THE NUCLEOPROTAMINE OF TROUT SPERM

The nucleoprotamine of trout sperm can be extracted completely with 1 M sodium chloride. On reducing the salt concentration to 0.14 M, physiological saline, the nucleoprotamine precipitates in long, fibrous strands. When the nucleoprotamine, dissolved in M NaCl, is dialyzed all the protamine diffuses through the membrane leaving behind highly polymerized, protein-free desoxyribose nucleic acid. The nucleoprotamine constitutes 91 per cent of the lipid-free mass of the sperm nucleus. While nucleoprotamine is being extracted by M NaCl a stage is reached at which the sperm chromosomes are clearly visible.     

CHROMOSIN,A DESOXYRIBOSE NUCLEOPROTEIN COMPLEX OF THE CELL NUCLEUS

A desoxyribose nucleoprotein complex, which we have referred to as a chromosin, has been prepared from a great variety of cells, mainly animal but also plant and bacterial. A chromosin is derived from the cell nucleus. In the course of preparation precautions have been taken to prevent contamination by cytoplasmic constituents. To assure the nuclear origin of all components of chromosin, nuclei have in several instances been isolated before extraction was begun. Because of the precautions taken, chromosins do not contain detectable quantities of ribose nucleoproteins; but, incidentally, extraction of ribose nucleoproteins, free of desoxyribose compounds, has also been described in this paper. A typical chromosin contains 3 components: desoxyribose nucleic acid, histone, and non-histone protein. The nucleic acid, being highly polymerized, is exceedingly viscous when dissolved and fibrous when precipitated. Histone and non-histone protein differ from each other in a number of ways, of which one of the most definite is that whereas a histone contains no more than traces of tryptophane, the non-histone protein of chromosin contains nearly 1 per cent of tryptophane. In neutral physiological saline both proteins can combine with nucleic acid. With the isolation of chromosins from so many different kinds of cells, it can now be seen that (contrary to the view expressed by Kossel) histones are present in most animal cells and at least in some plant and bacterial cells. Chromosin prepared from the Type III pneumococcus is active in transforming the type of a pneumococcus culture. It has been pointed out that it is not yet known whether or not protein is a necessary constituent of the transforming agent. To extract chromosin from a cell M NaCl is used. When dissolved in M NaCl the nucleic acid and histone components of a chromosin are to a considerable extent dissociated. They are not dissociated when the chromosin is dissolved in 0.02 M NaCl, but in this medium a partial depolymerization of the nucleic acid occurs. A chromosin should certainly not be considered to be a definite chemical compound. It is a complex extracted from chromatin, which is itself a complicated nuclear structure. And in the course of extraction, it need hardly be said, the structure of chromatin has been considerably changed. To avoid complications it has been considered an advantage in this work to begin with isolated nuclei, and it would clearly be a further simplification to begin chemical procedures only after the chromosomes themselves have been isolated. This is now being accomplished, and it is found that the methods described in this paper are of value in learning how the substances present in a chromosin are put together in a chromosome.     

COMPARATIVE STUDIES ON RESPIRATION : IX. THE EFFECTS OF ANTAGONISTIC SALTS ON THE RESPIRATION OF ASPERGILLUS NIGER.

1. In the presence of 0.05 per cent dextrose the respiration of Aspergillus niger is increased by NaCl in concentrations of 0.25 to 0.5M, and by 0.5M CaCl₂. 2. Stronger concentrations, as 2M NaCl and 1.25M CaCl₂, decrease the respiration. The decrease in the higher concentrations is probably an osmotic effect of these salts. 3. A mixture of 19 cc. of NaCl and 1 cc. of CaCl₂ (both 0.5M) showed antagonism, in that the respiration was normal, although each salt alone caused an increase. 4. Spores of Aspergillus niger did not germinate on 0.5M NaCl (plus 0.05 per cent dextrose) while they did on 0.5M CaCl₂ (plus 0.05 per cent dextrose) and on various mixtures of the two. This shows that a substance may have different effects on respiration from those which it has upon growth.     

