland extracts lead to the conclusion that 
still an additional chromatophorotropie princi- 
ple is present in some. Extracts of the elands of 
Palaemonetes and other shrimp, but not of Uea 
or other true crabs, will, upon injection, lighten 
the telson and uropods of black Crago. It is still 
unknown as to whether the shrimp sinus elands 
possess three hormones and the crab, only two, 
or whether the glands of both groups possess only 
two, with one of the two differing in properties 
between the two groups. 

The presence of chromatophorotropins within 
the central nervous system is of general occur- 
rence among the crustaceans. The actions of these 
principles may supplement or may antagonize 
those of the sinus glands, depending upon the 
chromatophore type. The general roles of these 
substances in color change may perhaps be most 
lucidly illustrated by a description of a few types 
of results obtained with the shrimp, Crago. When 
one removes the eyestalks with their included 
sinus glands from this sand shrimp, a very char- 
acteristic color change ensues. First, the whole 
shrimp becomes very pale, except for the telson 
and uropods which become intensely black. 
After about an hour the shrimp gradually as- 
sumes a typical coloration of eyestalkless speci- 
mens, a mottled body and a completely pale tail; 
this coloration is then retained indefinitely. One 
can reproduce the transitory color ¢hange just 
described, by strong eleétrical or other stimula- 
tion of the cut ends of the optic stalks in the eve- 
stalkless animals. The same transitory response 
can be obtained by injection of an aqueous ex- 
tract of the central nervous System of another 
specimen. Incidentally, this peculiar coloration 
involving a light body and a dark tail is so often 
observed in normal individuals of the genus, that 
one common Pacific coast species bears the spe- 
cific name, nigricauda. 


If instead of applying a strong stimulus to 
the cut ends of the optic stalks, a mild electrical 
stimulus is used, the response is quite different ; 
the whole animal now undergoes a transitory 
blackening. It can easily be seen that at least 
two active principles from somewhere within the 
central nervous svstem are here involved. A 
study of the various parts of the central nervous 
system reveals that whereas an extract of any 
major portion of the system will lighten the body 
of darkened Crago, only extracts of the minute 
tritocerebral commissure, connecting the two cir- 
cumoesophageal connectives just posterior to the 
oesophagus, will both hghten the body and black- 
en the telson and uropods. If one goes further 
and now extracts the tritocerebral commissure 
with alcohol, this fraction, like the other parts of 
the nervous system, will lighten the body but not 
darken the tail. The alcohol-insoluble residue, 
now freed from its body-lightenine activity, 
blackens not only the tail of Crago, but the whole 
body as well. The central nervous system of 
Crago, therefore, clearly appears to have two 
principles, one of whose activities is to lighten 
the body but not the tail (sinus gland extract 
lightens both), and the other darkens the whole 
body. The mild stimulation of the optic stalk 
therefore caused a selective liberation of only 
one of the principles; the strong stimulation pro- 
duced extensive liberation of both. 

A histological examination of the tritocerebral 
commissures has disclosed that the neurilemma 
of this region shows a greatly thickened area 
whose cells are filled with secretory granules. 

When one examines the nervous systems of 
other crustaceans for the presence of this Crago- 
hghtenine and this Crago-darkening activity, it 
is found that all of the decapod crustaceans ex- 
cept the true crabs possessed both of these prin- 
ciples though their distributions within the ner- 
vous systems differ from genus to genus. True 
crabs, such as Uca, do not possess the Crago- 
darkening activity in any part of their nervous 
system. It can readily be demonstrated, how- 
ever, that the nervous system of Uca possesses 
two chomatophorotropins. One of the latter is a 
white pigment concentrating principle, the other 
is a black pigment dispersing one, and these two 
show quite different distributions through the 
system. 

It is seen from the foregoing that sources dis- 
tributed within the central nervous system and 
the sinus glands are important in the control of 
the complex chromatophore systems of crusta- 
ceans. There is no reason to believe that these 
initial demonstrations of a few principles from 
these sources have provided a complete picture. 
Undoubtedly, more will be shown to exist. Fur- 
thermore, little or nothing is known of the nature 


November, 1949 | 


of joint actions of the various principles. 

There are three general kinds of responses of 
crustacean chromatophore systems to environ- 
mental factors. The first general type is a re- 
sponse to total illumination. In this response it 
seems to be rather venerally true that the greater 
the illumination the ereater the degree of dis- 
persion of all the pigments, both dark and heht. 
This is apparently the primitive response of the 
system and is probably quite comparable to the 
primary or embryonic one of the vertebrate. It 
can be demonstrated by such a technique as 
shielding a limited portion‘of the integument 
that this response is at least in good measure an 
‘““ndependent-effector’’ one in the crustaceans. 
On the basis of this response; crustaceans tend, 
other factors being equal, to become opaque in 
bright light and transparent in dim heht. 

A second general kind of response is one to 
the albedo, or in other words, a response to the 
ratio of incident to reflected light strikine the 
eye. This type of response is obviously depend- 
ent upon the possession of a complex eye. A 
eood white background diffusely reflects about 
1/3 of the ineident light and a good black back- 
eround diffusely reflects about 1/200 of the inci- 
dent light, hence the ratios in the two instances 
are 3 and 200 respectively. The albedo responses 
of the chromatuphore system are ones resulting 
in a mimicking of the shade of the background 
upon which the animal lies. By this response 
the dark pigments typically disperse and the 
white pigment concentrates when the animal is 
upon a black background. The pigments assume 
the opposite condition upon a white background. 
It is clear from what has been said that the al- 
bedo response and total illumination response 
may supplement one another (e.g. the white pig- 
ment on black and white backgrounds) or oppose 
one another (e.g. dark pigment upon black and 
white backgrounds). The imitating of the colors 
of backgrounds similarly is an albedo type of 
response, but here obviously there is involved a 
capacity for color perception as well. 

The third major type of response of the chro- 
matophore system to environmental factors in- 
volves temperature. Elevation of the tempera- 
ture above the normal range tends to disperse 
white pigment and to concentrate dark pigment. 
The result is that at these higher temperatures 
the body reffects more, and absorbs less, radiant 
enerey. This would appear to serve to some de- 
eree as a body-temperature regulating mechan- 
ism. 

All three of the kinds of responses are normal- 
ly operating simultaneously upon any given 
chromatophore system, but the relative influences 
of the three vary with the species, the chromato- 
phore type, and the magnitude of the intensity 


THE COLLECTING NET 


11 


factor for each in any given situation. It can 
be seen that the characteristics of the responses 
of the system that have just been described are 
all compatible with all of the four commonly 
postulated adaptive significances of color change 
in animals in general. A function of obliterative 
coloration appears to be fulfilled by the albedo 
response. <A function of protection of the body 
from injury due to excessive illumination ap- 
pears subserved by the total illumination re- 
sponse. The temperature response appears con- 
sistent with the hypothesis that the system helps 
protect the body from excessive elevation in: body 
temperature due to sunlight absorption. Both 
the temperature and total illumination responses 
appear to be such as to favor the absorption of 
heat through radiation within the viable tem- 
perature range. 

The chromatophore systems of crustaceans are 
in many instances also subject to persistent endo- 
eenous rhythms. The operation of such a rhythm 
is especially conspicuous in the fiddler crab, Uea, 
which tends to be dark in color by day and pale 
by night. This diurnal color change persists 
with remarkable regularity even when the ani- 
mal is kept in constant darkness and tempera- 
ture. Under such constant conditions:the rhythm 
has been observed to remain completely in phase 
with the solar day-night evcle for several weeks. 
The frequency of the rhythm is independent of 
temperature through a wide range. Uca may be 
taken from room temperature and placed in a 
constant temperatured darkroom at any tempera- 
ture between 6 and 26° C. and in every instance 
the rhythm retains very precisely its twenty-four 
hour frequency. Despite the temperature inde- 
pendence the rhythm appears to be based upon 
a metabolically operated mechanism. This is in- 
dicated by such an observation as the result of 
chilling animals in a darkroom for several hours 
at 0 to 3° C. When the animals are rewarmed 
the rhythm immediately returns as a twenty-four 
hour cycle, but one that is now permanently out 
of phase with the earlier rhythm by a length of 
time approximately equal to the period of chill- 
ing. 

The temperature-independence of the rhythm 
through such a wide range of temperatures is 
most remarkable, and, I believe, a unique situa- 
tion in biology. It is a phenomenon whose ex- 
planation ean scarcely be postulated at the pres- 
ent time. It seems obvious, however, that there 
must be within this poikilothermic crab, Uca, a 
very precise temperature-compensating mech- 
anism. 

Recent work has given us some information as 
to characteristics of response of the rhythmical 
mechanism and some interesting suggestions as to 
the nature of its organization. In constant illumi- 


12 THE COLLECTING NET 


[Vol. XIX, No. 1 


nation the rhythm of color change in Uca gradu- 
ally becomes weaker and weaker and after four or 
five days the rhythm is completely lost and the 
animal remains continuously dark. The rhythm 
in such inhibited animals will immediately reap- 
pear. upon placing the animals in constant dark- 
ness. The frequeney of this restored rhythm is 
exactly twenty-four hours but the phase is de- 
termined by the time the animals are placed in 
the darkness. If such inhibited animals are 
placed in darkness at 7 a.m. the rhythm will be 
approximately 6 hours out of phase with the 
normal day-night cycle; if, however, they are 
placed in darkness at 1 p.m., 7 p.m., or 1 a.m., 
they are completely in phase. These results in- 
dicate clearly that the light-inhibited crabs must 
still possess a twenty-four hour rhythmicity 
which is expressed here as a cycle of sensitivity 
change. Furthermore, it is seen that a single 
light change if administered during a critical 
period, can abruptly alter the cycle. 

The rhythm.of color change in Uca ean be re- 
versed by illumination at night and darkness by 
day. This reversal is characterized by a gradual 
inhibition of the rhythmic color changes over a 
two- or three-day period and then a gradual in- 
crease in amplitude of the rhythm in its new and 
reversed phase. This rhythm can be restored to 
its normal phase by returning the crabs to the 
normal day-night environment. The return is 
again characterized by an initial period of in- 
hibition and after a few days the resumption of 
the original rhythm. 

The responses of the rhythm to alternating 
six-hour periods of light and darkness are inter- 
esting and instructive ones. If the crabs are 
illuminated between 7 a.m. and 1 p.m. and be- 
tween 7 p.m. and 1 a.m. and darkened during the 
intervening periods, the cycle of color change is 
abruptly thrown six hours out of phase with the 
previous one. In this case there is no initial 
inhibition. The behavior of the rhythm upon 
placing these animals in constant darkness now 
depends upon the time of the last period of illu- 
mination. If the last illumination period was 
7 p.m. to 1 am. there is a gradual creeping of 
the rhythm to its original phase over a four- to 
five-day period in darkness. In previously re- 
versed animals, there is a comparable gradual 


return to the reversed rhythm when the last 
period of illumination was 7 a.m. to 1 p.m. On 
the contrary, if the last illumination was 7 a.m. 
to 1 p.m. ( or 7 p.m. to 1 a.m. in reversed ani- 
mals) the return to the earlier phase of the 
rhythm is immediate. 

These last two types of experiments appear to 
indicate two things: (1) the influence of one of 
the six-hour light periods is fully cancelled by 
the second, and therefore only a single light 
period induced the slowly transitory alteration, 
and (2) there are two centers of rhythmicity 
with only one of them altered here; an unaltered 
one gradually restores the original phase in this 
instance. 

All of the characteristics of responses of the 
rhythmical mechanism of Uca appear to be sim- 
ply explained in terms of such an hypothesis as 
the following. There are two rhythmical centers 
operating in the maintenance of the normal 
rhythm of color changes. One of the centers 
which might be referred to as Center I possesses 
a deep-seated twenty-four hour rhythmicity 
which persists throughout the life of the animal. 
A second center, Center II, also possesses a 
rhythmicity but this latter rhythm persists for 
only a few days in the absence of influence from 
Center I. Continuing with this hypothesis, light 
acts in such a manner as to inhibit the rhythmic 
influence of Center I on Center II. Center II, 
thus cut off from Center I, gradually loses its 
rhythm. Center II can not alter the phase of 
of Center I but center I can gradually alter Cen- 
ter II when the later is out of phase with the 
former. 

The evidence appears to indicate that each of 
the two centers may have its rhythm abruptly 
altered independently of the other by light stim- 
uli of specific sorts presented during sensitive 
periods in its eyele. Those stimuli such as re- 
versal of illumination, and placing animals which 
had been inhibited, into darkness at 7 a.m. must 
have induced an alteration in Center I. Transi- 
tory alterations such as that induced by a six- 
hour period of illumination from 7 p.m. to 1 
a.m., would be presumed to have effected an 
alteration in Center IT. 


Nore: Based on a lecture presented at the Marine 
Biological Laboratory. 


LABILE P IN NUCLEIC ACIDS 


By Apet LaJTHA 
Marine Biological Laboratory 


There is a striking parallelism between muscle 
and other organs, for example, kidney and liver. 
If we let rabbit muscle or any other kind of mus- 
cle stand at room temperature, the A7'P in it is 


eradually split; parallel with the decreasing 
ATP concentration, the elasticity of muscle fibers 
decreases, a stiffness gradually develops (rigor 
mortis), and the solubility of the highly viscous 


November, 1949 | 


THE COLLECTING NET 13 


muscle protein actomyosin decrease. 

The post mortem changes in kidney are anal- 
ogous. If we mix fresh minced kidney with 
strong salt solutions, a greatly viscous extract is 
obtained ; the sticky solution shows a strong dou- 
ble refraction of flow. If, before extraction of 
the kidney, we let it stand at room temperature 
for about half an hour, the viscosity of the solu- 
tion will be very small, there will be no DRF, 
and it does not appear sticky—showing that only 
very small amounts of the kidney structure-pro- 
tein went into solution, if any did at all. 

In the muscle the changes are mostly restored 
by adding physiological amounts of ATP. Even 
large amounts of ATP don’t restore fhe lost 
solubility of structure proteins in kidney. 

The analysis shows that compared with the 
muscle, there is only about one tenth as much 
ATP in the kidney. The question arises whether 
in kidney the nucleic acid plays the role plaved 
by ATP in muscle. The first question in ap- 
proaching this problem is whether nucleic acids 
contain labile P. 

Nucleic acids were prepared from kidney and 
liver in three different ways. In one set of ex- 
periments emphasis was laid on purity of the 
product, in another on quantitative yields, and 
in the third on mildness of the method avoiding 
all possibility of hydrolysis. 

To prepare pure nucleic acids the organs were 
washed with cold trichloracetic acid, then with 
hipoid solvents and finally with strong NaC1 solu- 
tion reprecipitated with acids several times and 
washed with lipoid solvents again several times 
at pH 2.5. As in the other methods, I followed 
the purification with pentose and desoxypentose 


tests and with ultra-violet absorption spectra. 
With this type of reprecipitation, we get pure 
nucleic acids very fast, and working at low tem- 
perature, we can retain almost all labile P. 

To get quantitative results, I extracted the or- 
gans with hot NaCl solution containing five per 
cent, NasCOs, extracting three times for about 
15 minutes. The analysis of the remainder 
showed that about 98 per cent of the P contain- 
ing compounds were dissolved. Precipitation 
was made complete with the combined action of 
acid and alcohol. This method, however, must 
be corrected, as experiments with nucleic acids 
prepared in another way and after being boiled 
in basic NaCl solution showed that about ten 
per cent of the labile P is split off by 45 minutes 
of boiling in a salt solution containing five per 
cent Na»COs. ; 

To work as fast as possible and retain all labile 
P groups, the organ was washed with cold alco- 
hol and then with water and then extracted in 
many ways. One of the methods used, for ex- 
ample, was just washing it with hot water. All 
the extracting solutions were then analysed for 
nucleoprotein and labile P afterwards. 

The result of these experiments is that the 
nucleic acids of kidney and liver contain a labile 
P which is hydrolysed in normal acid in ten min- 
utes and which amounts to about 20 per cent of 
the total P. 

This would show that for approximately every 
tetranucleotide unit there is one labile P in the 
nucleic acids. 

Nore: Based on a paper presented at the Marine Bio- 
logical Laboratory. 


