CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : I. EFFECTS OF GUAIACOL ON VALONIA

In normal cells of Valonia the order of the apparent mobilities of the ions in the non-aqueous protoplasmic surface is K > Cl > Na. After treatment with 0.01 M guaiacol (which does not injure the cell) the order becomes Na > Cl > K. As it does not seem probable that such a reversal could occur with simple ions we may assume provisionally that in the protoplasmic surface we have to do with charged complexes of the type (KX _I)⁺, (KX _II)⁺, where X _I and X _II are elements or radicals, or with chemical compounds formed in the protoplasm. When 0.01 M guaiacol is added to sea water or to 0.6 M NaCl (both at pH 6.4, where the concentration of the guaiacol ion is negligible) the P.D. of the cell changes (after a short latent period) from about 10 mv. negative to about 28 mv. positive and then slowly returns approximately to its original value (Fig. 1, p. 14). This appears to depend chiefly on changes in the apparent mobilities of organic ions in the protoplasm. The protoplasmic surface is capable of so much change that it does not seem probable that it is a monomolecular layer. It does not behave like a collodion nor a protein film since the apparent mobility of Na⁺ can increase while that of K⁺ is decreasing under the influence of guaiacol.     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : IV. INFLUENCE OF GUAIACOL ON THE EFFECTS OF SODIUM AND POTASSIUM IN NITELLA

In Nitella, as in Halicystis, guaiacol increases the mobility of Na⁺ in the outer protoplasmic surface but leaves the mobility of K⁺ unaffected. This differs from the situation in Valonia where the mobility of Na⁺ is increased and that of K⁺ is decreased. The partition coefficient of Na⁺ in the outer protoplasmic surface is increased and that of K⁺ left unchanged. Recovery after the action current is delayed in the presence of guaiacol and the action curves are "square topped."     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : II. THE ACTION OF GUAIACOL AS AFFECTED BY pH

The normal P.D. across the protoplasm of Valonia macrophysa is about 10 mv. negative (inwardly directed). On adding 0.01 M guaiacol to the sea water the P.D. becomes positive and then slowly returns approximately to the normal value. In many cases this behavior is not much affected by raising the pH and so increasing the concentration of the guaiacol ion but in other cases such an increase makes the P.D. somewhat more negative. But if we wait until the exposure to guaiacol has lasted 5 minutes (and the P.D. has returned to its normal value) before we raise the pH, the result is very different. The cell then behaves as though it had been sensitized to the action of the guaiacol ion which appears to be far more effective than undissociated guaiacol in making the P.D. more positive. This may be due in part to the high apparent mobility of the guaiacol ion and in part to alterations which it produces in the protoplasm (such alterations increase the P.D. across the protoplasm whereas ordinary injury would be expected to lower it and the cells live on after this treatment and show no signs of injury). This action of the guaiacol ion is in marked contrast to the behavior of other anions whose effect resembles that of Cl^-.     

CALCULATIONS OF BIOELECTRIC POTENTIALS : VI. SOME EFFECTS OF GUAIACOL ON NITELLA

Values have been calculated for apparent mobilities and partition coefficients in the outer non-aqueous layer of the protoplasm of Nitella. Among the alkali metals (with the exception of cesium) the order of mobilities resembles that in water and the partition coefficients (except for cesium) follow the rule of Shedlovsky and Uhlig, according to which the partition coefficient increases with the ionic radius. Taking the mobility of the chloride ion as unity, we obtain the following: lithium 2.04, sodium 2.33, potassium 8.76, rubidium 8.76, cesium 1.72, ammonium 4.05, ½ magnesium 20.7, and ½ calcium 7.52. After exposure to guaiacol these values become: lithium 5.83, sodium 7.30, potassium 8.76, rubidium 8,76, cesium 3.38, ammonium 4.91, ½ magnesium 20.7, and ½ calcium 14.46. The partition coefficients of the chlorides are as follows, when that of potassium chloride is taken as unity: lithium 0.0133, sodium 0.0263, rubidium 1.0, cesium 0.0152, ammonium 0.0182, magnesium 0.0017, and calcium 0.02. These are raised by guaiacol to the following: lithium 0.149, sodium 0.426, rubidium 1.0, cesium 0.82, ammonium 0.935, magnesium 0.0263, and calcium 0.323 (that of potassium is not changed). The effect of guaiacol on the mobilities of the sodium and potassium ions resembles that seen in Halicystis but differs from that found in Valonia where guaiacol increases the mobility of the sodium ion but decreases that of the potassium ion.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : III. THE EFFECTS OF AMMONIA