THE EFFECTS OF CAFFEINE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE FERTILIZED EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O₂ consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O₂ consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O₂ uptake had been depressed to 50 per cent or more of the normal controls. 6. O₂ consumption-time relationship data indicate an average depression, in O₂ consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O₂ consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O₂ uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.     

CHEMICAL ANTAGONISM OF IONS : III. EFFECT OF SALT MIXTURES ON GELATIN ACTIVITY.

1.25 per cent gelatin solutions containing enough NaOH to bring them to pH 7.367 (or KOH to pH 7.203) were made up with various concentrations of NaCl, KCl and MgCl₂, alone and in mixtures, up to molar ionic strength. The effects of these salts on the pH were observed. MgCl₂ and NaCl alone lower the pH of the Na gelatinate or the K gelatinate, in all amounts of these salts. KCl first lowers the pH (up to 0.01 M K⁺), then raises the pH. Mixtures of NaCl and KCl (up to 0.09 M of the salt whose concentration is varied) raise the pH; then (up to 0.125 M Na⁺ or K⁺) lower the pH; and finally (above 0.125 M) behave like KCl alone. Mixtures of MgCl₂ and NaCl raise the pH up to 0.10 M Na⁺, and lower it up to 0.15 M Na⁺ regardless of the amount of MgCl ₂. Higher concentrations of NaCl have little effect, but the pH in this range of NaCl concentration is lowered with increase of MgCl₂. Mixtures of MgCl₂ and KCl behave as above described (for MgCl₂ and NaCl) and the addition of NaCl plus KCl to gelatin containing MgCl₂ produces essentially the same effect as the addition of either alone, except that the first two breaks in this curve come at 0.07 M and 0.08 M [Na⁺ + K⁺] and there is a third break at 0.12 M. In this pH range the free groups of the dicarboxylic acids and of lysine are essentially all ionized and the prearginine and histidine groups are essentially all non-ionized. The arginine group is about 84 per cent ionized. Hence we are studying a solution with two ionic species in equilibrium, one with the arginine group ionized, and one with it non-ionized. It is shown that the effect of each salt alone depends upon the effect of the cation on the activity of these two species due to combination. The anomalous effects of cation mixtures may be qualitatively accounted for if one or both of these species fail to combine with the cations in a mixture in proportion to the relative combination in solutions of each cation alone. Special precautions were taken to ensure accuracy in the pH measurements. The mother solutions gave identical readings to 0.001 pH and the readings with salts were discarded when not reproducible to 0.003 pH. All doubtful data were discarded.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : III. OXYGEN CONSUMPTION AND CELL DIVISION OF FERTILIZED SEA URCHIN EGGS IN THE PRESENCE OF RESPIRATORY INHIBITORS

1. The effects of a number of respiratory inhibiting agents on the cell division of fertilized eggs of Arbacia punctulata have been determined. For eggs initially exposed to the reagents at 30 minutes after fertilization at 20°C., the levels of oxygen consumption prevailing in the minimum concentrations of reagents which produced complete cleavage block were (as percentages of the control): In 0.4 per cent O₂-99.6 per cent N₂, 32; in 0.7 per cent O₂-99.3 per cent CO, 32; in 1.6 x 10^–4 M potassium cyanide, 34; in 1 x 10^–3 M phenylurethane, 70; in 4 x 10^–3 M 5-isoamyl-5-ethyl barbituric acid, 20; in 3 x 10^–4 M iodoacetic acid, 53. 2. The carbon monoxide inhibition of oxygen consumption and cell division was reversed by light. The percentage inhibition of oxygen consumption by carbon monoxide in the dark is described by the usual mass action equation with K, the inhibition constant, equal to approximately 60, as compared to values of 5 to 10 for yeast and muscle. In 20 per cent O₂-80 per cent CO in the dark there was a slight stimulation of oxygen consumption, averaging 20 per cent. 3. Spectroscopic examination of fertilized and unfertilized Arbacia eggs reduced by hydrosulfite revealed no cytochrome bands. The thickness and density of the egg suspension was such as to indicate that, if cytochrome is present at all, the amount in Arbacia eggs is extremely small as compared to that in other tissues having a comparable rate of oxygen consumption. 4. Three reagents poisoning copper catalyses, potassium dithio-oxalate (10^–2 M), diphenylthiocarbazone (10^–4 M), and isonitrosoacetophenone (2 x 10^–3 M) produced no inhibition of division of fertilized Arbacia eggs. 5. These results indicate that the respiratory processes required to support division in the Arbacia egg may perhaps differ in certain essential steps from the principal respiratory processes in yeast and muscle.     