ON THE STRUCTURE OF FIBRIN CLOTS 


Dr. Evemeér MInALyI 


Research Assistant at the Institute of Muscle Research, Marine Biological Laboratory 


The mechanism of the transformation of fi- 
brinogen into fibrin is largely unknown. Several 
theories were formulated to explain the action 
of thrombin, but none of them proved to be satis- 
factory. Such a fundamental question as whether 
the fibrin molecules are bound together through 
co-valent bonds, or whether they are held only by 
weak forces, like the van der Waals forces, or 
hydrogen bonds is still unanswered. 

In the present investigation attempts were 
made to solve this question by studying the solu- 
bility of fibrin clots in urea solutions. 

Several investigators of the late nineteenth 
Century reported the solubility of fibrin clots in 
concentrated urea solutions..? Wohlisch and 


his co-workers,*: + however, could not confirm this 
finding. The problem has considerable import- 
ance, because the protein gels and coagula, where 
the particles are bound by weak secondary forces, 
are all soluble in urea solutions. Insolubility may 
thus be an indicator of co-valent bonds between 
the particles.° 

Lorand,® reinvestigating the problem, found 
fibrin resulting from the action of thrombin upon 
pure fibrinogen to be readily soluble in urea solu- 
tions. On the other hand, fibrinogen clotted by 
thrombin in the presence of serum and calcium 
ions, was insoluble. This finding may explain the 
contradictory results of different authors. 

In our investigations the solubility of pure 


14 


THE COLLECTING NET 


(Vol; XLX Now 


fibrin clots was confirmed, and the reversibility 
of the dissolution demonstrated. These facts 
seem to justify the conclusion that by the clot- 
ting process no co-valent bonds are formed. 

When urea is dialysed from a fibrin solution 
the clot is reconstituted. Similarly, if the fibrin 
solution is diluted with distilled water, the urea 
concentration being lowered in this way, the solu- 
tion suddenly undergoes gelification. The plIl 
of the fibrin solution during the dialysis decides 
whether a coarse or a fine type gel will be 
formed, 

The dissolving effect of urea depends on its 
concentration and the pH of the system. It is 
easier to investigate the interaction of these fac- 
tors by studying the inverse phenomenon 7.e. the 
gelation of a fibrin solution. The fibrin dissolved 
in urea solution was mixed with buffers of differ- 
ent pH and distilled water in order to have dif- 
ferent urea concentrations and pH. The viscosity 
of the solutions was determined. At 30% urea 
concentration, over the entire pH range studied, 
the viscosity of fibrin solutions did not differ 
from that of fibrinogen in similar conditions. The 
solutions had Newtonian viscositv and showed 
no double refraction of flow. At 20% urea con- 
centration the viscosity rose at pII values more 
alkaline than 7.6 and reached a maximum at pIl 
8.6; it then decreased again to the original value 
by further alkalinisation. At still lower urea 
concentrations the increase of viscosity started at 
a pl] which was the more acidic the lower the 
urea concentration was and finally led to a gela- 
tion of the solution. In the region of rising vis- 
cosity the solutions became thixotropic; at the 
same time a strone double refraction of flow 
appeared. 

Urea affects the electrostatic forces between 
charged groups by increasing the dielectric con- 
stant of the medium. Although this effect may 
have some importance, it is far from being the 
cause of the dissolution of fibrin. Dipolar ions, 
like elycine for example, have a much greater 
effect on the dielectric constant. In spite of this, 
a fibrin solution in 15% urea, when diluted with 
2/M elycine, gelified exactly at the same degree 
of dilution as when it was diluted with distilled 
water. 

Most probably urea affects the hydrogen bonds 
of the protein molecules formed between the 
-NH- and -CO- groups of adjacent polypeptid 
chains. If this mechanism is really the cause of 
the dislocation of the fibrin particles, the hydro- 
gen bonds must play a considerable role in the 
building of the gel. 

The viscosimetric behavior of fibrin solutions 
in 30% urea indicates that the particles, in re- 
spect to their shape and size, are probably identi- 
cal with those of fibrinogen. Thrombin does not 


alter tie shape and size of the fibrinogen mole- 
cules; it only modifies some of their physico- 
chemical properties. 

The solubility of fibrin in urea solutions makes 
it possible to investigate the electrophoretic mo- 
bility of this protein. If the action of thrombin 
involves some of the ionizine groups of fibrino- 
gen, a study of the differences in electrophoretic 
mobilities between fibrinogen and fibrin may give 
some information about the nature of the process 
of clotting. 

The electrophoretic mobility of fibrinogen and 
fibrin, dissolved in 20% urea, was determined at 
different pH in the Tiselius apparatus. It was 
found that the mobility curve of fibrin is always 
below that of fibrinogen, 7.c., in the region more 
alkaline than the isoelectric point the fibrinogen 
is the faster, whereas in the region acidic to the 
isoelectric point fibrin is the more rapid; the iso- 
electric points of the two proteins are very close 
together. Fibrinogen is isoelectric at pH 5.5, 
fibrin at 5.6. 

The mobility differences of the two proteins 
are very small. To exclude the possibility that 
the observed differences were due to experimen- 
tal error, control runs were made with a mixture 
of fibrinogen and fibrin at each pH studied. The 
two components separate slowly in conformity 
with the mobility differences obtained in runs 
with a single component. 


The results indicate that the net charge of 
fibrin in the zone of clotting is lower than that 
of fibrinogen. The lower charge favors the ap- 
proach of the fibrin particles and thus their 
binding. The decrease of the net charge may be 
the result of an increase in the free NH» groups 
of the protein. If we suppose that the fibrin 
particles are bound by hydrogen bounds between 
-NHo»- and -CO- groups, the increase in the num- 
ber of free NH.» groups will favor also the gela- 
tion process. 

The results are in accord with our earlier in- 
vestigations’ in which we were able to demon- 
strate the role of NH» groups in the process of 
clotting. 


References 


1. Ph., Limbourg Z. Physiol. Chem. 13 450. 1889. 
2. K., Spiro Z. Physiol. Chem, 30 182. 1900. 


3. I., Meissner, and E., Wohlis ch Biochem. Z. 293 
133. L937. 

4. W., Diebold, and L., Jihling Biochem. Z. 296 389. 
1538. 


5. J. D., Ferry, and P. R. Morrison J. Am. Chem. Soc. 
69 388. 1947. 

6. L., Lorind Hungarica Acta Physiol. 1 194. 1946-48. 

7. E., Mihdlyi, and L., Lorand Hungarica Acta 
Physiol. 1 243, 1946-48. 


Note: Based on a report presented at the Marine Bio- 
logical Laboratory, August 2, 1949. 


November, 1949 | 


THE COLLECTING NET 15 


INVESTIGATIONS ON MUSCLE FIBERS 


ANDREW G. SZENT-GYORGYI 


Research Assistant at the Institute for Muscle Research, Marine Biological 


The elycerinated muscle preparation described 
by Dr. A. Szent-Gyoreyi (Biological Bulletin 
36, 140, (1949) eives an excellent material for 
the study of muscle contraction itself, without 
being influenced by conductivity and transmis- 
sion. The question is how far the behaviour of 
this muscle preparation corresponds to that of 
the actomyosin thread. 

The material used consisted of thin bundles 
of the rabbit psoas washed with 50% elycerol 
for 2-9 days. The muscle preparation was made 
to contract by placing it in 0.2% ATP plus vary- 
ing NaCl concentrations at pH 7.0 in the pres- 
ence of 0.0015 MZ MeCls. There was no difference 
between Na or K. 

According to the experiments of our laboratory, 
in the presence of ATP, actomyosin can only be 
either in the contracted or in the dissociated 
state. At low NaCl concentrations (0.05 — 0.2 
M NaCl) the actomyosin thread is maximally 
contracted. As one increases the salt concentra- 
tion a critical point is reached, at which contrac- 
tion no longer occurs, instead the actomyosin 
dissolves and dissociates, as shown by its low 
viscosity. In the case of the actomyosin thread 
this critical point falls to 0.2-0.22 M NaCl con- 
centration. re 

Repeating the same experiment with, muscle 
fibers washed with glycerol the results are quite 
different. The contraction of the fibers is prac- 
tically independent of salt concentration and 
maximal contraction is obtained by varyine the 
salt concentration from 0.015 up to 0.5: M NaCl, 
the highest salt concentration used, The’ degree 
of contraction amounted to 60-70% of the oriei- 
nal leneth at every salt concentration investi- 
eated. The close packing and the prolonged 
washing;in glycerol stabilizes the actomyosin in 
the fibers so much that ATP could not dissociate 
it even at high salt concentrations. If the acto- 
myosin has existed in the musele fibers in the 
associated ‘state for some time it would become 
very stable and hence would contract in the pres- 
enee of ATP over a wide range of salt concentra- 
tion. 

If we want to start with a muscle where acto- 
myosin is dissociated we have to loosen the strue- 
ture by high concentration of ions which have a 
specific dissociatine action. This can be done for 
instance with 0.1 JJ Na-pyrophosphate at pH 7.5, 
or with 0.1 WM NaHCOsz in the presence of 0.5 M 
NaCl. or with 0.4 12 NaOCN at pH 8.8 or with 
0.05 M Na-tri-phosphate of pH 7.5. Treating the 


Laboratory 


fibers with these solutions there is a pronounced 
difference in physical appearance. The originally 
opaque, white, completely inelastic and brittle 
muscle becomes transparent and slightly elastic. 
The effect takes place within 1 minute, thoueh 
usually 4 minutes incubation time was used. (Im- 
mersine the fibers into these solutions brings 
about a slight shortening in the absence of A7'P 
too, which does not exceed 10-15% of the original 
leneth. This was kindly observed by Dr. A. G. 
Matoltsy under the microscope; it is most prob- 
ably due to the dissociation of the rigid acto- 
myosin structure. This shortening was not taken 
into account in these measurements.) The fibers 
after treatment with pyrophosphate, or with the 
solutions described above, behave like fresh acto- 
myosin or perhaps like fresh muscle. The con- 
traction depends on a critical salt concentration 
between 0.1 and 0.2 M NaCl. 

Below and above this salt concentration range 
there is no contraction. At 0.015 M NaCl con- 
centration the fibers remain relaxed in spite of 
the presence of ATP. Between 0.1 and 0.2 M 
NaCl the fibers contract maximally. The upper 
salt limit of contraction varies somewhat and is 
around 0.25 WM NaCl. The transition to contrac- 
tion is usually sharp, though the single points 
differ only by 0.05 M NaCl. ATP does not cause 
the contraction of the fibers treated with pyre- 
phosnbate at higher salt concentrations. The con- 
traction of the pyrophosphate treated fibers oc- 
curs thus in two steps. The first one is the acto- 
myosin formation, the second the contraction due 
to the effect of ATP. 

The dissociating action of pyrophosphate is re- 
versible. If we put the pyrophosphate treated 
muscle into 0.1 M NaCl for about 10 minutes 
in the absence of AT7'P, it begins again to behave 
as eglycerinated muscle untreated with pyrophos- 
phate. That means the fibers contract again at 
every salt concentration employed, though over 
0.4 M NaCl concentration the contraction is not 
maximal, showing that the actomyosin formed 
in 0.1 MW NaCl is not as stable as the actomvosin 
formed during the prolonged washing in ely- 
cerol. The same reversal effect can be obtained 
by puttine the muscle into 50% elvcerol after 
pyrophosphate treatment, though here a longer 
incubation time (about 30 minutes) is needed. 

Starting with the elycerinated muscle we have 
actomyosin in a very stabilized form, after pyro- 
phosphate treatment we have fibers where the 
actomyosin is dissociated. One can study separ- 


16 THE COLLECTING NET 


[Vol. XIX, No. 1 


ately the actomyosin formation and the eontrac- 
tion by the aid of these preparations. £.g., 
NaHCO; does not inhibit the contraction, even 
at 0.1 M concentration, the glycerinated fibers 
contract maximally. After pyrophosphate treat- 
ment the contraction is inhibited in the presence 
of 0.03 M NaHCOs, showing that NaHCO; in- 
hibits actomyosin formation. 

The results indicate at least two different steps 


in the contraction of muscle after pyrophosphate 
treatment. The first is actomyosin formation. 
That depends on salt concentration. The second 
is the contraction itself, which does not depend 
on salts. ATP causes contraction only after ac- 
tomyosin is formed or under conditions, where 
formation of actomyosin is favored. 


Nore: Based on a report presented at the Marine Bio- 
logical Laboratory. 


EVIDENCE FOR ACTIVITY OF DNAse IN MITOSIS BY USE OF d-USNIC ACID 


3y Dr. ALFRED MARSHAK 


Research Associate in Biochemistry, New York University School of Medicine 


Along the coast of California there is a good 
deal of fog during the summer. In the neighbor- 
hood of Monterey, the fog comes in almost every 
night and remains for a good portion of the day. 
A lichen (Ramalina reticulata) grows in abund- 
ance in this foe belt as an epiphyte, especially 
on oaks. It absorbs both moisture and nutri- 
ment from the air. About the surface of the 
lichen and between the hyphae of its fungal com- 
ponent there is a carbohydrate material which is 
very hygroscopic so that in the fog the lichen 
acquires an almost gelatinous consistency. Since 
this provides almost ideal conditions for the 
erowth of bacteria which would decompose the 
lichen, the very existence of the plant implies 
its possession of some chemical defense against 
bacterial invasion for it appears to have no physi- 
cal protection such as an epithelium. Explora- 
tory investigations confirmed this hypothesis; 
finally a crystalline substance with marked anti- 
bacterial properties was isolated from the lichen 
and was shown to be d-usnie acid. This substance 
is present in a good many of the Uneaceae found 
throughout the world. It was first isolated by 
Stenhouse in 1848. Its structural formula has 
been tentatively established although not con- 
clusively proved by Curd and Robertson and by 
Asanina in a series of papers, 1933-37. 

When tested against bacteria in vitro, it shows 
some activity against gram positive bacteria and 
a few gram negative ones but is most active 
against mycobacteria and especially active 
against the human tubercle bacillus. 

Work done at Woods Hole in 1947 with Miss 
Jane Harting showed that as little as 10 pg/ml 
would inhibit cleavage and 1 pg/ml would pro- 
duce very abnormal cleavage in Arbacia eggs. 
No effect on O» consumption could be observed 
with 100 pe/ml although this concentration com- 
pletely inhibited P*? uptake. 

The inhibition of cleavage was investigated 
further and it was found that in the presence of 


10 pg/ml the sperm nucleus reached the egg nu- 
cleus at the same time as in the controls, 1.e., be- 
tween 8-12 minutes after fertilization. Some eges 
were fixed in Bowin’s solution for eytological 
observation, sectioned and stained with hema- 
toxylin. Another portion was fixed in 5% citric 
acid, the remaining pigment extracted with 5% 
citric in 30% aleohol and finally lipids removed 
by boiling in acetone: alcohol. After the lipid 
extraction they were stained en masse by the 
Feulgen technique and examined with and with- 
out counterstain. 

The ege nucleus did not stain with the Feul- 
gen, neither did the nucleus of the unfertilized 
ege. Sperm were bright red as were the chromo- 
somes in metaphase and anaphase. In the two- 
cell stage and subsequent interphases, the nu- 
cleus had a faint but definite magenta color. 
Later interphase nuclei had more color than 
earlier ones. 

As the sperm nucleus approached the egg nu- 
cleus it became slightly larger and stained some- 
what less intensely. On fusion with the egg 
nucleus, the intensity decreased and finally be- 
came indistinguishable from the egg chromatin 
which was colorless. 

In the presence of usnic acid the sperm nu- 
cleus reaches the ege nucleus in the same time 
as in the controls but does not fuse with it; neith- 
er is there any decrease in intensity of its Feul- 
gen stain. It may remain in this condition for at 
least four hours. This observation suggests that 
usnic acid may interfere with whatever mechan- 
ism in the sperm or egg is concerned with dis- 
persion of desoxyribonucleic acid. To determine 
whether this might be desoxyribonuclease, ex- 
periments were conducted on the activity of 
DN Ase in the presence and absence of usnic acid. 

