The Organism as a Whole, from a Physicochemical Viewpoint

throws a different light upon the seemingly purposeful character of animal reactions. Heliotropism depends primarily upon the presence of photosensitive substances in the eye or the epidermis of the organism, and these substances are inherited regardless of whether they are useful or not. It is only a metaphor to call reactions resulting from the presence of photosensitive substances ' ' adaptation. ' ' In this book other examples are given which show that authors have too often spoken of adaptation to environment where the environment was not responsible for the phenomena. The blindness of cave animals and the resistance of certain marine animals to higher concentrations of sea water are such cases. Cuenot speaks of "preadaptation" to express this relation. The fact is that the "adaptations" often existed before the animal was exposed to surroundings where they were of use. This relieves us also of the necessity of postulating the existence of the inheritance of acquired characters, although it is quite possible that the future may furnish proof that such a mode of inheritance exists.

6. We have mentioned that according to Claude Bernard two groups of phenomena occur in the living organism: (i) the phenomena of vital creation or organizing synthesis (especially in the egg and during development); (2) the phenomena of death or organic destruction. These two processes are briefly discussed in the first and last chapters. These introductory remarks may perhaps make it easier for the reader to retain the thread of the main ideas in the details of experiments and tables given in this book.

I. Each organism is characterized by a definite form and we shall see in the next chapter that this form is determined by definite chemical substances. The same is true for crystals, where substance and form are definitely connected and there are further analogies between organisms and crystals. Crystals can grow in a proper solution, and can regenerate their form in such a solution when broken or injured; it is even possible to prevent or retard the formation of crystals in a supersaturated solution by preventing "germs" in the air from getting into the solution, an observation which was later utilized by Schroeder and Pasteur in their experiments on spontaneous generation. However, the analogies between a living organism and a crystal are merely superficial and it is by pointing out the fundamental differences between the behaviour cf crystals and that of living organisms that we can best

understand the specific difference between non-living and living matter. It is true that a crystal can grow, but it will do so only in a supersaturated solution of its own substance. Just the reverse is true for living organisms. In order to make bacteria or the cells of our body grow, solutions of the split products of the substances composing them and not the substances themselves must be available to the cells ; second, these solutions must not be supersaturated, on the contrary, they must be dilute; and third, growth leads in living organisms to cell division as soon as the mass of the cell reaches a certain limit. This process of cell division cannot be claimed even metaphorically to exist in a crystal. A correct appreciation of these facts will give us an insight into the specific difference between non-living and living matter. The formation of living matter consists in the synthesis of the proteins, nucleins, fats, and carbohydrates of the cells, from the split products. To give an historical example, Pasteur showed that yeast cells and other fungi could be raised on the following sterilized solution : water, loogm., crystallized sugar, 10 gm., ammonium tartrate, 0.2 gm. to 0.5 gm., and fused ash from yeast, o.i gm. T He undertook this experiment to disprove the idea that protein or organic matter in a state of decomposition was needed for the origin of new organisms as the defenders of the idea of spontaneous generation had maintained.

2. That such a solution can serve for the synthesis of all the compounds of living yeast cells is due to the fact that it contains the sugars. From the sugars organic acids can be formed and these with ammonia (which was offered in the form of ammonium tartrate) may give rise to the formation of amino acids, the "building stones" of the proteins. It is thus obvious that the synthesis of living matter centres around the sugar molecule. The phosphates are required for the formation of the nucleins, and the work of Harden and Young suggests that they play also a r61e in the alcoholic fermentation of sugar.

Chlorophyll, under the influence of the red rays of light, manufactures the sugars from the CO2 of the air. This makes it appear as though life on our planet should have been preceded by the existence of chlorophyll, a fact difficult to understand since it seems more natural to conceive of chlorophyll as a part or a product of living organisms rather than the reverse. Where then should the sugar come from, which is a constituent of the majority of culture media and which seems a prerequisite for the synthesis of proteins in living organisms?

