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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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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]
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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
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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
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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-
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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 |
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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
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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 |
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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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THE COLLECTING NET 37
4 new WILEY books — 4 practical subjects INTROGRESSIVE HYBRIDIZATION
By Edgar Anderson
This book in the Wiley Biological Research Series shows methods for studying hybridization in the field, so that one may measure its effects in natural populations. A unique feature is the sec- tion showing how to make detailed taxonomical descriptions of a species without seeing it. With
1949 109 pages
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SELECTED INVERTEBRATE TYPES
Largely written by staff members of the Woods Hole Laboratory, this is an informative— rather than tersely “directive’—laboratory guide to the study of about 100 common American in- vertebrate animals. It includes the most widely
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38 THE COLLECTING NET [ Vol. XIX, No. 1
Jus Published VERTEBRATE EMBRYOLOGY
Third Edition
ROBERT S. McEWEN, Oberlin College
In this revision of a distinguished text the whole book has heen rewritten and brought completely up to date, and an important new section on the Pig has been added to make it a comprehensive account of the embryology of chordate forms. The new edition contains an introductory section on developmental processes in general, especially as they occur in vertebrates. From this it proceeds to describe the early developments of Amphioxus, the Frog, the Fish and Gymnophiona. the Chick and the Pig. <A valuable feature is the inclusion of the most recent and significant experimental data. The many new and redrawn illustrations also add to the clarity and teachability of this new edition of a widely used book.
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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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