The DNA used was prepared by the method 
of Gulland, N/P = 1.66; another batch was pre- 
pared by the method of Greenstein, N/P = 1.87, 
containing some contaminating protein. Two 


November, 1949] 


THE COLLECTING NET 17 


batches of DN Ase were also used, one which was 
non-crystalline obtained from Dr. M. McCarty 
and another crystalline preparation obtained from 
the Worthington Chemical Company. The latter 
showed marked activity at 0.1 pe/ml; the former 
only at 1 pg/ml. The index of activity used was 
whether or not a minimal concentration of en- 
zyme would or would not decrease the viscosity 
of a solution of DNA. Measurements were made 
in veronal buffer at pH 7.8 containing .001 M. 

As little as 1 ng/ml of usnic acid will produce 
partial inactivation of the system DNA-DN Ase 
and 10 pg/ml gives complete inactivation. It was 
found also that the inactivation requires the pres- 
ence of cobalt in the reduced form (CoCl, .001- 
.002 M) and that this effect varies with the con- 
centration of usnate. In the absence of usnate 
cobalt has no effect on enzyme activity. The in- 
activation cannot therefore be due simply to re- 
moval or inactivation of Mg by Co, but is a con- 
sequence of formation of complexes involving 
Co, usnate and enzyme or substrate. 

Seymour Cohen showed that streptomycin 
forms a complex with desoxyribonucleie acid, re- 
sulting in turbidity of the solution. Sodium 
usnate was found not to produce such complexes 
as Cohen described. It was also found that strep- 
tomycin in concentrations up to 100 pg/ml in the 


presence or absence of cobalt will not inactivate 
the system DNA-DNAse. The results thus show 
that the usnate inactivation is produced by com- 
plex formation with the enzyme rather than the 
substrate. 

Since usnie acid will also inhibit further cell 
division if added after cleavage has begun, it 
follows that desoxyribonuclease or a similar en- 
zyme is involved in the mitotic process as well 
as in the fusion of the sperm and egg nuclei. 

It is perhaps significant that the concentration 
required to inactivate the enzyme is the same as 
that required to inhibit cleavage in sea urchin 
eges and to prevent the growth of tubercle bac- 
illi. Although it may seem a far ery from sea- 
urchins to lichens, these findings suggest a pos- 
sible role of usnic acid in the lichen in which it 
is found. The lichen is composed of an alga and 
fungus growing together, neither of which can 
outgrow the other without destroying the sym- 
biont relationship. From the experiments de- 
scribed it is apparent that usnic acid could serve 
as a growth inhibitor of the alga, while the rate 
of fungal growth could of course be limited by 
the aleae photosynthetic activity. 


Nore: Based on a paper presented at the Marine Bio- 
logical Laboratory. 


COLD AS A MEANS OF COMBATTING ASPHYXIA IN NEWBORN GUINEA PIGS 


JAameES A. MILLER, JR. 
Emory University 


Maternal mortality statistics associated with 
childbirth have been reduced in the last few 
years to a negligible figure. The medical pro- 
fession has not succeeded quite as well in pre- 
venting death of the baby during the first year 
of its life, and in fact there has been no real im- 
provement in mortality statistics for infants 
during the first 24 hours after birth. 

The largest single cause of death of full term 
infants found at autopsy is asphyxiation with 
frequencies up to 68% reported by various au- 
thors. Therefore, any means which will enable 
fetuses and newborn babies to resist anoxia may 
result in an appreciable saving of lives during 
the most hazardous period of postnatal existence. 

There are clinical indications and experimental 
proof in guinea pigs (Windle and Becker, 1942a, 
19426) that the sequel of neonatal asphyxia are 
not transitory but may persist into adulthood. 
They range from patent neurological symptoms 
to mental dullness which may be too subtle to be 
detected except in carefully controlled experi- 
ments. Accordingly, any treatments which enable 
the newborn to resist the effects of anoxia may 


aid in reducing the numbers both of individuals 
with frank neurological symptoms and of men- 
tally subnormal individuals. 

The standard treatment for anoxic babies in- 
cludes, among other procedures, the maintenance 
of body temperature by placing them in a 
warmed bassinet or incubator until normal res- 
piration is established. So far as they have been 
studied, all newborn mammals (including the 
human) are poikilothermic, with body temper- 
ature varying with ambient temperature. Since 
there is a close relationship between temperature 
and rate of chemical reactions, and since, accord- 
ing to van’t Hoff’s rule, rates of most reactions 
are doubled or trebled with each 10°C rise in 
temperature, it appeared to the writer that 
changing the temperature of the anoxic fetus or 
newborn might alter the metabolism of vital cen- 
ters sufficiently to appreciably affect survival. 
This line of reasoning raised grave doubts con- 
cerning the rationale for maintaining the anoxic 
baby at a high body temperature and thereby in- 
creasing its need for oxygen at a time when its 
energy supplies are so low as to threaten life 


18 THE COLLECTING NET 


[Vol. XEX, No. 1 


itself. Accordingly, experiments were instituted 
to test the leneth of survival of anoxic animals 
at various temperatures. 

Material: Guinea pigs were used because of 
their size and advanced condition at birth (more 
nearly resembling the human than do rats, mice, 
rabbits. or hamsters) and because they were used 
by Windle and Becker in their demonstration 
that asphyxia at birth produced lesions and gen- 
eralized atrophy in the nervous system. 

Methods: Litter mates 24 hours old or less were 
tested either at room temperature, after warming 
in an incubator, or after cooling by the evapora- 
tion of alcohol or by immersion to the neck in ice 
water. Cooling usually required five minutes or 
less. Temperatures were determined with a 
U.M.A. skin thermocouple modified so that is 
could be inserted 2 em. into the colon. Animals 
were tested in pairs in a bell jar through which 
95% No + 5% COs mixture was flowing. Com- 
plete records of the behavior of each animal were 
kept, including the time of each gasp. Autopsies 
were performed on all animals which died. 

Results: Preliminary experiments (Miller, 
1949) had shown that is was possible by cooling 
to’save the lives of littermates of animals killed 
by 414 minutes exposure to 95% Ne + 5% COs. 
Likewise, when all animals in a litter were ex- 
posed until death the cooled lived longer than the 
untreated while the warmed animals died in the 
shortest time. 

Next, 160 animals were tested in order to de- 
termine effects of temperature upon anoxic sir- 
vival over a wider range than was used in the 
preliminary experiments. When averaged to- 
gether in the 3° classes, a 185% increase in mean 
survival time is noted between 37.5°C and 
11.0°C. This represents an increase of 7.1% for 
each degree or 71% for each 10° decrease in 
temperature. However, when the data are re- 
corded in three groups according to the time of 
year during which they were obtained, there are 
striking differences in survival of animals at the 
same temperature. The averages in experiments 
21 through 51 (when mean daily temperatures 
were approximately 80°F.) at every temperature 
were ereater than either those in experiments 
1 through 10 or 11 through 20. Similarly, ex- 
periments 1 through 10 (performed between 
March 5 and 28 when mean daily temperatures 
were in the low fifties) gave the shortest survival 
times. The means of experiments 11 through 20 
in general were intermediate, as were the mean 
daily temperatures. These results are of interest 
in the light of the well known fact that the thy- 
roid is larger and more active in cool weather, 
and that basal metabolism is higher in winter 


and lower in summer. Below 11°C cold itself 
begins to be lethal under conditions of these ex- 
periments. 

Since it seemed possible that the temperature 
regulating mechanisms of the adult might be 
rendered inoperative by anoxia as are all visible 
reflexes, a series of 34 experiments was per- 
formed to test the effects of temperature upon 
young adult animals (291.4 - 321.2 ems.), At the 
temperatures tested these animals lived approxi- 
mately one half as lone as the day old animals. 
Decreasing colonic temperatures was not quite 
as effective in prolonging anoxic survival as it 
was In newborn animals. <A 10° decrease in tem- 
perature increased survival almost 50%, a 16° 
decrease increased survival 75%. These results 
are in accord with the recent report on adult rats 
by Blood and d’Amour (1949). They found that 
the highest incidence of recovery from anoxia 
produced by simulated high altitudes was in the 
group subjected to low temperature. Thus, we 
may conclude that in spite of the presence of well 
developed temperature regulating mechanisms, 
adult mammals subjected to severe anoxia behave 
like poikilothermic animals, and reduction of 
temperature may be expected to prolong life. 

Cooline a conscious animal stimulates ereat 
motor activity which reduces the supply of com- 
pounds available for anwerobic metabolism during 
a subsequent period of anoxia. To reduce this 
activity nembutal was given one half hour before 
testing. In a series of 20 neonatal animals the 
mean seconds survival of nembutal treated ani- 
mals was longer for each 2° class than that of the 
untreated series. This suggests that cold may be 
especially effective in protecting the asphyxiated 
human infant that is often partially anesthetized 
in addition. 

A similar series of experiments to test the 
effects of temperature on survival of day old 
rabbits which was started this summer has heen 
showing even more striking results. Another ani- 
mal in which cold is effective in prolonging life 
of the anoxic newborn is the rat (Adolph, Dee. 
1948). Neonatal animals of this species tolerate 
a temperature of 10°C and at this temperature 
will survive two hours exposure to nitrogen. 

Though still not quite complete, these experi- 
rents are being reported at this time because 
they suggest that the present treatment of warm- 
ing the anoxic baby may explain in part the lack 
of improvement, during recent years, in mor- 
tality statistics of infants during the first day or 
two after birth. 

This work has been supported by a grant (#RG-281) 


from the Division of Research Grants and Fellowships of 
the National Institutes of Health. 


Nore: Based on a report presented at the Marine Bio- 
logieal Laboratory. 


November, 1949 | 


THE COLLECTING NET 19 


A SIMPLE, NON-INJURIOUS METHOD FOR INDUCING REPEATED SPAWNING OF SEA 
URCHINS AND SAND-DOLLARS 


Dr. ALBERT TYLER 
California Institute of Technology, Pasadena, California 


This note is for the purpose of acquainting 
investigators, usine sea urchins or sand-dollars, 
with a simple rapid method of obtaining eggs 
and sperm repeatedly without injury to the ani- 
mals or the gametes. It involves simply inject- 
ing isosmotie KCl into the animals. 

Palmer (1937), at the suggestion of L. V. 
Heilbrunn, tested solutions of various salts and 
extracts of various tissues of Arbacia and other 
animals for their ability to cause the gonads of 
ripe Arbacia to shed their gametes. Her method 
consisted of making two slits in the peristome 
and introducing measured amounts of the solu- 
tions through one of these slits. She found that 
isosmotie solutions of KCl and CaCl, were effee- 
tive. E. B. Harvey (1940) described a method 
of determining the sex of Arbacia that involved 
injecting a drop of sea water saturated with 
KCl into one of the gonopores by means of a 
fine hypodermic needle (No. 27). Eges or sperm 
exude almost immediately from the injected 
gonopore but the shedding can be stopped at 
once by placing the animal in a jar of still sea 
water. So, males and females can be separated 
and be made available for later use. 

I have, for the, past 12 years, used a slight 
modification of the above methods for the pur- 
pose of inducing spawning in various echinoids ; 
including Arbacia and Echinarachnius at Woods 
Hole and Strongylocentrotus (2 species), Lyte- 
chinus (2 species), Dendraster and Lovenia, on 
the West Coast. The method involves simply in- 
jecting isosmotic KCl with a hypodermic syringe 
into the body cavity. It is preferable that the 
needle be inserted into the lantern coelom since 
it may pick up eggs or sperm if it enters the 
gonad in the perivisceral coelom and then re- 
quire washing before use on another animal. A 
single injection of 0.5 ec. of 0.5 M KCl into an 
average size Arbacia, of about 30 cc. volume will 
induce shedding of virtually all the ripe eggs or 
sperm. For larger or smaller animals the dose 
should be proportionately larger or smaller. The 
shedding starts within a few seconds(and is com- 
pleted in five to fifteen minutes. nly a few 
eggs or a small amount of sperm are desired, 
correspondingly smaller amounts of KCl should 
be injected. The same animals will then be avail- 
able for further material at later times. The 
eggs can be collected readily by immersing the 
aboral surface of the animal in a dish of sea 


water. The sperm can be removed ‘‘dry’’ or by 
similar shedding in sea water. 

Sand dollars are conveniently injected by in- 
serting the needle through the mouth in a direc- 
tion as nearly parallel as possible to the oral 
surface of the animal. 

The gametes are not injured by this method 
of inducing spawning. Also, the animals can re- 
evenerate egos and sperm after the treatment. I 
have, with Lytechinus and Strongylocentrotus, 
been able to obtain successive new batches of eggs 
at two-week intervals for four weeks during their 
breeding seasons, after initial forced shedding of 
practically all their ripe eggs (as determined by 
opening control animals) and feeding them on 
eel erass, kelp, mussels and other marine plants 
and animals. 

This method of obtaining the gametes is less 
troublesome than that of opening the animals. 
Also the eges do not need to be strained from 
eonadal tissue and very few, if any, unripe ege's 
are obtained. Unripe animals generally fail to 
respond. The injection is conveniently made 
with a 2 ml syringe and the needles should be 27 
to 25 gauge and 1% to 34 inch. 

It has been shown by Oshima (1921), Harvey 
(1939) and Pequegnat (1948) that the test of sea- 
urchins contains material that is inhibitory to 
fertilization. This material is liberated by wash- 
ine the animals in fresh water, by drying the 
surface or by other procedures that injure the 
delicate epithelial covering of the test. The 
eametes, however, are not injured by this agent. 
Even when the material is present in high con- 
centration (as indicated by a_reddish-yellow 
color in Arbacia), a single washing of the eges 
removes it sufficiently to permit 100 per cent 
fertilization and normal development. 

At laboratories, such as the Marine Biological 
Laboratory at Woods Hole, where it is sometimes 
difficult to provide the tremendous numbers of 
sea-urchins that are requested, and where slaugh- 
ter of the animals might lead to a local deple- 
tion, this procedure would alleviate supply prob- 
lems by permitting repeated use of the same 
animals and their return to the natural habitat 
at the end of the season. 

During the current summer Arbacia has also 
shown itself to be capable of regenerating eggs 
and sperm and of yielding, after two weeks of 
feeding on sea weeds, about as many gametes as 
upon initial forced shedding. Smaller amounts 


20 THE COLLECTING NET 


[Vol. XIX, No. 1 


are obtained if the time interval is less than two 
weeks. One lot of Arbacia has furnished four 
successive batches of eggs in nearly original 
quantity in a period of six weeks, with most of 
the animals responding each time. 


References 
Harvey, E. B. 1940 Biological Bulletin, 79: 363. 
Harvey, E. B. 1939 The Collecting Net, 14: 180-181. 
Oshima, H. 1921 Science, 54: 578-580. 
Pequegnat, W. 1948 Biological Bulletin, 95: 69-82. 
Palmer, Louise. 1937 Physiol. Zool. 10: 352-367. 


BIOLOGICAL SPECIFICITY AND PROTEIN STRUCTURE* 


Dr. DorotHy WrRINCH 
Lecturer in Physics, Smith College 


Studies on the relation between biological 
function and biological form are already so far 
advanced that it is universally conceded that all 
biological specificities belong to the world of 
angstroms. There is no longer any doubt that 
the fundamental questions can be formulated, 
and subsequently studied and elucidated, only 
by recourse to the nature of atomic patterns and 
electron density distributions. 

The extent to which biological specificity de- 
pends upon the minutiae of atomic architecture 
(cp. Wrinch, Australian J. of Sci. 8:103. 1946) 
is well illustrated by the work of Zentmeyer 
(Science 100:214. 1944) on the inhibition of 
erowth of micro-organisms whose enzyme sys- 
tems depend upon the presence of metals. In this 
work use is made of the power of 8-hydroxyquin- 
oline to form chelate salts with a number of 
metals. It is known that this compound loses 
this power at lower pH’s and it is elegantly 
shown that its power to inhibit growth is re- 
stricted in a Gorresponding manner. 

A second example may be taken from the work 
of Rubbo, Albert and Maxwell (Brit. J. Lap. 
Path. 23:69. 1942). It is found that the anti- 
septic action of the 2-,3-,4- and 5- mono amino 
acridines is not present in the 1-mono amino 
acridine. The reason? The fact that in this one 
alone of the 5 compounds, an internal NH...N 
hydrogen bridge makes the amino group inop- 
erative. 