COMPARATIVE STUDIES ON RESPIRATION : XV. THE EFFECT OF BILE SALTS AND OF SAPONIN UPON RESPIRATION.

1. The addition of Na taurocholate produces an increase in the rate of respiration at a concentration of 0.0000125 M, and a decrease at 0.001 M and in higher concentrations. 2. NaCl is antagonized by Na taurocholate, the most favorable proportion being 14,375 parts of NaCl to 1 part of Na taurocholate (molecular proportions). 3. Solutions of saponin, at concentrations from 0.00005 M to 0.001 M, decrease the rate of respiration: lower concentrations produce no effect.     

CHEMICAL ANTAGONISM OF IONS : IV. EFFECT OF SALT MIXTURES ON GLYCINE ACTIVITY.

The pH of a 0.01 molar solution of glycine, half neutralized with NaOH, is 9.685. Addition of only one of the salts NaCl, KCl, MgCl₂, or CaCl₂ will lower the pH of the solution (at least up to 1 µ). If a given amount of KCl is added to a glycine solution, the subsequent addition of increasing amounts of NaCl will first raise the pH (up to 0.007 M NaCl). Further addition of NaCl (up to 0.035 M NaCl) will lower the pH, and further additions slightly raise the pH. The same type of curve is obtained by adding NaCl to glycine solution containing MgCl₂ or CaCl₂ except that the first and second breaks occur at 0.015 M and 0.085 M NaCl, respectively. Addition of CaCl₂ to a glycine solution containing MgCl₂ gives the same phenomena with breaks at 0.005 M and 0.025 M CaCl; or at ionic strengths of 0.015 µCaCl₂ and 0.075 µCaCl₂. This indicates that the effect is a function of the ionic strength of the added salt. These effects are sharp and unmistakable. They are almost identical with the effects produced by the same salt mixtures on the pH of gelatin solutions. They are very suggestive of physiological antagonisms, and at the same time cannot be attributed to colloidal phenomena.     

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM : I. EFFECTS OF FORMALDEHYDE ON NITELLA

When protoplasm dies it becomes completely and irreversibly permeable and this may be used as a criterion of death. On this basis we may say that when 0.2 M formaldehyde plus 0.001 M NaCl is applied to Nitella death arrives sooner at the inner protoplasmic surface than at the outer. If, however, we apply 0.17 M formaldehyde plus 0.01 M KCl death arrives sooner at the outer protoplasmic surface. The difference appears to be due largely to the conditions at the two surfaces. With 0.2 M formaldehyde plus 0.001 M NaCl the inner surface is subject to a greater electrical pressure than the outer and is in contact with a higher concentration of KCl. In the other case these conditions are more nearly equal so that the layer first reached by the reagent is the first to become permeable. The outer protoplasmic surface has the ability to distinguish electrically between K⁺ and Na⁺ (potassium effect). Under the influence of formaldehyde this ability is lost. This is chiefly due to a falling off in the partition coefficient of KCl in the outer protoplasmic surface. At about the same time the inner protoplasmic surface becomes completely permeable. But the outer protoplasmic surface retains its ability to distinguish electrically between different concentrations of the same salt, showing that it has not become completely permeable. After the potential has disappeared the turgidity (hydrostatic pressure inside the cell) persists for some time, probably because the outer protoplasmic surface has not become completely permeable.     