The investigations of Winogradsky on nitrifying,1 sulphur and perhaps also on iron bacteria have to all appearances pointed a way out of this difficulty. It seemed probable that there were specific micro-organisms which oxidized the ammonia formed in sewage or in the putrefaction of living matter, but the attempts to prove this assumption by raising such a nitrifying micro-organism on one of the usual culture media, all of which contained organic compounds, failed. Led by the results of his observations on sulphur bacteria it occurred to Winogradsky that the presence of organic compounds stood in the way of raising these bacteria, and this idea proved correct. The bacteria oxidizing ammonia to nitrites were grown on the following medium ; I gm. ammonium sulphate, I gm. potassium phosphate, I gm. magnesium carbonate, to I litre of water. From this medium, which is free from sugar and contains only constituents which could exist on the planet before the appearance of life, the nitrifying bacteria were able to form sugars, fatty acids, proteins, and the other specific constituents of living matter. Winogradsky proved, by quantitative determination, that with the nitrification an increase in the amount of carbon compounds takes place. "Since this bound carbon in the cultures can have no other source than the CO2 and since the process itself can have no other cause than the activity of the nitrifying organism, no other alternative was left but to ascribe to it the power of assimilating CO,."1 ''Since the oxidation of NH3 is the only source of chemical energy which the nitrifying

organism can use it was clear a priori that the yield in assimilation must correspond to the quantity of oxidized nitrogen. It turned out that an approximately constant ratio exists between the values of assimilated carbon and those of oxidized nitrogen." This is illustrated by the results of various experiments as shown in Table I. It is obvious that I part of assimilated carbon corresponds to about 35.4 parts oxidized nitrogen or 96 parts of nitrous acid.

These results of Winogradsky were confirmed in very careful experiments by E. Godlewski, Sr. x The nitrites are further oxidized by another kind of micro-organisms into nitrates and they also can be raised without organic material. the hydrogen sulphide which is formed as a reduction product from CaS04 or in putrefaction by the activity of certain bacteria can be oxidized by certain groups of bacteria, the sulphur bacteria. Such bacteria, e. g., Beggiatoa, are also commonly found at the outlet of sulphur springs. They utilize the hydrogen sulphide which they oxidize to sulphur and afterwards to sulphates, according to the scheme :

Winogradsky assumes that the oxidation of H2S by the sulphur bacteria is the source of energy which plays the same r61e as the oxidation of NH3 plays in the nitrifying bacteria, or the oxidation of carbon compounds — sugar and others — in the case of the other lower and higher organisms. Winogradsky has made it very probable that sulphur bacteria do not need any organic compounds and that their nutrition may be accom- W plished with a purely mineral culture medium, like that of the nitrite bacteria. On the basis of this assumption they should also be able to form sugars from the CO a of the air.

of bacteria which oxidize thiosulphate to sulphuric acid. They will develop if some Na2S203 is added to sea water. These bacteria can only develop if C0a from the air is admitted or when carbonates are present. For these organisms the CO2 cannot be replaced by glucose, urea, or other organic substances. Such bacteria must therefore possess the power of producing sugar and starch from C02 without the aid of chlorophyll. Similar observations were made by Beijerinck on a species of fresh-water bacteria. x

Finally the case of iron bacteria may briefly be mentioned though Winogradsky's views are not accepted by Molisch. We may, therefore, consider it an established fact that there are a number of organisms which could have lived on this planet at a time when only mineral 7 constituents, such as phosphates, K, Mg, S04, CO2, \ and O2 besides NH3, or SH2, existed. This would lead us to consider it possible that the first organisms on this planet may have belonged to that world of micro-organisms which was discovered by Winogradsky.

If we can conceive of this group of organisms as producing sugar, which in fact they do, they could have served as a basis for the development of other forms which require organic material for their development. In 1883 the small island of Krakatau was destroyed by the most violent volcanic eruption on record. A visit to the islands two months after the eruption showed that "the three islands were covered with pumice and layers of ash reaching on an average a thickness of thirty metres and frequently sixty metres." z Of course all life on the islands was extinct. When Treub in 1886 first visited the island, he found that blue-green algss were the first colonists on the pumice and on the exposed blocks of rock in the ravines on the mountain slopes. Investigations made during subsequent expeditions demonstrated the association of diatoms and bacteria. All of these were probably carried by the wind. The algas referred to were according to Euler of the nostoc type. Nostoc does not require sugar, since it can produce that compound from the C02 of the air by the activity of its chlorophyll. This organism possesses also the power of assimilating the free nitrogen of the air. From these observations and because the Nostocacecz generally appear as the first settlers on sand the conclusion has been drawn that they or the group of Schizophycece to which they belong formed the first settlers of our planet.2 This conclusion is not quite safe since in the settlement of Krakatau as well as in the first colonizing of sand