A third example may be taken from the studies 
of Chen and others on the elyco8ides, bufagins 
and bufotoxins. Such compounds share a com- 
mon steroid ring system with a lactone ring on 
Ci7z. It is recognized that the unsaturated lae- 
tone rine and the hydroxyl on Cy4 are essential 
features and it is now found that local stero- 
chemical changes in the ring system Have a re- 
markable influence on the pharmacological ac- 
tion of these substances. Only in this way can 
the fact that any digitalis-like action is missing 
in such compounds as allocymarin and allstroph- 
anthidin be explained. 

These examples show the kind of issues which 
present themselves in these specific biological ac- 


*This work is supported by the Office of Naval Research under 
contract NS8onr—579. 


tivities: chelate rings, hydrogen bridges, delicate 
stereochemical variations. They also point the 
way to a conclusion which accumulating evidence 
makes more and more inescapable: the view that 
the ultimate repository of biological specificity 
lies in the native proteins. The nature of the 
atomic patterns of native proteins must today 
be regarded as the major unsolved problem of 
biology and medicine—a problem whose solution 
is the necessary preliminary to any deep and 
satisfying progress in such vast fields as the ef- 
fects of radiation on living matter, virus diseases 
and the cancer problem. 

While it seems necessary to emphasize the im- 
portant fact that the atomic pattern of proteins 
is today unknown, equal emphasis should be 
given to the fact that there is a great store of 
factual information in other fields of work which 
is available to guide protein studies. For the 
native protein, unique in its functions and in 
the subtlety and precision of its architecture, car- 
ries on its surface and largely operates through 
end groupines which occur throughout organie 
and inorganic chemistry. Practically everyone 
of these has already been the subject of studies 
in various domains of structure chemistry and 
erystallography. The main issue throughout 
seems to be the matter of favorable environments 
for various atomie groupings and for the various 
foreign components, e.g. water, sugars and metal 
ions which play so important a role in protein 
specificity. It so happens that a great deal is 
known about the atomic environments favored 
by many ions of the ‘first importance in biological 
processes. What are the studies of the mica’ and 
clay groups of the silicates but a strikingly com- 
plete and vivid picture of the requirements of 
many such types? Isomorphous substitutions in 
the micas may well prove a valuable model for 
studies on the interchangeability (or the lack of 
it) of ions in systems involving enzyme systems. 
Permeability of cell membranes, primarily made 
up of native proteins in orderly network asso- 
ciations, attains new status, as a concept, when 
the data available are studied in close relation 
to knowledge of such framework structures as 
the ultramarines and noselite, with their vari- 
able ionic populations. Well understood today 


November, 1949 | 


THE COLLECTING NET 21 


are the lithium and thallium ultramarines with 
a warm violet hue, the nearly colorless calecium- 
and zine-containing structures, the silver vari- 
ants, which are yellow or grey, the blood-red 
selenium-containing and the yellow tellurium- 
containing ultramarines (cp. Brage’s ‘‘ Atomic 
Structure of Minerals’’). In all these cases, the 
framework is the common background. In a simi- 
lar manner, we may see the protein framework 
as the unifying theme in all the physiological 
situations which we have in mind. 

With this viewpoint, the oligodynamiec phe- 
nomena, so long studied at Woods Hole, fall into 
place. Perhaps the most striking case is that of 
the pining disease of sheep which for a century 
or more plagued farmers in Scotland and New 
Zealand. Extensive investigations finally uncoy- 
ered the cause—a cobalt deficiency in the soil— 
and the simple cure, a result of great interest in 
connection with the new vitamin of the B group. 
The amount of cobalt responsible for the differ- 
ence between a healthy and an unhealthy soil was 
found to be so small as to defy spectroscopic 
analysis. How then were such minute amounts de- 
tected? The answer is interesting: they were 
detected by the use of organic reagents, such as 
o-nitrophenol, capable of taking up cobalt ions. 
The balance to be maintained for health proved 
to be very delicate with a slight excess causing 
the sheep to contract polycythemia. But how can 
such minute amounts of cobalt be visualized as 
playing so vital a role? Evidently in terms of 
the concept of proteins having highly specific 
cavities or nests on their surfaces, which for sta- 
bility must be suitably tenanted by a very few 
specific foreign components including vitamins 
and metal ions (cp. Glaser, Am. Sci. 33:175. 
1945). 

For a fuller understanding of the nature of 
such active patches on native protein surfaces, 
at which key reactions are localized, we may turn 


to studies of molecular erystals which abound 
with examples of specific associations (ep. 
Wrinch, Wallerstein Comm, 11:175. 1948). I 
would particularly stress the symmetry elements 
present in many such situations. It seems inevi- 
table that the associations of the protein mega- 
molecules should require what physiologists (ep. 
Findlay et al. Biochem. J. 36:1. 1942) have 
called multipoint groupings. But multipoint 
groupings in association must in general depend 
upon a series of coincidences, since favorable en- 
vironments for each of the atoms involve definite 
conditions. When a symmetry element is pres- 
ent, multipoint groupings are automatically per- 
mitted. 

To understand biological specificity, not only 
problems of enzyme action but also problems of 
morphology must be elucidated by following them 
down through microscopic and sub-microscopic 
levels to the Angstrom world. Here the work of 
many pioneers will finally attain its greatest 
fruitfulness. We may particularly bear in mind 
the work of Moll whose concepts are the forerun- 
ners of modern ideas on morphogenetic fields and 
Frey-Wyssling whose early use of crystallo- 
graphic concepts in morphology opened new 
vistas. In the center of the picture today stand 
studies on the relations of symmetry in the de- 
veloping embryo (cp. Harrison, Trans. Conn. Ac. 
Arts and Sci, 36:277. 1945), and much work on 
polarity in general (cp. the invaluable review 
of botanical literature by Block, Bot. Rev. 9:261. 
1945). The major task in the next decades is the 
unfolding of the stages by which atomic patterns 
in biological materials, particularly the native 
proteins, determine both the gross morphology 
and the functions of living matter. Only when 
the atomic patterns of native proteins are known 
and this sequence has been established step-by- 
step will the nature of biological specificity be 
truly understood. 


MOTION PICTURES SHOWING THE REACTIONS OF CELLS IN FROG TADPOLES TO 
IMPLANTS OF TANTALUM 


Dr. Cart C. SPEIDEL 
Professor of Anatomy, University of Virginia’ 


In recent years the metal tantalum has been 
increasingly used in various surgical procedures. 
It is regarded as being especially ‘‘kind to tis- 
sues.’’ It provokes a minimum of inflammatory 
response. Surgeons have used it in the form of 
plates, wire, foil, gauze, and powder. Tiny tanta- 
lum bolts are used in the technique for the ob- 
servation of living cells in the mouse (G. Algire). 

Implants of tantalum in the form of both 
powder and fine caliber wire have been watched 
for long periods. The same individual implants 


have been observed and the day-to-day changes 
recorded by cine-photomicrography. Cellular 
movements are revealed best by pictures taken at 
low speeds. 

In the implants of.tantalum powder the chief 
features of interest are the early response of 
leukocytes, endothelial cells of lymph vessels and 
to a less extent those of blood vessels, fibroblasts 

1Aided by a grant from the American Cancer Society 
(Committee on Growth). The tantalum was furnished 


by the Ethicon Suture Laboratories, New Brunswick, 
INSWole 


22 THE COLLECTING NET 


[Vol. XIX, No.1 


The Collecting Net 


A publication devoted to the scientific work at marine 
biological laboratories 
Edited by Ware Cattell with the assistance of Aleida 
C, Thompson. 


and epithelium. Often there ave developed out- 
erowths of the skin at or near the site of the im- 
plant. Such cutaneous papille may include a 
fair portion of the implanted powder. The papil- 
le become separated from the animal by pinching 
off at the base after a week or two. 

A feature of cell behavior is the transportation 
of tantalum powder by macrophages. Tantalun- 
laden macrophages may enter either lymph ves- 
sels or blood vessels and thus be carried away 
from the site of the implant. Such activity may 
start as early as the second day and continue for 
many weeks. Other tantalum-laden  macro- 
phages, however, remain at the implant site for 
long periods (the motion pictures record this for 


as long as 84 days). Such cells exhibit continual 
slow movements and changes in their positions. 

Frequently a process of encapsulation takes 
place. After about 12 days in some implants a 
closely packed group of tantalum-laden macro- 
phages becomes encased by a definite capsule. In 
later stages the surrounding capsular substance 
may develop a fibrous appearance. Movements 
of the encapsulated macrophages are reduced to 
a minimum. Such capsules may persist indefi- 
nitely with little further change. 

Implanted tantalum wire (of about 50 micra 
caliber) is surrounded within a few hours by a 
thin layer of leukocytes. This layer effectively 
isolates it from the adjacent tissues. There is 
very little disturbance of closely situated nerve 
fibres or other tissues. Short leneths of tantalum 
wire, thus walled off, persist indefinitely. Wher- 
ever the wire implant presents rough or jagged 
edges the ensheathine leukocyte shell becomes 
somewhat thicker. 

Nove: Based on a paper presented at the Marine Bio- 
logical Laboratory. 


GROWTH AND METAMORPHOSIS OF THz PLUTEUS OF ARBACIA PUNCTULATA 


ETHEL BROWNE HARVEY 
Princeton University 


The name Pluteus paradoxrus was given by 
Johannes Miiller in 1846 to what he thought was 
a new animal found in the North Sea near Hel- 
evoland, because it resembled an easel (pluteus) 
when drawn, as he drew it, upside down. In the 
same year he discovered his mistake that it was 
really the larval form of an Ophiurid, later iden- 
tified as Ophiura albida. 

The pluteus of Arbacia punctulata which is 
known to most of the investigators at the M.B.. 
is the early pluteus of two or three days. This 
has four arms, two lone anal arms and two short 
oral arms and bright red pigment spots. The 
later stages in its development are practically 
unknown to the investigators at Woods Hole. The 
reason for this is simple. For the later stages, 
it is necessary to feed the plutei. The best food 
has been found to be the diatom, Nitzschia clos- 
terium, and this must be cultured on Miquel’s 
solution, a combination of a number of. salts 
(Allen and Nelson 1910). Many years ago, in 
1882, W. K. Brooks and two of his students, Gar- 
man and Colton (1883), raised the plutei of Ar- 
bacia punctulata at Beaufort, North Carolina, 
without any special feeding. The sea water there 
is rich in diatoms, and the plutei can probably 
obtain what they require for growth from the 
sea water, but this is apparently not the case at 
Woods Hole. This work was published in Brooks’ 
“Handbook of Invertebrate Zoology.’’ 


Since the photographs shown at the Seminar 
cannot be given in the present article, a brief 
description of the development must suffice. In 
about ten days after fertilization, a new pair of 
arms, with red tips, grows out toward the base 
of the pluteus, and in about three weeks, another 
pair of arms, without red tips, grows out between 
the red tipped ones and the original pair of long 
anal arms. The animal now swims about by 
means of its cilia and tumbles about on its arms. 
All the arms grow much longer, and the body of 
the adult Arbacia is apparent as a yellowish 
ereen mass inside the pluteus. By six weeks, the 
pluteus has become quite complicated, with two 
pairs of additional short (oral) arms and two 
pairs of tubular processes (auricles), one pair 
dorsal and one ventral. Soon afterwards the 
five primitive ambulaeral feet, with suckers at 
their ends, appear at one side of the body. These 
are continually contracted and expanded. In 
about two and a half months after fertilization, 
three flattened plates appear between each two 
ambulacral feet; these are the primitive spines. 
The pluteus has now reached its full develop- 
ment, and the arms their maximal leneth, about 
1.6 mm. Now, or sometimes before this, the 
arms begin to go to pieces; the flesh peels off, 
leaving the bare skeleton. Several arms may be 
cast off together like a shell. The pluteus is meta- 
morphosing into the adult Arbacia, the greenish 


November, 1949] 


mass becoming larger, all the arms being eradu- 
ally lost. The animal measures about a half milli- 
meter in diameter. These last stages take place 
rapidly ; the whole process of growth and meta- 
morphosis took over four months in my cultures 
(July 12 to November 17). The animals now re- 
quire another diet, a protozoon, Trichospherimn, 
or a red alga, Corallina, these providing the cal- 
careous matter needed for the development of 
the test and spines. This food was not available 
to me, and the four small Arbacia left in my eul- 
tures died. The further development has, how- 
ever, been described by Miss Gordon (1929), with 
especial reference to the test. 

The pluteus from the centrifuged ege develops 
in just the same way. The pigment spots which 
are at first unevenly distributed, become evenly 
distributed after three or four days, so that one 
cannot distinguish between the pluteus from a 
centrifuged egg and that from a normal ege. If 
the plutei are fed Nitzschia, they acquire the 
extra arms; continuing to develop lke normal 
plutei. 

The pluteus from the white half-ege, obtained 
by centrifuging, is at first colorless and smaller 
than that from the whole ege. It acquires the 
pigment spots in three or four days, and if fed, 
develops the first pair of new arms in about ten 
days. These are as heavily pigmented at the tips 
as in the normal pluteus; the animals are also of 


THE COLLECTING NET 23 


the same size at the same stages. The second pair 
of new arms (unpigmented at the tips) develops 
at the normal time. Owine to lack of material, 
these were carried no further, but presumably, 
since they are just like the normal, they would 
develop into normal <Arbacia. Even normal 
plutei are difficult to raise; out of thousands of 
plutei fed from an early stage, comparatively 
few were alive after two months and only four 
survived through metamorphosis. 

The complete paper is to be published in the 
Biological Bulletin for December, 1949. 


References 

Allen, E. J. and E. W. Nelson, 1910. On the artificial 
culture of marine plankton organisms. Quart. Jour. 
Mier. Sc., 55: 361-431. Also in Jour. Marine Biol. 
Assoc., 8: 421-474. 

Brooks, W. K., 1882. ‘‘ Handbook of Invertebrate Zo- 
ology.’’ Cassino, Boston. 

Garman, H. and B. P. Colton, 1883. Some notes on the 


development of Arbacia punctulata, Lam. Studies 
Biol. Lab. Johns Hopkins Univ., 2: 247-255. 
Gordon, I. 1929. Skeletel development in Arbacia, 


Echinarachnius and Leptasterias. Phil. Trans. Roy. 
Soc. London B 217: 289-334. 

Miiller, J. 1846a. Bericht iiber einige neue Thierforme 
der Nordsee. Arch. f. Anat. und Physiol., 1846. 
101-110. 

Miiller, J., 1846b. Uber die Larven und die Metamor- 
phose der Ophiuren und Seeigel. Abhandl. d. Kon. 
Akad. d. Wissen, zu Berlin, 1846. 273-312. 


Nore: Based on a paper presented at the Marine Bio- 
logical Laboratory. 


REVERSIBLE ENZYMIC REDUCTION OF RETINENE TO VITAMIN A 


Dr. AurRED F., BLIss 
Associate Professor of Physiology, Tufts College Medical School 


An adequate intake of vitamin A is necessary 
for vision in dim light. Vitamin A deficiency 
results in a failure in the synthesis of visual pig- 
ments. The visual pigment of retinal rod cells, 
called visual purple or rhodopsin, bleaches in the 
light with the release of a yellow carotenoid 
named retinene by Wald. Retinene has been 
identified as vitamin A aldehyde by Morton. 

If a freshly-excised retina is allowed to stand 
for an hour after bleaching, the vitamin A alde- 
hyde is reduced to vitamin A independently of 
light. The speaker has shown (Biological Bul- 
letin, 1946) that an enzymic process in fresh 
solutions of bleached visual purple likewise forms 
vitamin A. Morton’s group has recently shown 
that vitamin A is also formed when synthetic 
retinene (vitamin A aldehyde) is fed or injected 
into rats. Since vitamin A is an alcohol, and can 
be dehydrogenated by the intact retina to a typi- 
cal aldehyde, it seemed possible that the enzvme 
involved might be the well-known reversible 
DPN—specific alcohol dehydrogenase. 


This hypothesis was tested with acetone and 
ammonium sulfate precipitates of rabbit. liver 
prepared according to Lutwak-Mann, and show- 
ine high ethyl alcohol dehydrogenase activity. 
Since the equilibrium of the dehydrogenation is 
far toward the alcohol side, it is customary to 
drive the reaction toward the aldehyde side by 
removing the aldehyde as fast as it is formed. 
This may be accomplished by various substances 
which combine with the carbonyl group of the 
aldehyde, e.g., evanide and bisulfite. 