BIOELECTRIC POTENTIALS IN HALICYSTIS : VII. THE EFFECTS OF LOW OXYGEN TENSION

The potential difference across the protoplasm of impaled cells of Halicystis is not affected by increase of oxygen tension in equilibrium with the sea water, nor with decrease down to about 1/10 its tension in the air (2 per cent O₂ in N₂). When bubbling of 2 per cent O₂ is stopped, the P.D. drifts downward, to be restored on stirring the sea water, or rebubbling the gas. Bubbling 0.2 per cent O₂ causes the P.D. to drop to 20 mv. or less; 1.1 per cent O₂ to about 50 mv. Restoration of 2 per cent or higher O₂ causes recovery to 70 or 80 mv. often with a preliminary cusp which decreases the P.D. before it rises. Perfusion of aerated sea water through the vacuole is just as effective in restoring the P.D. as external aeration, indicating that the direction of the oxygen gradient is not significant. Low O₂ tension also inhibits the reversed, negative P.D. produced by adding NH₄Cl to sea water, 0.2 per cent O₂ bringing this P.D. back to the same low positive values found without ammonia. Restoration of 2 per cent O₂ or air, restores this latent negativity. At slightly below the threshold for ammonia reversal, low O₂ may induce a temporary negativity when first bubbled, and a negative cusp may occur on aeration before positive P.D. is regained. This may be due to a decreased consumption of ammonia, or to intermediate pH changes. The locus of the P.D. alteration was tested by applying increased KCl concentrations to the cell exterior; the large cusps produced in aerated solutions become greatly decreased when the P.D. has fallen in 0.2 per cent O₂. This indicates that the originally high relative mobility or concentration of K⁺ ion has approached that of Na⁺ in the external protoplasmic surface under reduced O₂ tension. Results obtained with sulfate sea water indicate that Na⁺ mobility approaches that of SO₄ ^— in 0.2 per cent O₂. P.D. measurements alone cannot tell whether this is due to an increase of the slower ion or a decrease of the faster ion. A decrease of all ionic permeability is indicated, however, by a greatly increased effective resistance to direct current during low O₂. Low resistance is regained on aeration. The resistance increase resembles that produced by weak acids, cresol, etc. Acids or other substances produced in anaerobiosis may be responsible for the alteration. Or a deficiency of some surface constituent may develop. In addition to the surface changes there may be alterations in gradients of inorganic or organic ions within the protoplasm, but there is at present no evidence on this point. The surface changes are probably sufficient to account for the phenomena.     

COMPARATIVE STUDIES ON RESPIRATION : VIII. THE RESPIRATION OF BACILLUS SUBTILIS IN RELATION TO ANTAGONISM.

1. In relatively low concentrations of NaCl, KCl, and CaCl₂ the rate of respiration of Bacillus subtilis remains fairly constant for a period of several hours, while in the higher concentrations, there is a gradual decrease in the rate. 2. NaCl and KCl increase the rate of respiration of Bacillus subtilis somewhat at concentrations of 0.15 M and 0.2 M respectively; in sufficiently high concentrations they decrease the rate. CaCl₂ increases the rate of respiration of Bacillus subtilis at a concentration of 0.05 M and decreases the rate at somewhat higher concentrations. 3. The effects of salts upon respiration show a well marked antagonism between NaCl and CaCl₂, and between KCl and CaCl₂. The antagonism between NaCl and KCl is slight and the antagonism curve shows two maxima.     