1 Ernst, A., The New Flora of the Volcanic Island of Krakatau, Cambridge, 19-8. areas the nature of the first settler is determined chiefly by the carrying power of wind (or waves and birds). We may now return from this digression to the real object of our discussion, namely that the nutritive solutions of organisms must be very dilute and consist of the split products of the complicated compounds of which the organisms consist. The examples given sufficiently illustrate this statement.

The nutritive medium of our body cells is the blood, and while we take up as food the complicated compounds of plants or animals, these substances undergo a digestion, i. e., a splitting up into small constituents before they can diffuse from the intestine into the blood. Thus the proteins are digested down to the amino acids and these diffuse into the blood as demonstrated by Folin and by Van Slyke. From here the cells take them up. The different proteins differ in regard to the different types of amino acids which they contain. While the bacteria and fungi and apparently the higher plants can build up all their different amino acids from ammonia, this power is no longer found in the mammals which can form only certain amino acids in their body and must receive the others through their food. As a consequence it is usually necessary to feed young animals on more than one protein in order to make them grow, since one protein, as a rule, does not contain all the amino acids needed for the manufacture of all

the proteins required for the formation of the material of a growing animal. x 3. The essential difference between living and nonliving matter consists then in this: the living cell synthetizes its own complicated specific material from indifferent or non-specific simple compounds of the surrounding medium, while the crystal simply adds the molecules found in its supersaturated solution. This synthetic power of transforming small "building stones" into the complicated compounds specific for each organism is the "secret of life" or rather one of the secrets of life.

What clew have we in regard to the nature of this synthetic power? We know that the comparatively great velocity of chemical reactions in a living organism is due to the presence of enzymes (ferments) or to catalytic agencies in general. Some of these catalytic agencies are specific in the sense that a given catalyzer can accelerate the reaction of only one step in a complicated chemical reaction. While these enzymes are formed by the action of the body they can be separated from the body without losing their catalytic efficiency. It was a long time before scientists succeeded in isolating the enzyme of the yeast cell which causes the alcoholic fermentation of sugar; and this gave rise to the

1 This fact was thoroughly established by Mendel and Osborne. A summary of their work is given in Underhill, F. P., Physiology of the Amino Acids, 1916. premature statement that it was not possible to isolate this enzyme since it was bound up with the life of the yeast cell. Such a statement was even made by a man like Pasteur, who was usually a model of restraint in his utterances, and yet the work of Buchner proved him to be wrong. The general mechanism of the action of the hydrolyzing enzymes is known. The old idea of de la Rive, that a molecule of enzyme combines transitorily with a molecule of substrate; the further idea, which may possibly go back to Engler, that the molecule of substrate is disrupted in the "strain" of the new combination and that the broken fragments fall off or are easily knocked off by collision from the ferment molecule which is now ready to repeat the process, seems to be correct. On the assumption that the velocity of enzyme reaction is proportional to the mass of the enzyme and that de la Rive's idea was correct, Van Slyke and Cullen were able to calculate the coefficients of the velocity of enzyme reactions for the fermentation of urea and other substances, and the agreement between calculated and observed values was remarkable. x

While the hydrolytic action of enzymes is thus clear the synthesis in the cell is still a riddle. An interesting suggestion was made by van't Hoff, who in 1898 expressed the idea that the hydrolytic enzymes should also act in the opposite direction, namely synthetically. Thus it should not only be possible to digest proteins with pepsin but also to synthetize them from the products of digestion with the aid of the same enzyme. This expectation was based on the idea that the enzyme did not alter the equilibrium between the hydrolyzed and non-hydrolyzed part of the substrate but only accelerated the rate with which the equilibrium was reached. Van't Hoff's idea omitted, however, the possibility that in the transitory combination between enzyme molecule and substrate a change in the molecular configuration of the substrate or in the distribution of intramolecular strain may take place. The first apparently complete confirmation of van't HofFs suggestion appeared in the form of the synthesis of maltose from grape sugar by the enzyme maltase, which decomposes maltose into grape sugar. By adding the enzyme maltase from yeast to a forty per cent, solution of glucose Croft Hill1 obtained a good yield of maltose. It turned out, however, that what he took for maltose was not this compound but an isomer, namely isomaltose, which has a different molecular configuration and cannot be hydrolyzed by the enzyme maltase.