In the present experiments crystalline vitamin 
A, dissolved with a detergent, Tween 80, was the 
substrate and coenzyme J, the hydrogen acceptor. 
At the end of the reaction the aldehyde formed 
was released by dilution or alkaline destruction 
of the addition compound (absorption maximum 
ca, 330 mu) and extracted with petroleum ether. 

Experiments to date have shown up to 40% 
conversion to the aldehyde. Complete reversi- 
bility of the dehydrogenation was easily accom- 
plished in the presence of enzyme and reduced 
co-enzyme, 


24 THE COLLECTING NET 


[Vol. XIX, No. 1 


The reversibility of the retinal dehydrogenase 
has likewise been tested in this laboratory. Wald 
has reported that retinene in retinal rods and 
extracts of whole retinas is irreversibly reduced 
by the retinene reductase of the rods in the pres- 
ence of reduced coenzyme 7. We have confirmed 
the activity of isolated rods. However, the re- 
ductase activity of extracts of whole retinas ap- 
pears to be an artefact due to the large amount 
of reductase in the non-visual portion of the 
retina. Furthermore, we have found that vita- 


min A formation by isolated rods is freely re- 
versible in the presence of cyanide. 

We therefore need no longer assume a closed 
visual cycle to explain the formation of retinene 
from vitamin A. Instead, it is probable that the 
dehydrogenation is accomplished by alcohol de- 
hydrogenase with the formation of visual purple 
which acts as the physiological trapping com- 
pound for retinene. 


Nore: Based on a paper presented at the Marine Bio- 
logical Laboratory. 


SOME EFFECTS OF ULTRA-VIOLET LIGHT ON THE CATALASE ACTIVITY AND ON 
PHOTOSYNTHESIS OF CHLORELLA PYRENOIDOSA 


Dr. ALBERT FRENKEL 
Assistant Professor of Botany, University of Minnesota 


It had been shown by Arnold (1) that ultra- 
violet light (A = 2537 A) inhibits the light re- 
action of photosynthesis. The percentage inhibi- 
tion was observed to be the same in flashing light 
and in continuous light; thus the effect is similar 
to the one produced by narcotics. 

In an attempt to obtain an action spectrum of 
the inhibition of photosynthesis by ultra-violet 
light, some observations were made to elucidate 
the mechanism of the inactivation process. 

On the assumption that ultra-violet light could 
have inactivated the oxygen-liberating catalyst 
in photosynthesis, the catalase activity of Chlo- 
rella cells was tested before and after irradiation 
with ultra-violet light. In each case an increase 
in the catalase activity was found (Table I), a 


TABLE I 

Measurements were performed with .026 em? of Chlorella 
cells suspended in 3 ml. of .035 M KHCO, and .065 M 
NaHCO, at 25°C. 

Exposures to ultra-violet light were made using a quartz 
vessel, the gaseous phase was either air or nitrogen. 

Time of exposure Rate constants of 
to ultra-violet light. decomposition of 
Incident intensity H:H:z by 1 em? of 
1.3 ergs/mm2. sec. Chlorella cells. 


Photosynthesis 
in % of control 


(Sec-1) 
0 minutes 100 Apes} 
3 he 80 1.5 
6 he 50 2.5 
ala ay 0 9.8 


phenomenon which had been observed by Euler 
(2) in irradiated yeast. No catalase was released 
by the cells as the suspending fluid showed no 
activity after the cells had been centrifuged off. 

At the present time we are not able to state 
if this enhanced catalase activity is due to an in- 
creased permeability of the cells to hydrogen 
peroxide (3), or to a photochemical activation of 
the catalase system (4), or due to the release of 
enzyme molecules from a structural configura- 
tion in which they were either inactive or only 


very slightly active (5). 

According to Arnold the inactivation of photo- 
synthesis by ultra-violet light follows a first order 
reaction. When we attempted to duplicate these 
results using a General Electric resonance lamp 
we found that the effect of this ultra-violet light 
(emission at 2537 A rated at 95% +) was more 
effective when a definite amount of radiation of 
constant energy flux was given in one dose, than 
when given in several doses interspersed by 
dark periods or exposures to visible light. Thus 
the inactivation process appears to be complex, 
and not always of the first order. 

Arnold had observed that the absorption spec- 
trum of chlorophyll does not change after irradi- 
ation of Chlorella suspensions by ultra-violet 
light. However, it was noticed that in intact 
cells the transmission of the red chlorophyll peak 
increased after the ultra-violet light treated cells 
were exposed to visible light. This bleaching of 
chlorophyll is a function of the intensity and of 
the time of exposure to visible ight. In some 
way the energy transferring mechanism in photo- 
synthesis has become uncoupled and the lght 
energy, directly, or through some photoperoxide, 
produces the bleaching of chlorophyll. 

A test was made to determine if hydrogen per- 
oxide would give results similar to those pro- 
duced by ultra-violet light, but in agreement 
with Gaffron (6) it was found that inhibition of 
photosynthesis with hydrogen peroxide (10 to 
10° moles per liter) was only observed at high 
light intensities and not at low light intensities, 
and thus hydrogen peroxide affects one or more 
of the dark reactions of photosynthesis in con- 
trast to the inactivation of the light reaction by 
ultra-violet light. 

Thus, to summarize, it can be stated that an 
amount of ultra-violet radiation which only im- 
perceptibly affects the respiratory rate of Chlo- 
rella cells will not only decrease the photosyn- 


November, 1949] 


THE COLLECTING NET 


25 


thetic ability of these cells, but will also sensitize 
these cells so that bleaching of chlorophyll will 
occur subsequently in visible light. It also will 
cause, directly or indirectly, an acceleration of 
the catalase activity of intact Chlorella cells. 


References 
1. Arnold, W. 1933. Jour. Gen. Physiol. 17: 135. 
2. Euler, H. & G. Giinther 1933. Zeitschr. f. physiol. 
Chemie. 220: 69. 


Yamafuji, K. 1937. Enzymologia 2: 147. 
3. Penrose, M. & J. H. Quastel 1930. Proc. Roy. Soc. 
B. 107: 168. 
4. Burge, W. E. & E. L. Burge 1924. Bot. Gaz. 77: 220. 
5. Keilin, D. & E. F. Hartree 1945. Biochem, Jour. 39: 
293. 
6. Gaffron, H. 1937. Biochem. Zeitschr. 292: 241. 
Marine 


Nore: Based on a paper ‘presented at the 


Biological Laboratory, July 19, 1949. 


SYNTHESIS OF ACETYLCHOLINE 


D. NACHMANSOHN, S. HestrRiIn AND H. VorRIPAIEFF 
Columbia University, Department of Neurology, College of Physicians and Surgeons 


The ionic concentration gradients which exist 
between the inside of the nerve fiber and its outer 
environment have long been supposed to be the 
source of the electromotive force for the electric 
currents which propagate the nerve impulse. It 
has been postulated that during the passage of 
the impulse the resistance of the active mem- 
brane surrounding the axon breaks down. Due 
to the increased permeability, the ions can move 
freely and the concentration gradients may be- 
come effective. Recent investigations with radio- 
active sodium and potassium ions carried out in 
Cambridge, England, and in our laboratory, 
have shown that there are continuous ion move- 
ments across the axonal membrane even in rest. 
During activity the influx of sodium ions into 
the interior and the leakage of potassium ions 
to the outside are greatly accelerated. This could 
be expected from the respective concentration 
gradients of these two ion species in case of in- 
creased permeability. Some change in the active 
membrane must then obviously occur which is 
responsible for the rapid ion movement during 
the passage of the impulse and this change must 
be quickly reversible, since the resting condition 
is restored in a period of time less than one milli- 
second. A quickly reversible chemical process 
producing the change: in the active membrane 
appears to be-the most likely assumption for the 
interpretation of the mechanism underlying 
conduction. 

During the last 12 years a great variety of 
facts have accumulated supporting the assump- 
tion that the release and removal of acetylcho- 
line. are essential events in the neuronal surface 
membrane and inseparably associated with the 
electrical manifestations during the conduction 
of the nerve impulse. The essential results have 
been recently reviewed and need not be discussed 
here (1, 2). For the understanding of the pre- 
cise role of the ester, it appeared necessary to 
integrate the breakdown and the formation of 
the ester into the metabolism of the nerve cell 
and to correlate it with the electric currents. The 


extremely small amount of energy involved in 
conduction — the initial heat is of the order of 
magnitude of 1 & 10~* ecal per gram nerve per 
impulse—offered a great obstacle to the correla- 
tion of electrical and chemical events. The diffi- 
culty was overcome by the use of electric organs, 
where by the special arrangement of a great 
number of cellular units in series chemical and 
electrical events occur on a scale which is within 
the range of measurement. In investigations us- 
ing this material a whole chain of chemical reac- 
tions has been established and associated with 
the action potential. It was found that the en- 
ergy released by the breakdown of phosphocrea- 
tine, a compound rich in energy, accounts for the 
total electrical energy released during the action 
potential (3). It is known from muscular physi- 
ology that the breakdown of adenosinetriphos- 
phate (ATP) precedes that of phosphocreatine. 
The same sequence of reactions was assumed to 
occur in the conductive membrane. On the basis 
of the work of Meyerhof and Lohmann, Engel- 
hardt and Lubimova, Needhams and their asso- 
ciates, Szent-Gyérgyi and his associates, it is to- 
day generally believed that in muscle ATP 
reacts directly with protein, this reaction being 
the primary process of contraction. However, it 
is difficult to conceive, for many reasons, that the 
reaction of A7’P: with the proteins or lipopro- 
teins of the nerve membrane is the primary proc- 
ess In conduction.. It was assumed that the re- 
lease and breakdown of acetylcholine precedes 
that of ATP and that in the conductive mem- 
brane the breakdown of ATP is:-the primary re- 
covery process yielding the energy for the syn- 
thesis of acetylcholine. 

In accordance with this hypothesis an enzyme, 
choline acetylase, was discovered, which forms 
acetylcholine using the energy of ATP (4). The 
enzymatic system is rather complex but has been 
reconstructed in vitro during the last few years 
(5, 6). The enzyme requires for full activity, in 
addition to ATP and:the substrates, acetate and 
choline, adequate amounts of a coenzyme, cys- 


26 THE COLLECTING NET 


(Vol. XIX, No. 1 


teine, and the following ions: potassium, cal- 
cium and magnesium. An inhibitor of acetylcho- 
line esterase is always added to inactivate this 
enzyme completely, since a small fraction may 
be still present even if most of it is inactivated 
or removed. 

By fractional ammonium sulfate precipitation 
a highly active and concentrated solution of cho- 
line acetylase has been recently obtained from 
acetone dried powder of rabbit brains (7). The 
fraction between 16 and 35 per cent ammonium 
sulfate was found to contain most of the enzyme 
protein. The yield of acetylcholine obtained with 
this enzyme solution was equivalent to about 2 
micromoles of acetylcholine per ml. (350-400 
pe), as tested with the usual bio-assay technique. 
The amount of acetylcholine formed per gram 
protein per hour was close to 200 milligrams. 

It is remarkable that, in spite of the great 
physiological importance of acetylcholine, no 
chemical methods of determining the ester were 
available except after isolation with very tedious, 
time-consuming procedures. By necessity in- 
vestigators were compelled to use bioassays of 
questionable specificity. Recently a very rapid 
and simple chemical method has been introduced 
by Hestrin (8). It is based upon the reaction of 
acetylcholine with hydroxylamine in alkaline 
medium. If the mixture is subsequently acidified 
and ferric chloride added, a brown-purple color 
develops. With the Beckman spectrophotometer 
less than 1/10 of a micromole of acetylcholine 
per ml. may be easily determined. The underly- 
ing process is a reaction of the acyl group with 
hydroxylamine forming stoichiometrically hy- 
droxamie acid. The determination of acetylcho- 
line may therefore be performed in excess of 
acetate and choline. It may be noted that the 
method may be applied to other short chain 
O-acyl derivatives and its usefulness may there- 
fore be extended—if properly adapted—to other 
compounds containing O-acyl groups. 

When the method was apphed to the highly 
active enzyme preparation described it was 
found, surprisingly, that less than 50 per cent of 
the activity obtained in the bioassay can be ac- 
counted for by the chemical method. More than 
50 per cent of the effect obtained must be attrib- 
uted to a substance which appears to have the 
same biological activity as acetylcholine, but may 
be distinguished by chemical procedures. Acetyl- 
choline added at the beginning or the end of the 
incubation was analytically recovered by both 
chemical procedure and bioassay. The substance 
is synthesized in the reaction mixture in the ab- 
sence of added choline. In contrast, acetylcholine 
is not found to be formed by the chemical method 
if choline is omitted in the reaction mixture. 
In presence of added choline the synthesis of the 


compound occurs at a higher rate than in its 
absence. In the absence of acetate neither acetyl- 
choline nor the compound are formed. Propionate 
and butyrate, but not formate and valerate may 
substitute acetate. The pharmacological proper- 
ties of the enzymatically formed compound have 
been tested by Middleton and Middleton (9). 
These investigators found that the compound de- 
creases the arterial blood pressure of eats and 
the amplitude of the isolated frog heart in the 
same way as acetylcholine. Atropine regularly 
suppressed both actions in the same concentra- 
tions as it suppressed the action of acetylcholine. 

The chemical method of acetyleholine deter- 
mination may be used for measuring acetylcho- 
line esterase activity. The simplicity and rapid- 
ity of the technique make it most suitable for 
clinical investigations. Compared with the wide- 
ly used manometric method it has the advantage 
that it may be readily applied over a wide range 
of pH. Using the colorimetric method with a 
highly purified acetylcholine esterase obtained 
from electric tissue Hestrin (10) was able to 
demonstrate an equilibrium of the following 
formula : 

|acetyleholinet | [water | 

= IX 


|acetic acid| {choline | 


A considerable synthesis of acetylcholine is ob- 
tained in this system at pH 5.0. With the varia- 
tion of pH K remains constant, thus confirming 
the assumption that acetic acid rather than ace- 
tate ion is the reactant species. From the data 
obtained the free energy of acetylcholine hy- 
drolysis was caleulated to be approximately 
—3100 geals. 

This figure is of considerable physiological in- 
terest. It has been recently shown that in frog 
sciatic nerves exposed to di-isopropyl filuoro- 
phosphate (DFP)—a potent inhibitor of cho- 
linesterase—more than 90 per cent of the enzyme 
may be inactivated without affecting conduction. 
The remaining enzyme activity, however, is es- 
sential for conduction, which by further decrease 
is impaired and finally abolished. The minimum 
activity corresponds to 400-500 pg. of acetylcho- 
line split per gram nerve per hour. Since the 
initial heat developed per gram per impulse is 
about 1 & 107-8 geal., at most 0.0006 pg of acetyl- 
choline could be split per gm. nerve per impulse. 
This figure is obtained on the basis of the free 
energy of acetylcholine hydrolysis if the initial 
heat is attributed exclusively to acetylcholine 
hydrolysis—a most unlikely assumption. It ap- 
pears more probable that the release and _ re- 
moval of acetylcholine act as a trigger in a chain 
of reactions. On the assumption that one third 
of the initial heat may be ascribed to acetylcho- 

(Continued on page 32) 


November, 1949 | 


THE COLLECTING NET 20 


DIRECTORY OF THE MARINE BIOLOGICAL LABORATORY 
INVESTIGATORS AND ASSISTANTS 


Abrams, R., asst. prof. biochem. radiobiol. Chicago, Nu- 
cleic acid and protein metabolism in Arbacia em- 
bryos. i 

Abramsky, Tess, res. asst. phys. Georgetown Med. Po- 
tassium transport in invertebrate nerve. 

Adler, F. H., prof. opthal. Pennsylvania Med. 

Allen, E., prof. biol. Stetson. Degenerating spermato- 
eytes in early postnatal rats. 

Allen, M. Jean, instr. zool. New Hampshire. 
ology of the marine annelid, Diopatra. 
Alscher, Ruth P., instr. phys. Manhattanville. 
chemistry of blood cells of marine annelids. 
Amberson, W. R., prof. phys. Maryland Med. Extrac- 

tion of invertebrate muscle proteins. 