NATIVE AND REGENERATED BOVINE ALBUMIN : I. PREPARATION AND PHYSICOCHEMICAL PROPERTIES

1. Whole bovine albumin, homogeneous in diffusion and sedimentation, and essentially homogeneous in electrophoresis, has been prepared by a method involving ammonium sulfate precipitation of the globulins in the cold and of the albumin at room temperature, isoelectric precipitation of the euglobulins, and reprecipitation of the albumin. 2. The product has been characterized by chemical analysis and by viscosity, diffusion, sedimentation, and electrophoresis measurements. The carbohydrate content is 0.38 per cent, the nitrogen content, 15.2 per cent. The molecular shape approximates that of a prolate ellipsoid with an axial ratio of 3.1, assuming 33 per cent hydration; the average molecular weight is 65,000. 3. Bovine albumin is readily denatured by concentrated solutions of urea or guanidine hydrochloride, gross changes in molecular shape resulting. 4. Regeneration of bovine albumin denatured in solutions of 8 M urea or guanidine hydrochloride yields a material closely resembling the native in carbohydrate content, in molecular size and shape, and in electrophoretic properties. However, the regenerated protein differs from the native in susceptibility to tryptic digestion, and, in this respect, appears to be in a denatured state. 5. In 8 M solutions of guanidine hydrochloride a limiting yield of regenerated albumin equivalent to 95 per cent of the original protein is approached. 6. Bovine crystalbumin, a crystalline carbohydrate-free fraction of the whole albumin, appears to be more susceptible to denaturation than whole bovine albumin.     

PROLYTIC ION EXCHANGES PRODUCED IN HUMAN RED CELLS BY METHANOL,ETHANOL, GUAIACOL,AND RESORCINOL

When the washed red cells of heparinized human blood are exposed at 4°C. to methanol, ethanol, guaiacol, or resorcinol in hypolytic concentrations in isotonic NaCl, the prolytic loss of K at the end of 20 hours varies from about 25 per cent of the initial K content of the cells in the case of 3.1 M methanol to about 55 per cent of the initial K in the case of 0.04 M resorcinol. As in the case of the prolytic losses observed with other lysins, the K loss is rapid at first and then slows down so that what appears to be a new steady state is reached logarithmically. The K lost from the cells during the period of the prolytic loss is replaced by an approximately equivalent amount of Na, derived from the isotonic NaCl in which the cells are suspended. The Na which enters can be replaced by K by washing the cells in isotonic KCl, and this K can again be replaced by Na by washing the cells in isotonic NaCl. The remainder of the cell K., i.e. the K which was not lost during the period of the prolytic loss, is retained in the cell unaffected by these washing procedures. The capacity of red cells for undergoing disk-sphere transformations is scarcely affected by their having been exposed to hypolytic concentrations of methanol, ethanol, guaiacol, or resorcinol in isotonic NaCl, and their resistance to osmotic hemolysis and to lysis by saponin and digitonin is altered only in minor respects even when as much as 50 per cent of the cell K has been exchanged for Na. Some restriction to the movement of K between the cell and its environment is apparently modified irreversibly when the cell is exposed to hypolytic concentrations of lysins, and the modification is such that only a fraction of the cell K is affected, the fraction being a function of the lysin concentration, the duration of its action, and other factors. A modification of some part of the cell structure and of the properties dependent on its integrity is probably involved: K may be lost more readily from some cells than from others, from some parts of the cell more readily than from other parts, or the explanation may lie in changes in the extent to which Hb binds ions or in modifications of metabolic processes.     

RESTORATION OF THE POTASSIUM EFFECT BY MEANS OF ACTION CURRENTS

Treatment with distilled water removes from Nitella the ability to give the large potential difference between 0.01 M KCl and 0.01 M NaCl which is known as the potassium effect. The potassium effect may be restored by action currents. This might be explained by saying that distilled water removes from the surface a substance, R, which is responsible for the potassium effect and which moves into the surface during the action current and thereby restores the potassium effect.     

THE PHYSIOLOGICAL EFFECTS OF l-TYROSINE AND l-PHENYL-ALANINE ON THE RATE AND QUALITY OF PULSATION IN THE SEVENTY-TWO HOUR EXTIRPATED EMBRYONIC CHICK HEART

1. 72 hour isolated chick hearts show an increase in pulsation rate when placed in M/1000, M/10,000, and M/50,000 l-tyrosine solutions. The optimal effect is seen in M/10,000 and M/50,000 l-tyrosine. 2. All hearts show disturbance of rhythm either in the form of irregular rhythm or heart block. 3. 62 hour isolated chick hearts are not susceptible to l-tyrosine while 96 hour hearts are markedly sensitive. 4. 72 hour isolated chick hearts placed in 1 part in 10,000 and 1 part in 50,000 l-epinephrine show approximately the same effects as were seen with l-tyrosine. 5. 72 hour isolated chick hearts placed in M/1000 and M/10,000 l-phenylalanine show an initial depression followed by an l-tyrosine effect.     