Lactose is hydrolyzed from kephyr by an enzyme lactase into galactose and glucose; by adding this enzyme to galactose and glucose a synthesis was obtained not of lactose but of isolactose; the latter, however, is not decomposed by the enzyme lactase. E. F. Armstrong has worked out a theory which tries to account for this striking phenomenon by assuming "that the enzyme has a specific influence in promoting the formation of the biose which it cannot hydrolyze."1 The theory is very ingenious and seems supported by fact. This then would lead to the result that certain hydrolytic enzymes may have a synthetic action but not in the manner suggested by van't Hoff.

The principle enunciated by Armstrong, that in the synthetic action of hydrolytic enzymes not the original compound but an isomer is formed which can not be hydrolyzed by the enzyme, may possibly be of great importance in the understanding of life phenomena. It shows us how the cell can grow in the presence of hydrolytic enzymes and why in hunger the disintegration of the cell material is so slow. It was at first thought that the formation of isomers contradicted the idea of the reversible action of enzymes, but this is not the case ; on the contrary, it supports it but makes an addition which may solve the riddle of what Claude Bernard called the creative action of living matter. We shall come back to this problem in the last chapter.

Kastle and Loevenhart demonstrated the synthesis of a trace of ethylbutyrate by lipase if the latter enzyme was added to the products of the hydrolysis of ethylbutyrate, ethyl alcohol, and butyric acid by the same enzyme.1 Taylor2 obtained the synthesis of a slight amount of triolein by the addition of the dried fat-free residue of the castor bean to a mixture of oleinic acid and glycerine. . . . No synthesis occurred with acetic, butyric, palmitic, and stearic acids with glycerine, mannite, and dulcite, and the experiments with the last two alcohols and oleinic acid likewise yielded no synthesis.

This suggests possibly a specific action of the enzyme. If this slight reversible action had any biological significance (which might be possible, since in the organism secondary favourable conditions might be at work which are lacking in vitro) there should be a parallelism between masses of lipase in different kinds of tissues and fat synthesis. Loevenhart indicated that this might be a fact, but a more extensive investigation by H. C. Bradley has made this very dubious. 3

Very little is known concerning the reversible action of the hydrolytic protein enzymes. A. E. Taylor digested protamine sulphate with trypsin and found that after adding trypsin to the products of digestion a precipitate was formed after long standing; and we may also refer to experiments of Robertson with pepsin on the products of caseinogen to which we shall return in the next chapter. It therefore looks at present as if van't Kofi's idea of reversible enzyme action might hold in the modification offered by Armstrong. It remains doubtful, however, whether this reversibility can explain all the synthetic processes in the cell. No objection can be offered at present if any one makes the assumption that each cell has specific synthetic enzymes or some other synthetic mechanisms which are still unknown.

The mechanisms for the synthesis of proteins must have one other peculiarity: they must be specific in their action. We shall see in the next chapter that each species seems to possess one or more proteins not found in any other but closely related species. Each organism develops from a tiny microscopic germ and grows by synthetizing the non-specific building stones (ammo acids) into the specific proteins of the species. This must be the work of the yet unknown synthetic enzymes or mechanisms. The elucidation of their character would seem one of the main problems of biology. Needless to say crystallography is not confronted with problems of such a nature.