Anderson, R. S., prof. phys. South Dakota. X-ray ef- 
feets on proteins. 
Atwood, K. C., res. assoc. 

netics of neurospora. 

Bacon, C. R. T., Pennsylvania. Cell physiology. 

Baily, N. A.. res. radiol. Columbia. Supervision of prob- 
lems utilizing radioactive material. 
Ball, E. G., prof. biol. chem. Harvard Med. 
and pigments of marine organisms. 

Ballard, W. W., prof. zool. Dartmouth. 

Barron, E. S. G., assoc. prof. biol. chem. Chicago. The 
effect of ionizing radiations on cell metabolism. 

Bartlett, J. H., prof. physics. Illinois. Transient effects 
in electrolytic systems—theoretical. 

Battley, E. H., grad. zool. Harvard. Eyestalk hormone 
and intermedin in invertebrates. 

Batty, T. V., instr. anat. Kansas. Development neural 
mechanisms in Opsanus tau. 

Bauer, M. H., grad. stud. phys. Princeton. Temperature 
effect on production of antibodies. 
Benson, Elenore, res. assoc. zool. Missouri, 

of desoxyribonueleie acid. 

Berger, C. A., dir. biol. lab. Fordham. 
embryology of tunicates. 

Bernatowicz, Albert J., bot. Michigan. Plant ecology. 

Bernstein, M. H., grad. asst. zool. Washington. De- 
velopment of ribonuclease in eggs of Arbacia. 

Bliss, A. F., assoc. prof. physics. Tufts Med. Physi- 
ology of vision. 

Bloch, R., asst. prof. bot. Yale. 
plant morphogenesis. 

Blum, H. F., lect. phys. Nat’l. Cancer Inst. Princeton. 
Effect of ultraviolet and X-ray on sea-urchin eggs. 

Blumenthal, Gertrude, res. assoc. phys. Missouri. Ef- 
feet of radiation on enzyme substrate films. 

Bodian, D., assoc. prof. empidemiology. Johns Hopkins. 
Phosphorus metabolism of regenerating nerve cells. 

Boyle, E. Marie, science teacher. Baldwin School. Ecol- 
ogy of marine algae. 

Bridgman, Anna J., prof. biol. Limestone. Cytological 
study of protozoa and mating reactions. 

Bronk, D. 

Brooks, Matilda M., res. assoc. biol. California. Oxida- 
tion-reduction studies on fertilization of eggs. 
Brown, F. A., Jr., prof. zool. Northwestern. Endoe- 

trine mechanisms of crustacea. 

Browning, I., Nat. Res. fel. zool. Pennsylvania. Physi- 
ology of cellular nuclei, especially viscosity. 
Brumm, Anne F., res. asst. biol. New York. Biology 

and life history of parasitic worms. 

Brust, M., res. asst. phys. Chicago. Action potential 
in perephenial nerve. 


Embry- 


Histo- 


microbiol. Columbia. Ge- 


Enzymes 


Specificity 


Cytology and 


Work on textbook of 


Bulloch, Jane A., grad. asst. phys. Oklahoma. 
of baeterial toxins on cell respiration. 
Burbanck, W. D., chrm. biol. dept. Drury. 
survey of Rand Harbor, Megansett. 
Burk, D., prin. chemist Nat. Cancer Inst. 
Photosynthesis. 
Butler, E. G., prof. biol. 
phibian regeneration. 
Cantoni, G. L., pharmacology. New York Med. 
Cantor, Nancy J., res. asst. phys. Vermont Med. 
duction in voluntary and cardiae muscle. 
Carlson, F. D., instr. biophys. Johns Hopkins. 
metabetism and conduction. 

Case, J. F., grad. biol. Johns Hopkins. 
Cattell, W., editor, The Collecting Net. 
electric current on cell division. 

Chambers, R. 

Chase, A. M., assoc. prof. biol. 
of bioluminescence; vision. 

Cheney, R. H., prof. gen. phys. Brooklyn. Influence of 
methylated purines on cellular physiology. 

Claff, C. L., res. fel. surgery. Harvard Med. Cartesian 
diver technique, Protozoan metabolism. 

Clark, A. M., asst. prof. biol. Delaware. 
in Habrobracon. 

Clark, E. R., prof. anat. Pennsylvania Med. 
and behavior of living cells and tissues. 

Clark, L. B., prof. biol. Union. Biological effects of 
high voltage x-ray. 

Clark, Lenore. 

Clement, A. C., prof. biol. 
embryology. 

Clendenning, K. A., assoc. res. 
Labs. Photosynthesis. 

Clowes, G. H. A., emerit. res. dir. Lilly Research Labs. 
Particulate systems of Arbacia eggs. 

Cohen, A. I., asst. zool. Minnesota. 
in embryogenesis. 

Cohen, I., assoc. prof. biol. American International. 
Fixation and staining of plant and animal nuclei. 

Cole, K. A., scientific dir. Naval Med. Res. Inst. Elee- 
trical responses of squid axon membrane. 

Collier, A., marine biol. Gulf Oil Corporation. Oyster 
mortality. 

Colwin, A. L., asst. prof. biol. Queens. Development of 
Dolichoglossus and Thyone. = 

Colwin, Laura H., instr. biol. Queens. Development of 
Dolichoglossus, Tunicata and Thyone. 

Conklin, E. G., emerit. prof. biol. Princeton. 
phology of the eggs of different phyla. 

Conklin, Ruth E., prof. phys. Vassar. 

Cookson, B. A., cancer res. fel. Pennsylvania. Studies 
on cleavage. 


Effect 
Eeologieal 

3ethesda. 
Prineeton. 


Studies on am- 


Con- 
Nerve 
Effect of direct 
Princeton. 


Chemistry 


Gene action 


Growth 


Charleston. Experimental 


biol. Nat. Res. Couneil 


Apyrase enzymes 


Promor- 


Cooperstein, S. J., instr. anat. Western Reserve. Ex- 
perimental diabetes, cytochemistry of nervous 
tissue. 

Costello, D. P., chrm. dept. zool. North Carolina. Ex- 


perimental embryology of Nereis and Caetopterus. 

Cotzias, G. C., asst. physician, Rockefeller Inst. Effect 
of regeneration on conditioned reflex. 

Coyle, Elizabeth E., assoc. prof. biol. Wooster. Segre- 
gation of Enteromorpha species on rock surfaces. 

Croasdale, Hannah, res. assoc. zool. Dartmouth. Fresh- 
water Algae of Woods Hole. 

Crowell, Sears, asst. prof. zool. Indiana. 

Csaky, T. Z., res. assoc. Duke Hospital, N. C. 


28 THE COLLECTING NET 


[Vol. XIX, No. 1 


Curtis, P., student Oberlin. Experimental diabetes 
cytochemistry of nervous tissue. 

Curtis, W. C., emerit. prof. zool. Missouri. Regenera- 
tion in Hydroids and Planaria. 

Dahl, A. O., prof. and chrm. bot. Minnesota. Compara- 
tive pollen morphology. 

Davidson, Margaret E., curator demonstrator zool. 
MeGill. National history of marine invertebrates. 

DeLamater, E. D., assoc. prof. dermatol. Pennsylvania. 
Nuclear cytology of yeasts and fungi. 

Dent, J. N., assoc. prof. biol. Virginia. Effects of radi- 
ation on regenerating amphibian limbs. 

Diller, Irene C., assoc. member Inst. for Cancer Res. 
Ctyology and chemotherapy of cancer. 

Diller, W. F., asst. prof. zool. Pennsylvania. Cytology 
of protozoa. 

Dixon, June A., grad. asst. zool. Washington. Nutrition 
of slime molds. 

Donovan, Joanne, res. asst. Yale. Role of specific sub- 
stances in fertilization. 

Doty, M. §S., asst. prof. biol. Northwestern. System- 
atics, physiology and ecology of marine algae. 


Driscoll, Dorothy H., instr. zoo]. Smith. Histogenesis 
of adrenal gland. 
Duryee, W. R., cytologist. Nat. Cancer Inst. Cell 


fragment culture; radiation damage mechanism. 

Edsall, G., prof. bact. Boston Med. General bacteri- 
ology; immunology. 

EBichel, B., asst. prof. biochem. Rutgers. 
zymes in carbohydrate metabolism. 

Eichel, H. J., asst. phys. Rutgers. Enzymes in earbo- 
hyrate metabolism. 

Essner, E. A., asst. instr. phys. Pennsylvania. Physical 
properties of cytoplasmic particles. 

Evans, Jeanne, student, Pennsylvania. 
in invertebrates. 

Fahey, Elizabeth M., grad. Boston. Systematics, physi- 
ology and ecology of marine algae. 

Failla, G., physicist, Columbia Med. X- 
rays. 

Fass, J. S., res. asst. 
permeability. 
Ferguson, F. P., asst. prof. phys. Maryland Med. Ex- 
traction of musele proteins from invertebrates. 
Flagler, Elizabeth A., biochem. res. asst. Princeton. 
Flood, Veronica, M., Jr., biochem. Argonne Nat. Lab. 
Irradiation, respiration of Amoeba — Cartesian 

Diver. 

Fogelman, M. J., fel. neurosurgery, Southwestern Med. 
Woter exchange in fish. 
Foley, Mary T., res. asst. Yale. 
stances in fertilization. 
Frenkel, A., asst. prof. bot. Minnesota. Photosynthesis 

and photoreduction in algae. 
Freund, J., chief, div. appl. immun. P. H. Res. Inst. 


Book on en- 


Blood clotting 


and Gamma 


Rockefeller Inst. Studies on 


Role of specific sub- 


(N. Y.) Immunology. 

Friedberg, F., instr. biochem. Howard. Protein syn- 
thesis. 

Friedler, Gladys, instr. zool. Pennsylvania. Andro- 


genesis in Mormoniell. 

Fromberg, Vivian, Pennsylvania Women’s Med. Radi- 
ation induced mutations in bacteria. 

Gabriel, M. L., asst. prof. biol. Brooklyn. 


Gaffron, H., assoc. prof. biochem. Chicago. Photo- 
synthesis. 
Gagnon, A., res. asst. zool. ‘Pennsylvania. Viscosity 


and calcium release in nerve cells. 

Garnic, Justine, grad. asst. bot. Northwestern. Culture 
of algae. 

Garrey, W. E., emerit. prof. phys. Vanderbilt Med. 
Comparative cardiac physiology and neurogenic 
hearts. 


Gasvoda, Betty, Jr. biochem. Argonne Nat. Lab. Ir- 
radiation and respiration in Arbacia. 

Gates, R. R., res. fel. biol. Harvard. Book on geneties.. 

Gennaro, J. F. Jr., investigator biol. Pittsburgh. Dis- 
tribution of p-32 in metamorphosing tadpoles. 


Gilman, L. C., assoc. prof. zool. Miami. Intervarietal 
mating of Paramecium caudatum. 
Glaser, O. C., emerit. prof. biol. Amherst. Copper,. 


Vanadium in Aseidian physiology. 

Goldring, Roberta, grad. zool. Vassar. 
elasmobranch kidney. 

Goldstein, L., grad. zool. Pennsylvania. 
maturation of Chaetopterus eggs. 
Gombas, P., Inst. for Muscle Research. Quantum theory 

of proteins. 

Goodchild, C. G., prof. biol. Southwest Missouri State. 
Studies on digenetic trematodes. 

Gopalkrishnan, K. S., res. fel. bot. Notre Dame. Marine 
fungi and algae. 

Gould, H. N., prof. biol. Newcomb. Variations in sexual 
development of Crepidula Plana. 

Gourevitch, H. G., grad. phys. Chicago. Metabolism of 
the sperm of Arbacia. 

Grand, C. G., invert. biol. New York. Effects of dyes 
on marine eggs. 

Green, J. W., asst. prof. phys. Rutgers. Permeabliity 
of fish erythrocytes to acid compounds. 

Greenberg, R., instr. phys. Ohio State. Dynergy rela- 
tion between acetyl choline adrenaline. 

Grimm, Madelon R., res. asst. bact. Amherst. Radiation 
induced mutations in bacteria. 

Grosch, D. S., asst. prof. zool. North Carolina State. 
Cytological aspects of development of Habro- 
bracon. 

Grundfest, H., assoc. prof. neurol. Columbia Med. 
Nature of bioelectric potentials of squid nerve. 
Gudernatsch, F., phys. Cornell Med. Comparative anat- 

omy and phylogeny of endocrines. 

Gurewich, V., asst. visit. physician Bellevue Hosp. 
(N. Y.). Cireulation. se 

Halaban, A., grad. phys. Pennsylvania. Parthenogene- 
sis and viscosity changes by Vitamin K. 

Harding, C. V., asst. instr. zool. Pennsylvania. Ultra- 
violet radiation on starfish germinal vesicle. — 

Harding, Drusilla, asst. zool. Pennsylvania. Artificial 
parthenogenesis. 

Harris, D. L., asst. prof. phys. Chicago. Properties of 
cytoplasmic granules. 

Harvey, Ethel B., investigator biol. Princeton. 
trifuging experiments on Arbacia eggs. 
Harvey, E. N., prof. phys. Princeton. Bioluminescence. 
Hausler, H., dean med. faculty Graz (Austria). Phar- 

cology and related sciences. 

Haxo, F., instr. biol. Johns Hopkins. Photosynthesis— 
physiology of plant pigments. ‘ 

Hay, Elizabeth, Johns Hopkins. Role of epithelium in 
amphibian regeneration. 

Haywood, Charlotte, prof. phys. Mt. Holyoke. Nitro- 
gen and helium pressures on Arbacia eggs. i 

Heidenthal, Gertrude, assoc. prof. biol. Russell Sage. 
X-ray induced recessive lethals in Habrobracon. 

Heilbrunn, L. V., prof. phys. Pennsylvania. Physiology 
of cell division. 

Henley, Catherine, res. asst. North Carolina. Mitosis: 
salamander epithelium and chaetopterus eggs. 
Hickson, Anna K., chem. Eli Lilly. Particulate systems 

of Arbacia eggs. 

Himmelfarb, Sylvia, res. asst. phys. Maryland Med. 
Extraction of proteins from invertebrate muscle. 

Hirshfield, H. I., res. assoc. Missouri. Studies in radia- 
tion and respiration of Amoebae. 

Hobson, L. B., assoc. med. dir. Squibb. Infectious dis- 
eases and antimicrobial therapy. 


Physiology of 


Nature of 


Cen- 


November, 1949] 


THE COLLECTING NET 29 


Hodes, R., prof. exper. neurol. Tulane Med. Studies of 
conduction velocity in squid axon. 

Hoffman, J. F., grad. phys. Princeton. 
bility. 
Holland, B., lab. asst. Inst. Radiobiology 
Physiology of nerve conduction, 
Honneger, Carol M., instr. phys. Temple. 
and physiology of Pelomyxas. 

Hopkins, A., grad. stud. Pennsylvania. Cell physiology. 

Hopkins, H. S., assoc. prof. phys. New York. Effects 
of ionic composition of sea water on clams. 

Houlihan, R., grad. asst. biol. Boston College. Enzyme 
systems in Protozoa. 

Howard, R. S., asst. instr. zool. Miami. 
invertebrate zoology course. 

Hsiao, S. C., asst. prof. biol. New York. 
zoology. 

Hsu, Dorothy L., grad zool. Pennsylvania. 

Hunter, F. R., assoc. prof. phys. Oklahoma. Effect of 
bacterial toxins on cells. 

Hutchings, Lois M., res. asst. Sloan-Kettering Inst. 
Ultra violet irradiation. 

Inone, S., grad. biol. Princeton. Study of cell structures 
with polarized light. 

Jacobs, B. R., Pennsylvania Med. Effects of antico- 
agulants of protoplasmic coagulation. 

Jacobs, M. H., prof. phys. Pennsylvania. 
of erythrocytes. 
Jaffee, O., res. asst. zoal. New York. 
on fertilization and cleavage. 
Jaskoski, B. J., Notre Dame. Literature in general 
zoology and embryology. 

Jenkins, G. B., emerit. prof. anat. George Washington. 
Early embryogenesis. 

Jepps, Margaret W., lect. zool. Glasgow. 
squids and other Cephalopods. 


Cell permea- 
(Chicago). 