I. THE RELATION BETWEEN ATTACHMENT TO THE SUBSTRATUM AND INGESTION BY AMEBA IN STRYCHNINE SULFATE SOLUTION AND CONDITIONED MEDIA : II. BIOLOGICAL ASSAY OF CONDITIONED MEDIA

1. Strychnine sulfate 0.000069 M decreased percentage attachment to the substratum by Amoeba proteus in 0.0029 M NaCl from 77.3 to 1.3, in 0.0029 M KCl from 40.8 to 2.5, in 0.002 M CaCl₂ from 73.3 to 68.0, in 0.002 M MgCl₂ from 85.5 to 83.3. 2. Frequency of ingestion of chilomonads by Amoeba proteus is increased by adding strychnine sulfate to solutions of NaCl, KCl, or CaCl₂. Frequency of ingestion is increased in NaCl solution from 1.3 to 2.3, in KCl from 0.75 to 2.25, and in CaCl₂ from 1.1 to 1.9 chilomonads per minute. Ingestion is not significantly increased by the addition of strychnine to MgCl₂ solution. 3. Frequency of ingestion of food by Amoeba proteus is not closely correlated with attachment to the substratum in NaCl and KCl solutions to which strychnine sulfate is added. 4. Chilomonads adhere to the plasmalemma of Amoeba proteus in solutions of NaCl, KCl, or CaCl₂ containing strychnine, but in MgCl₂ plus strychnine only a few adhere to it. Strychnine appears to make the surface of the amebae and chilomonads sticky in the former but not in the latter. Frequency of ingestion is apparently correlated with adherence of chilomonads to the plasmalemma. 5. Attachment to the substratum and ingestion by Pelomyxa carolinensis is increased by dead Chilomonas, Colpidium, and Paramecium in aqueous solutions, by materials obtained from paramecia by alcoholic-ether extraction, and by solutions in which these organisms have lived. 6. Attachment to the substratum by Pelomyxa carolinensis is not closely correlated with kind or concentration of inorganic salts used in this study. 7. Materials were found in extracts of paramecia which had certain characteristics in common with choline esters. There is no reason to doubt that under certain conditions materials are present in aqueous and alcoholic extracts which are pharmacologically similar to choline and acetylcholine. 8. Aqueous suspensions of paramecia when subcutaneously injected into young mice for 21 days inhibit the gonadotropic luteinizing hormone of the pituitary. Ovaries from injected mice showed no corpora lutea, and the seminal vesicles from injected males were smaller and contained less fluid than those of the controls.     

SODIUM CHLORIDE AND SELECTIVE DIFFUSION IN LIVING ORGANISMS

1. It is shown that NaCl acts like CaCl₂ or LaCl₃ in preventing the diffusion of strong acids through the membrane of the egg of Fundulus with this difference only that a M/8 solution of NaCl acts like a M/1,000 solution of CaCl₂ and like a M/30,000 solution of LaCl₃. 2. It is shown that these salts inhibit the diffusion of non-dissociated weak acid through the membrane of the Fundulus egg but slightly if at all. 3. Both NaCl and CaCl₂ accelerate the diffusion of dissociated strong alkali through the egg membrane of Fundulus and CaCl₂ is more efficient in this respect than NaCl. 4. It is shown that in moderate concentrations NaCl accelerates the rate of diffusion of KCl through the membrane of the egg of Fundulus while CaCl₂ does not.     