The fact that the living cell grows after taking up food has given rise to curious misunderstandings. Traube has shown that drops of a liquid surrounded with a semipermeable membrane may increase in volume when put into a solution of lower osmotic pressure. This has led and is possibly still leading to the statement that the process of growth by a living cell has been imitated artificially. Only one feature has been imitated, the increase in volume; but the essential feature of the process in the living cell, i. e., the formation of the specific constituents of the living cell from non-specific products, has of course not been imitated. 4, The constant synthesis then of specific material from simple compounds of a non-specific character is the chief feature by which living matter differs from non-living matter. With this character is correlated another one; namely, when the mass of a cell reaches a certain limit the cell divides. This is perhaps most obvious in bacteria which on the proper nutritive medium take up food, grow, and divide into two bacteria, each of which takes up food, divides, and grows ad infinitum, as long as the food lasts, provided the harmful products of metabolism are removed. If it be true that specific synthetic ferments exist in each cell it follows that the cell must synthetize these also,1

1 This would lead to the idea that the enzymes in the cell also synthetize molecules of their own kind, or that, in other words, the synthetic processes in the cell are of the nature of autocatalysis. Loeb, Der chemische Character des Befruchtungsvor gangs, Leipzig, 1908. Ro- bertson, T. B., Arch. f. Entwicklngsmech., 1908, xxv., 581; xxvi., 108; 1913, xxxvii., 497; Am. Jour. Physiol., 1915, xxxvii., i; Robertson and Wasteneys, H., Arch. f. Entwicklngsmech., 1913, xxxvii., 485. Ostwald, Wo., Uber die zeitlichen Eigenschaften der Entwicklungsvorgdnge, Leipzig,

as otherwise the synthesis of specific proteins would have to come to a standstill. This problem of synthesis leads to the assumption of immortality of the living cell, since there is no a priori reason why this synthesis should ever come to a standstill of its own accord as long as enough food is available and the proper outside physical conditions are guaranteed. It is well known that Weismann has claimed immortality for all unicellular organisms and for the sex cells of metazoa, while he claimed the necessity of death for the body cells of the latter. Leo Loeb was led by his investigations on the transplantation of cancer to assume immortality not only for the cancer cell but also for the body cell of the organism. He had found in transplanting a malignant tumor from one individual to another that the tumor grew; that it was not the cells of the host but the transplanted tumor cells of the graft which grew and multiplied, and that this process could be repeated apparently indefinitely so that it was obvious that the transplanted tumor cells outlived the original animal. Such experiments have since been carried on so long that we may now say that an individual cancer cell taken from an animal and transplanted from time to time on a new host lives apparently indefinitely. Leo Loeb had found that these tumor cells are simply modified somatic cells. He therefore suggested that the somatic cells might be considered immortal with the same right

as we speak of the immortality of the germ cells of such animals. l This view receives its support first from the fact that certain trees like the Sequoia live several thousand years and may therefore be considered immortal ; and second, from the method of tissue culture. The method of cultivating tissue cells in a test tube, in the same way as is done for bacteria, was first proposed and carried out by Leo Loeb, in i897,2 but his test-tube method did not permit the observation of the transplanted cell under the microscope. This was made possible by a modification of the method by Harrison, who established the fact that the axis cylinder grows out from the ganglionic cell. Harrison and Burrows then perfected the method for the cultivation of the cells of warmblooded animals, and with the aid of these methods Carrel succeeded in keeping connective-tissue cells of the heart of an early chick embryo alive more than four years, and these cells are still growing and dividing.3 Only very tiny masses of cells can be kept alive in this way since all the cells in the centre of a piece die on account of .lack of oxygen;

2 Loeb, Leo, Uber die Entstehung von Bindegewebe, Leucocyten und rotlien Blutkorperchen aus Epithet und uber eine Methode isolierte Gewebsteile zu zuchten. Chicago, 1897. 3 While this has been demonstrated thus far only for connectivetissue cells it may be true also for other cells. and every two days a few cells from the margin of the piece have to be transierred to a new culture medium. This effect of lack of oxygen explains also why the immortality of the somatic cells is not obvious. Death in a human being consists in the stopping of heart beat and respiration, which also terminates the action of the brain or at least of consciousness. Immediately after the cessation of heart beat and respiration the cells of muscle and of the skin and probably many or most other organs are still alive and might continue to live if transferred to another body with circulation and respiration. As a consequence of the lack of oxygen supply in the dead body they will, however, die comparatively rapidly. It may be stated that hearts taken out of the body after a number of hours can still beat again when put into the proper solutions and upon receiving an adequate oxygen supply.