Taxonomy 


Instruetion in 


Experimental 


Physiology 


Pressure effects 


Parasites of 


Kaan, Helen W., res. assoc. zool. Nat. Res. Coun. 
Galactose cataract in the albino rat. 

Kabat, E. A., assoc. prof. bact. Columbia. Preparation 
of book on immunology. 

Kaplan, Ann E., grad. phys. Mt. Holyoke. Os con- 


sumption of frog liver. 
Karush, F., fel. biophys. Sloan-Kettering Inst. Binding 
properties of serum albumins and antibodies. 
Kayhart, Marion, A. E. C. fel. Pennsylvania. Effect 
of radioactive isotopes on Mormoniella. 

Keefe, Mary. 

Keller, R., dir. of res. Madison Found. for Biochem. 
Res. Microscopie electrobiology. 
Kelly, Elizabeth M., grad. asst. biol. Delaware. X-radi- 
ation effects on development in Harbobracon. 
Kelly, J. W., grad. phys. Pennsylvania. Acid polyac- 
charides of marine eggs. 

Kempton, R. T., chrm. zool. Vassar. 
elasmobranch kidney. 

Keosian, J., prof. biol. Rutgers. Cellular physiology. 

Keston, A. S., asst. prof. chem. New York. Quantita- 
tive study of the denaturation of proteins. 

Kind, C. A., asst. prof. chem. Connecticut. Survey of 
marine invertebrate phosphatases. 

Kindred, J. E., prof. anat. Virginia. Effects of poisons 
on degeneration of testis. 


Physiology of 


Kirschner, L. B., res. asst. phys. Wisconsin. Studies 
on the neuromuscular junction in Squid. 
Kisch, B., prof. chem. Yeshiva and Fordham. Inves- 


tigations on fish. 

Kitchen, I. C., assoc. prof. biol. Georgia. 
amphibian neural tissues. 

Kleinholz, L. H., assoc. prof. biol. Reed. 
hyperglycemia in crustaceans. 

Klotz, I, M., assoc. prof. chem. Northwestern. Chemis- 
try of hemocyanin. 

Kopac, M. J., prof. biol. New York. 


Explants of 


Experimental 


Korey, S. R., res. assoc. neuro. Columbia. 
in central nervous system. 

Kozam, G., instr. anat. New York. 

Krahl, M. E., assoc. prof. biochem. Washington Med. 
Oxidative phosphorylation in marine eggs. 

Krasnow, Frances, dir. res. Guggenheim Dental Found. 


Acetlyation 


Significance of salivary constituents in Homo- 
sapiens. 
Kuff, E. L., instr. cyt. Washington, Lipids in cell 


division. 

Kuffler, S. W., asst. prof. phys. optics, Johns Hopkins. 
Synaptie transmission and nerve conduction. 
Kun, E., res. assoc. phys. Chicago. Cellular metabolism. 
Lajtha, A., res. asst. Inst. of Muscle Research. Nucleic 

acids in muscular contraction. 

Lansing, A. I. assoc. prof. anat. Washington Med. Cal- 
cium binding by cell surfaces. 

Lazarow, A., assoc, prof. anat. Western Reserve. Ex- 
perimental diabetes; eyto-chemistry of nerve tis- 
sue. 

Lee, Lois E., asst. zool. Southwest Missouri State. 
LeFevre, P. G., asst. prof. phys. Vermont Med. Com- 
plications of excitation theory in Squid axon. 
Leikind, M. C., head biol. & med. unit, Lib. of Congress. 

Guide to the literature of the history of science. 

Leonard, L., Lab. asst. chem. Haverford. Jon antago- 
nism. 

Levy, M., assoc. prof. biochem. New York Med. Quan- 
titative study of denaturation of proteins. 

Lillie, R. S., emerit. prof. phys. Chicago. Physiology 
of activation processes in marine eggs. 

Ling, G. N., fel. phys. Chicago. 

Litt, M., Rochester Med. School. 
potentials of squid nerve. 

Lochhead, J. H., asst. prof. zool. Vermont. 
of calcium for newly molted Crustacea. 

Loeffler, R., grad. asst. bot. Wisconsin. Study of flower 
primordia in commercial orchids. 

Loewi, O., res. prof. phys. New York Med. 
in physiology; library reader. 

Loud, A. V., grad. biophysics M. I. T. Ultra-strueture 
of axoplasm. 

Love, Lois, instr. phys. Pennsylvania. 
erythrocytes. 

Love, W. E., asst. instr. physiol. Pennsylvania. 
ology of erythrocytes. 

Lovelace, Roberta, adjunct. prof. biol. South Carolina. 
Fertilization and cleavage. 

Lucké, B., prof. path. Pennsylvania Med. Cell permea- 
bility; tumors in amphibia. 
Luyet, B., prof. biophysics St. Louis. 
at low temperatures. 
Lynn, F., res. asst. Stanford. 

studies. 

Lynn, W. G., prof. biol. Catholic. Thyroid funetion in 
cold blooded vertebrates. 

Malan, Martha, invest. genetics, Pennsylvania. Ge- 
netic studies on Mormoniella. 

Marmont, G. H., asst. prof. phys. Chicago. 
nerve condition in Squid. 

Marshak, A., res. assoc. biochem. New York Med. 
Nueleic acid metabolism of Arbacia eggs. 

Marsland, D., prof. biol. New York. Temperature- 
pressure effects on fert. and cleavage. 

Matoltsy, A., res. asst. Inst. Muscle Research. Histol- 
ogy and physiology of muscular contraction. 

Matzke, E., prof. bot. Columbia. 

Mavor, J. W., emerit. prof. biol. Union. Textbooks in 
general biology; invertebrate morphology. 

Mazia, D., prof. zool. Missouri. Specificity of deso- 
xyribonucleie acid. 

McCay, P. B., grad. asst. zool. Oklahoma. 
bacterial toxins on cell respiration. 


Nature of bioeleetrie 


Sources 


Consultant 


Physiology of 


Physi- 


Life and death 


Oxidotion-reduction 


Nature of 


Effect of 


30 THE COLLECTING NET 


| Vol. XLX, No. 1 


McColl, J. D.. res. fel. biochem. Western Oitario Med. 
Comparative biochemistry of Myelin. 

McCulloch, D., fel. embryology M.I.T. Techniques in 
connection with mitosis problem. 

McDonald, Sister Elizabeth, prof. biol. Mt. St. Joseph- 
on-the-Ohio. Comp. biochem. body fluids of marine 
inverts. 

McIntyre, Patricia, Johus Hopkins Med. 
tems of Arbacia. 

McKeehan, M. S., asst. zoo!. Chicago. 
phases of embryonic induction. 

McLean, D., instr. phys. Vassir. 

Menkin, V., assoc. prof. exp. path. Temple. Cytologi- 
cal work on Arbacia eggs. 

Metz, C. B., asst. prof. zool. Yale. 
substances in fertiHzation. 

Meyerhof, O., res. prof. biochem. Pennsylvania. 

Mihalyi, E., res. asst. phys, Inst. of Muscle Res. Elec- 
trochemistry of myosin and actin. 

Miller, Faith, res. asst. Emory. Temperature effects on 
anoxie newborn guinea pigs. 
Miller, J. A., assoc. prof. anat. Emory. 

studies on Tubularia. 

Miller, T. D., grad. bact. Amherst. 
mutations in bacteria. 

Milne, L. J., assoc. prof. zool. New Hampshire. 
physiology of invertebrates. 

Mitchell, Constance J., instr. biol. Delaware. 
mental studies on Habrobracon. 

Mitchell, R., grad. res. worker, Columbia. Biochemistry. 

Moore, G. M., chrm. zool. New Hampshire. Structure 
and natural history of Nudibranchs. 

Moos, C., res. asst. neur. M.I.T. Nature of bioelectric 
potentials of squid nerve. 

Morrison, D., Flight Safety Foundation. Psychology. 

Moskovic, S., fel. phys. New York. Temperature pres- 
sure effects on fert. and cleavage. 

Moul, E., asst. prof. bot. Rutgers. Botany instruction; 
fresh water and marine algae. 

Moulton, J. M., fel. biol. Harvard. 
Menidia and Fundulus. 

Mudd, H., student Harvard Med. Pigments in Homarus 
blood. 

Musacchia, X. J., instr. biol. St. Louis. 
in animals. 

Nachmansohn, D., asst. prof. neurology, Columbia. 
Chemical mechanism of nervous function. 

Nadeau, L. V., grad. stud. phys. Dominican House of 
Studies (Ill.). 

Nelson, L, instr. phys. Nebraska. 
urchin sperm. 

Neurath, H., prof., physical biochem. Duke. Crystalline 
proteolytic enzymes of the pancreas. 

Noland, J. L., fel. biochem. Wisconsin. Determination 
of amino acids in invertebrate’s b'ood. 2 

O’Brien, J. A., Jr., asst. prof. biol. Catholic. Plastid de- 
velopment in germinating grains. 

O’Brien, J. P., asst. prof. zool. Marquette. 
effects of X-radiation. 

O’Malley, B., grad. zool. Fordham. 
the protozoa. 

Orsi, E. V., fel. Cancer Inst. Fordham. “Butter-yellow” 
and develop. of Fundulus and Arbacia. 

Orski, Barbara M., Harvard Med. Rate of respiration 
of protozoa by cartesian diver. 

Osterhout, W. J. V., emerit. phys. Rockefeller Inst. 
Behavior of marine eggs and algae. 

Padykula, Helen, instr. zool. Wellesley. 
shell pigment by land gastropods. 

Palay, S. L., instr. anat. Yale Univ. Sch. Med. 
ological aspects of neurosecretion. 

Parmenter, C., prof. zool. Pennsylvania. Chromosomes 
in frog eggs. 


Enzyme sys- 


Cytological 


Role of specific 


Mieroinjection 
Radiation indueed 
Visual 


Develop- 


Embryology of 


Role of lipids 


Physiology of sea 


Biological 


Growth factors in 


Formation of 


Physi- 


Parport, A. K., chim. biol. Princeton. 
properties of cells. 

Parshley, H. M., chrm. zool. Smith. Comparison of 
north and south New England Hemiptera. 

Perkins, J. F., asst. prof. phys. Chicago. Temperature 
regulation in smooth muscle. 

Pfister, R. R., asst. zoo]. Columbia Med. 

Pick, J., assoc. prof. anat. New York Med. 
physiology of autonomie nervous system. 

Pierce, Madelene, assoc. prof. zool. Vassar. 
survey of Rand Harbor, Megansett. 

Plough, H. H., prof. biol. Amherst. Radiation induced 
mutations in bacteria. 


Permeability 


Anatomy, 


Ecological 


Plummer, Jewel, fel. biol. New York. Arbacia egg 
antimitotic studies. 
Proctor, N. K., grad. phys. Pennsylvania. Effects of 


various reagents on arthropod muscle. 

Prosser, C. L., prof. zool. Illinois. Comparative physi- 
ology of muscle. 

Provasoli, L., chrm. biol. St. Francis. 
eulture of flagellates. 

Quastel, J. H., prof. biochem. MeGill. 

Rawley, June instr. zool. Kent State. 
terial toxins on cell respiration. 

Reichart, Ruth, asst. biochem. Radcliffe. 

Reid, W. M., chim. biol. dept. Monmouth. Physiology 
of marine nemerteans, tapeworm parasites 

Reiner. J. M., res. assoc. Tufts Med. Intermediates of 
peptide bond synthesis in egg. 

Renn, C. E.. assoc. prof. sanitary eng. 
Toxicity of industrial wastes. 

Rice, Mary, res. asst. phys. Oberlin. 
effects on mineral composition. 

Rieser, P., res. asst. phys. Pennsylvania. 
problems in cell physiology. 

Root, R. W., assoc. prof. biol. City of New York. 
toplasmic ultra-structure. 

Rose, S. M., assoc. prof. zool. Smith. Cellular trans- 
formations during regeneration of limbs. 

Rosenbluth, Raja, res. asst. Rockefeller Inst. 

Rosenthal, T., assoc. anat. zool. Washington Med. 

Rossi, H. H., biophysics, Columbia. Biological effects 
of alpha radiation. 
Roth, J. S., asst. prof. biochem. Rutgers. Uptake of 
radioactive phosphate by Tetrahymena geleii. 
Rothenberg, M. A., res. asst. Columbia. Chemistry of 
nerve transmission. 

Roy, S. C., lect. Caleutta. Muscular contraction. 

Roys, C., grad. zool. Iowa. Insect physiology, senses. 

Rudenberg, F. H., A.E.C. fel. phys. Harvard. Uptake 
ard localization of Ca-45 in Arbacia eggs. 

Rugh, R., assoc. prof. radiology, Columbia. Effeets of 
Beta and X-rays on the embryo. 

Saltz, M., med. student, Amherst. Radiation 
mutations in bacteria. 

Sandeen, Muriel I., asst. zool. Northwestern. Compara- 
tive physiology of crustacea. 

Sarkar, N. K., lect. chem. Caleutta. Muscle contraction. 

Schaeffer, A. A., prof. biol. Temple. Leucoeytes of rab- 
hits. 

Schallek, W. B., asst. prof. biol. Oregon. Glycogen in 
nerves of invertebrates. 

€chmitt, F. O., head biol. dept. M. I. T. Nerve structure 
in squid axon. 

Schmitt, O. H., prof. zoo). biophysics, Minnesota. Nerve 
electrophysiology. 

Scholander, P. F., res. assoe. Swarthmore. Heat regula- 
tion in aretie and tropical homo sapiens. 

Schreibman, I.. instr. phys. Pennsylvania. Physiology 
of cell division. 

Sclufer, Evelyn, grad. biol. Bryn Mawr. Embryology. 

Scott, A. C., assoc. prof. biol. Union. Cytology of An- 
oxia in early development of marine eggs. 


Tsolation and 


Effect of bae- 


Johns Hopkins. 
Eneuronimental 
Micrurgieal 


Pro- 


induced 


November, 1949] 


Scott, Sister Florence Marie, prof. biol. Seton Hill. 
Embryology ot Amaroeciun constellatum. 

Scott, G. T., assoc. prof. zool. Oberlin, Enviroumental 
effects on mineral composition. 

Seaman, G. R., fel. phys. Fordham. Enzyme systems in 
Protozoa. 

Seki, S. Louise, grad. asst. phys. Mt. Holyoke. 
of nitrous oxide on Arbacia eggs. 

Shanes, A. M., assoc. prot. phys. & biophys. George- 
town Med. Potassium transport in invertebrate 
nerve. 

Shwartzman, G., bacteriologist, Mt. Sinai Hosp. 

Sheng, T. C., grad. zool. Columbia. Neurospora genetics. 

Sichel, F. J. M., prof. phys. Vermont Med. Conduction 
in voluntary and cardiae muscle. 

Slattery, L. F., electronic tech. Chicago. 
tion in squid. 

Slifer, Eleanor, asst. prof. zool. lowa. Cytology of wax 
secretion—grasshopper eggs. 

Speidel, C. C., prof. anat. Virginia. Cellular behaviour 
in vivo; cine-photomicrography. 

Stein, O. L., grad. asst. Minnesota. Pollen ontogeny. 

Steinbach, H. B., prof. zool. Minnesota. Distribution of 
enzymes in muscle anid nerve tissue. 

Stieglitz, Alice A., grad. phys. Pennsylvania. General 
physiology. 

Stokey, Alma G., emerit. prof. bot. Mt. Holyoke. Ga- 
metophyte of homosporous ferns. 

Stoudt, H. N., asst. prof. biol. & plant morph. Temple. 
Vegetative propagation in Oxalic ortgiesi. 

Stout, Caroiyn M., res. asst. Pennsylvania. Permeabhil- 
ity of red blood cells. 

Straus, W. L., assoc. prof. anat. Johns Hopkins. Somites 
and lateral plate contribution to body wall. 

Atrittmatter, C. F.. Harvard. Particulate systems of 
Arbacia eggs. 

Stunkard, H. W., prof. biol. New York. 
life history of parasitic worms. 
Suckling, E. E.. instr. phys. Long Island Med. 

fiber recording of nerve impulses. 

Sulkin, S. E., prof. bacteriology, Southwestern Med. 
Studies on the epidemiology of virus encephali- 
tides. 

Sutro, P. J., grad. phys. Harvard. Reading on problems 
in vision. 