THE ACTIVATION OF STARFISH EGGS BY ACIDS

1. Exposure of unfertilized starfish eggs to dilute solutions of weak acids (fatty acids, benzoic and carbonic acids) in isotonic balanced salt solution causes complete activation with the proper durations of exposure. For each acid the rate of activation (reciprocal of optimum duration) varies with concentration and temperature; at a given temperature and within a considerable range of concentrations (e.g. 0.00075 to 0.004 M for butyric acid), this rate is approximately proportional to concentration. We may thus speak of a molecular rate of action characteristic of each acid. 2. In general the molecular rate of action increases with the dissociation constant and surface activity of the acids. In the fatty acid series (up to caproic), formic acid has the most rapid effect, acting about four times as rapidly as acetic; for the other acids the order is: acetic = propionic ≦ butyric < valeric < caproic. Carbonic acid acts qualitatively like the fatty acids, but its molecular rate of action is only about one-fourteenth that of acetic acid. 3. Hydrochloric and lactic acids are relatively ineffective as activating agents, apparently because of difficulty of penetration. Lactic acid is decidedly the more effective. The action of both acids is only slightly modified by dissolving in pure (isotonic NaCl and CaCl₂) instead of in balanced salt solution. 4. The rate of action of acetic acid, in concentrations of 0.002 M to 0.004 M is increased (by 10 to 20 per cent) by adding Na-acetate (0.002 to 0.016) to the solution. The degree of acceleration is closely proportional to the estimated increase in undissociated acetic acid molecules. Activation thus appears to be an effect of the undissociated acid molecules in the external solution and not of the ions. Acetate anions and H ions acting by themselves, in concentrations much higher than those of the solutions used, have no activating effect. The indications are that the undissociated molecules penetrate rapidly, the ions slowly. Having penetrated, the molecules dissociate inside the egg, yielding the ions of the acid. 5. When the rate of activation is slow, as in 0.001 M acetic acid, the addition of Na-acetate (0,008 M to 0.016 M) has a retarding effect, referable apparently to the gradual penetration of acetate ions to the site of the activation reaction with consequent depression of dissociation. 6. An estimate of the C_H of those solutions (of the different activating acids) which activate the egg at the same rate indicates that their H ion concentrations are approximately equal. On the assumptions that only the undissociated molecules penetrate readily, and that the conditions of dissociation are similar inside and outside the egg, this result indicates (especially when the differences in adsorption of the acids are considered) that the rate of activation is determined by the C_H at the site of the activation reaction within the egg.     

NOTE ON THE NATURE OF THE CURRENT OF INJURY IN TISSUES

Leading off from two places on the same cell (of Nitella) with 0.001 M KCl we observe that a cut produces only a temporary negative current of injury. If we lead off with 0.001 M KCl from any cell to a neighboring cell we find that when sap comes out from the cut cell and reaches the neighboring intact cell a lasting negative "current of injury" is produced. This depends on the fact that the intact cell is in contact with sap at one point and with 0.001 M KCl at the other (this applies also to tissues composed of small cells). If we employ 0.1 M KCl in place of 0.001 M the current of injury with a single cell is positive (and is more lasting when a neighboring cell is present). Divergent results obtained with tissues and single cells may be due in part to these factors.     

EFFECTS OF GUAIACOL AND HEXYLRESORCINOL IN THE PRESENCE OF BARIUM AND CALCIUM

Guaiacol was applied at two spots on the same cell of Nitella. At one spot it was dissolved in 0.01 M NaCl, at the other in 0.01 M CaCl₂ or BaCl₂. The effect was practically the same in all cases, i.e. a similar change of P.D. in a negative direction, involving a more or less complete loss of P.D. (depolarization). When hexylresorcinol was used in place of guaiacol the result was similar. That Ca⁺⁺ and Ba⁺⁺ do not inhibit the effect of these organic depolarizing substances may be due to a lack of penetration of Ca⁺⁺ and Ba⁺⁺. The organic substances penetrate more rapidly and their effect is chiefly on the inner protoplasmic surface which is the principal seat of the P.D.