Szent-Gyorgyi, A., res. asst. Inst. of Muscle Res. Ionic 
influence on the contractile proteins. 

Talpey, W. B., Washington Med. Calcium binding by 
cell surfaces. 

Tannenbaum, S., grad. biol. Columbia. 

Taylor, L. S., chief biophysics A.E.C. Nat. Bureau 
Standards. High energy radiation measurement. 

Taylor, W. R., prof. bot. Michigan. Woods Hole ant 
Bermuda marine algae. 

Terry, R., asst. prof. biol. Union. Physiology of Arbacin 
eggs. 

TeWinkel, Lois E., assoc. zool. Smith. Development of 
muscle in dog fish embryos. 

Therman, P. O., physician, Inst. of Pa. Hospital. Prop- 
erties of motor-nerve fibers. 

Thomson, Betty F., asst. prof. bot. Connecticut Col. 

Tietze, F., res. fel. biochem. Northwestern. Chromatog- 
raphy of hemocyanin. 


Effect 


Nerve fune- 


Biology and 


Single 


THE COLLECTING NET 31 


Ting, T., res. assoc. biophys. Amherst. Studies on cal- 
cium uptake by Arbacia eggs. 

racy, H., prof. anat. Kansas. Development of neural 
mechanisms in Opsanus tau. 

Trinkaus, J. P., instr. zool. Yale. 
lation ia teleosts. 

Truant, A. P., asst. prof. pharm. George Washington. 
Distribution of procaine in squid axon. 
Tyler, A., assoc. prof. embr. Calif. Inst. Tech. 

ology of fertilization. 

Varga, L., res. asst. phys. Inst. for Muscle Res. Ther- 
modynamies of muscular contraction. 
Vincent, W. S., A.E.C. fel. biol. Pennsylvania. 

chemistry of nucleoli. 
Vinson, C. A., grad. asst. zool. North Carolina. Cold 
treatment of fertilized eggs of Nereis limbata. 
Vogel, M. L., res. asst. bact. Amherst. Bio-chemical 
varieties in Salmonella typhimurium. 

Wainio, W. W., assoc. res. specialist, Rutgers. 

Wald, G., prof. biol. Harvard. Chemistry, physiology of 
light reactions of organisms. 

Walters, C. Patricia, res. asst. Lilly Res. Labs. Particu- 
late systems of Arbacia eggs. 

Warner, RB. C., asst. prof. chem. New York Med. Rate 
of denaturation of proteins. 

Webb, H. Marguerite, asst. zool. Northwestern. 
parative physiology of crustacea. 

Weber. Patricia, res. asst. biol. St. Louis. Invertebrate 
biochemistry. 

Wenr:ch, D. H., prof. zool. Pennsylvania. 

West, Alice, Radcliffe. Oxidation-reduction studies. 

Whiting, P. W., prof. zool. Pennsylvania. Genetics of 
Hymenoptera. 

Wichterman, R., assoc. prof. biol. Temple. X-ray effect 
on mating in Paramecium. 

Wilber, C. G., dir. biol. lab. St. Louis. Effect 
vironment on invertebrate body fluids. 

Willier, B. H., dir. biol. labs. Johns Hopkins. 

Wilson, Marie, asst. zool. Northwestern. Assistant on 
the invertebrate zoology course. 

Wilson, T. H., instr. phys. Pennsylvania. 
molysis of human erythrocytes. 

Wilson, W. L., res. assoc. phys. Pennsylvania. Radia- 
tion on cell division and clotting. 

Winblad, J. N., instr. anat. Kansas Med. Development 
of Lehaviour and motility in Opsanus tau. 

Wittenberg, J., grad. biochem. Columbia. 

Wood, R. D., instr. bot. Rhode Island. Instruction in 
botany. 

Woodward, A. A., asst. prof. zool. Brown. Localization 
of enzymes in protoplasmic granules. 

Woodward, A. E., asst. prof. zool. Michigan. Effects of 
some vitamins on Hichinoderms. 

Wrinch, Dorothy, lect. physics, Smith. Structure of na- 
tive proteins. 

Wulf, V. J. asst. prof. physiol. Illinois. Physiology of 
optic pathway of Limulus and Loligo. 

Zolokar, M., res. fel. Calif. Inst. Tech. Fertilization 
studies on Nereis. 

Zeuthen, E., lect. Copenhagen. Respiratory metabolism 
of cell division. 

Zorzoli, Anita, asst. prof. Sch. of Dentistry, Washing- 
ton. 


Mechanism of gastru- 


Physi- 


Cyto- 


Com- 


of en- 


Osmotic he- 


THE M.B.L. CLUB IN 1949 


Dr. Roperts RueH 
President, M.B.L. Club for 1949; Columbia University 


The Marine Biological Laboratory Clubhouse 
has a long history, dating back to when it was a 
yacht club. Never has it been so used and useful 
as this summer. Some 483 full-term members 


have entered its portals and have benefited by 
its existence. In addition there were 53 weekly 
members. 

The M.B.L. Club is maintained for the pleas- 


32 THE COLLECTING NET 


[Vol. XIX, No. 2 


ure, convenience and recreation of laboratory 
personnel. Its membership is therefore limited to 
those connected with the laboratory or their im- 
mediate adult relatives. Workers at the Ocean- 
ographic Institution or the United States Bureau 
of Fisheries are admitted to special membership 
and a very limited number of patrons are ad- 
mitted, upon favorable action of the Executive 
Committee. The dues in these special cases are 
somewhat more than for the M.B.L. personnel, as 
it rightly should be. Unfortunately, the physical 
facilities of the Clubhouse make it necessary to 
limit the membership to adults, but it is hoped 
that the Laboratory will soon provide recrea- 
tional facilities for the younger people as well. 

The Club sponsors four regular weekly events : 
the Sunday Evening Home Talent Concert, the 
onday Evening Classical Record Concert, Thurs- 
day Evening Square Dancing with instruction, 
and Saturday Evening Ballroom Dancing with 
refreshments. The dances were under the direc- 
tion of the Social Chairman, Miss Ruth Alsher. 
These events have all been very well attended— 
in the case of one of the concerts fifty-five mem- 
bers were seen lying on the grass around the 
building within hearing distance. All floor and 
stair space was occupied. For the square dances 
Dr. Bartlett and Connie Mitchell have had as 
many as six groups for instruction and dancing. 

The Home Talent Concerts were under the di- 
rection of Dr. Walter Wainio, the president of 
the Club for 1950. These concerts attract some 
members who may not participate in any other 
activity of the Club. Fortunately, the club had 
the talented service of Max Pepper, a most ae- 
complished pianist who not only carried a heavy 
burden of accompanying but also of solo work. 
Miss Withrow, Mrs. Kisch and Dr. Wainio sang, 
and there were violin, flute and other instrumen- 
tal solos. 

The Monday Evening Classical Record Con- 
cert was of very high caliber due to the gener- 
osity of townspeople and friends of the Ocean- 


ographic Institution who loaned their valuable 
records. Mrs. Daniel Mazia organized the con- 
certs and Dr. M. Brust directed them. 

The dances were so well attended that the 
new Vice-President, Dr. Bartlett, is beginning a 
campaign to raise funds for the construction of 
a hall measuring about twenty by fifty feet 
which would adjoin the present building along 
the waterfront to the West. This large room 
would be used for the dances, relieving the pres- 
ent quarters for the more sedentary activities 
such as bridge, chess, checkers, reading, ete. It 
will probably also have a large sundeck on top. 
There is no question but that the membership 
has far outgrown the physical facilities of the 
present Clubhouse. 

During the present season the Executive Com- 
mittee purchased a piano aided by a gift from 
Mrs. Frost. They also purchased a Sonomaster 
record player and speaker for both regular and 
long-playing records, equipped with a micro- 
phone for calling at the square dances and for 
announcements. With a slight increase in the 
dues and four boat excursions (around the Is- 
lands, Edgartown Regatta, Gay Head, and Cut- 
tyhunk) for which there was a nominal charge, 
the Executive Committee left several hundred 
dollars in the treasury for the use of the incom- 
ing officers, to the pleasure of the new Secretary- 
Treasurer, Dr. Ralph Cheney. 

The major credit for the very successful sea- 
son goes to the Hostess, Miss Mary Lou Failla. 
She opened the Club on June 15 and closed it on 
September 15; during the interim she became ac- 
quainted with everyone who used the Clubhouse. 
Never before has the Club had such an atmos- 
phere of friendly welcome as it has through her 
good humor and fine sense of balance. It was 
Miss Failla’s generous spirit and her whole- 
hearted cooperation, far beyond the requirements 
of her position, that made the M.B.L. Club such 
an integral part of the lives of so many labora- 
tory people this summer. 


SYNTHESIS OF ACETYLCHLORINE 


(Continued from page 26) 


line hydrolysis the minimum amount of cholines- 
terase would be adequate to split the acetylcho- 
line released by two to three million impulses 
per gm per hour. Both the chemical and the 
thermodynamic data are thus in agreement with 
the assumption of the necessity of the acetylcho- 
line-esterase system for conduction. 

Nore: Based on a paper presented at the Marine Bio- 
logical Laboratory. 

References 


(1) Naechmansohn, D., Bull. Johns Hopkins Hosp., 83: 
463 (1948). 


(2) Nachmansohn, D., Biochimica et Biophysica Acta, 
Meyerhof Festschrift, 4: 78 (1950). 

(3) Nachmansohn, D., Cox, R. T., Coates, C. W., and 
Machado, A. L., J. Neurophysiol., 6: 383 (1943). 

(4) Naechmansohn, D., and Machado, A. L., J. Neuro- 
physiol., 6: 397 (1943). 

(5) Nachmansohn, D., and Berman, M., J. Biol. Chem., 
165: 551 (1946). 

(6) Nachmansohn, D., and Weiss, M. S., J. Biol. Chem., 
172: 677 (1948). 


(7) Nachmansohn, D., Hestrin, S., and Voripaieff, H., 
J. Biol. Chem., 180: 875 (1949). 


(8) Hestrin, S., J. Biol. Chem., 180: 249 (1949). 

(9) Middleton, S. and H. H., Proc. Soc. exp. Biol., 
N. Y., 71: 523 (1949). 

(10) Hestrin, S., J. Biol. Chem., 180: 879 (1949). 


November, 1949] 


THE COLLECTING NET 33 


THE REACTIVATION OF THE NARRAGANSETT MARINE LABORATORY 


Dr. Donaup J. ZINN, Research Associate 


In July of this year, the Narragansett Marine 
Laboratory of Rhode Island State College, newly 
reactivated under the directorship of Dr. Charles 
J. Fish, opened its doors for the first time since 
the summer of 1941, to scientists interested in re- 
search in marine biology. The Laboratory is situ- 
ated about three miles from the entrance to the 
western passage of Narragansett Bay, at Fort 
Kearney. It is less than thirty-five nautical miles 
from Woods Hole. 

Working with Dr. Fish on the life history of 
Venus mercenaria in Narragansett Bay are Dr. 
David M. Pratt, assistant professor of marine 
biology at Rhode Island State College, and Dr. 
Donald J. Zinn, assistant professor of zoology at 
the State College. Marie P. Fish is in charge of 
a Navy project on the production of sound in 
marine animals, and is being assisted by Alton 
Kelsey of the Woods Hole Oceanographic Insti- 
tution and John Kelley of Rhode Island State 
College. Dr. D. Eugene Copeland of Brown 
University is continuing work started last sum- 
mer at the Marine Biological Laboratory on the 


methods of adaptation of anadromous and eata- 
dromous fish. The U.S. Fish and Wildlife Serv- 
ice is working on a problem concerning the varia- 
tion in population of Venus mercenaria in Nar- 
ragansett Bay. Warren Landers, formerly of 
the Marine Biological Laboratory, is in charge of 
this project and is assisted by Thomas Kane. 

The Laboratory, through Rhode Island State 
College, has established in cooperation with the 
Woods Hole Oceanographic Institution a gradu- 
ate student training program in biological ocean- 
ography and marine fisheries biology. Instruction 
and supervision of research will be provided by 
members of the staffs of the two institutions, em- 
phasis being placed on open ocean investigations. 
Arrangements are being made for a limited num- 
ber of students who will enroll as candidates for 
the degree of Master of Science in marine bi- 
ology. The first year will be spent in Kingston 
and at the Narragansett Marine Laboratory. It 
is expected that students will spend the second 
year either at the Woods Hole Oceanographic 
Institution or at the Woods Hole Station of the 
U.S. Fish and Wildlife Service. 


THE INVERTEBRATE ZOOLOGY COURSE AT THE MARINE BIOLOGICAL LAEORATORY 


Dr. F. A. Brown, JR. 
Instructor in Charge; Professor of Zoology, Northwestern University 


The invertebrate zoology course, the oldest of 
the courses at Woods Hole, began another season 
Tuesday evening, July 26. Fifty-six students 
were enrolled. The demand for this course con- 
tinues to climb upwards, with the number of ap- 
plications now exceeding the number of available 
places nearly threefold. This pressure has re- 
sulted in a gradual reduction in the number of 
undergraduates admitted. 

In charge of the course was Dr. F. A. Brown, Jr., 
professor of zoology at Northwestern University. 
Other members of the Senior Staff were Dr. W. 
D. Burbanck, professor of biology, Drury Col- 
lege; Dr. C. G. Goodchild, professor of biology, 
Missouri State College, Southwest; Dr. Libbie 
H. Hyman, the American Museum of Natural 
History; Dr. L. H. Kleinholz, associate professor 
of biology, Reed College; Dr. J. H. Lockhead, 
assistant professor of zoology, the University of 
Vermont; Dr. Madelene E. Pierce, associate pro- 
fessor of zoology, Vassar College; Dr. W. M. 
Reid, professor of biology, Monmouth College, 
and Dr. T. H. Waterman, assistant professor of 
biology at Yale University. Members of the 


Junior Staff were Robert S. Howard, assistant in 
zoology, University of Miami and Marie Wilson, 
assistant in zoology, Northwestern University. 

The course opened with a lecture by Dr. G. L. 
Clarke, entitled ‘‘The Sea as an Environment,’” 
which began a series of six lectures on oceanog- 
raphy presented by members of the staffs of the 
Woods Hole Oceanographic Institution and the 
Woods Hole United States Bureau of Fisheries. 
Other lectures in the series were: ‘‘Tides’’ by 
Dr. William S. von Arx; ‘‘Chemical Problems of 
the Sea’’ by Dr. A. C. Redfield; ‘‘Oceanic Plank- 
ton’’ by Dr. Mary Sears; ‘‘Geographical Distri- 
bution of Marine Animals’’ by Dr. Louis W. 
Hutchins; ‘‘ Natural Resources of the Sea’’ by Dr. 
Paul S. Galtsoff. These lectures provided the stu- 
dents with an unusual opportunity to learn basic 
problems of the ocean from individuals especial- 
ly qualified to speak about them. 

The more conventional portion of the course 
involved a series of about thirty lectures and ap- 
proximately an equal number of laboratory pe- 
riods presented by members of the regular staff. 
In addition there were nine field expeditions. 


34 THE COLLECTING NET 


| Vol. XLX, No. 1 


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36 


THE COLLECTING NET [Vol. XTX, No. 1 


Outstanding MCGRAW-HILL Books 


ISOTOPIC TRACERS AND NUCLEAR RADIATIONS WITH APPLICATIONS TO 
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By WictiamM Sirti. With contributions by ELLswortH C. DouGHERTY, CoRNELIus A. 
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University of California. 653 pages, $12.50 
Bridges the gap between those books intended solely for the nuclear physicist and those 
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NATURAL HISTORY OF MARINE ANIMALS 
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A practical, yet thoroughly scientific treatise on the ocean as an environment, and of the 
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PLANT AND SOIL WATER RELATIONSHIPS 
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Gives an integrated discussion of the various factors which affect the absorption of water by 
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THE COLLECTING NET 37 


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INTROGRESSIVE HYBRIDIZATION 


By Edgar Anderson 


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1949 109 pages 


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38 THE COLLECTING NET [ Vol. XIX, No. 1 


Jus 
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40 THE COLLECTING NET [ Vol. XIX, No. 1 


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42 THE COLLECTING NET [ Vol. XLX, No. 1 


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THE COLLECTING NET 43 


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44 THE COLLECTING NET [Vol. XIX, No.1 


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