The nature and origin of the large "protoplasmic" potential in Halicystis must be studied by altering conditions, not only in external solutions, but in the sap and the protoplasm itself. Such interior alteration caused by the penetration of ammonia is described. Concentrations of NH₄Cl in the sea water were varied from 0.00001 M to above 0.01 M. At pH 8.1 there is little effect below 0.0005 M NH₄Cl. At about 0.001 M a sudden reversal of the potential difference across the protoplasm occurs, from about 68 mv. outside positive to 30 to 40 mv. outside negative. At this threshold value the time curve is characteristically S-shaped, with a slow beginning, a rapid reversal, and then an irregularly wavering negative value. There are characteristic cusps at the first application of the NH₄Cl, also immediately after the reversal. The application of higher NH₄Cl concentrations causes a more rapid reversal, and also a somewhat higher negative value. Conversely the reduction of NH₄Cl concentrations causes recovery of the normal positive potential, but the threshold for recovery is at a lower concentration than for the original reversal. A temporary overshooting or increase of the positive potential usually occurs on recovery. The reversals may be repeated many times on the same cell without injury. The plot of P.D. against the log of ammonium ion concentration is not the straight line characteristic of ionic concentration effects, but has a break of 100 mv. or more at the threshold value. Further evidence that the potential is not greatly influenced by ammonium ions is obtained by altering the pH of the sea water. At pH 5, no reversal occurs with 0.1 M NH₄Cl, while at pH 10.3, the NH₄Cl threshold is 0.0001 M or less. This indicates that the reversal is due to undissociated ammonia. The penetration of NH₃ into the cells increases both the internal ammonia and the pH. The actual concentration of ammonium salt in the sap is again shown to have little effect on the _P.D. The pH is therefore the governing factor. But assuming that NH₃ enters the cells until it is in equilibrium between sap and sea water, no sudden break of pH should occur, pH being instead directly proportional to log NH₃ for any constant (NH₄) concentration. Experimentally, a linear relation is found between the pH of the sap and the log NH₃ in sea water. The sudden change of P.D. must therefore be ascribed to some system in the cell upon which the pH change operates. The pH value of the sap at the NH₃ threshold is between 6.0 and 6.5 which corresponds well with the pH value found to cause reversal of P.D. by direct perfusion of solutions in the vacuole.     

EFFECTS OF POTASSIUM ON THE POTENTIAL OF HALICYSTIS

Sea water in which sodium has been replaced by potassium produces about the same degree of negativity in Halicystis and in Valonia. With increasing dilution of this sea water up to 1 ÷ 16 the degree of negativity steadily falls off in Halicystis. This differs from the situation in Valonia where Damon finds that with increasing dilution the negativity passes through a minimum after which increasing dilution produces increasing negativity. But conditions in the two organisms differ so greatly that a comparison is of rather doubtful significance.     

BIOELECTRIC POTENTIALS IN HALICYSTIS : VIII. THE EFFECTS OF LIGHT

The effects of light upon the potential difference across the protoplasm of impaled Halicystis cells are described. These effects are very slight upon the normal P.D., increasing it 3 or 4 per cent, or at most 10 per cent, with a characteristic cusped time course, and a corresponding decrease on darkening. Light effects become much greater when the P.D. has been decreased by low O₂ content of the sea water; light restores the P.D. in much the same time course as aeration, and doubtless acts by the photosynthetic production of O₂. There are in both cases anomalous cusps which decrease the P.D. before it rises. Short light exposures may give only this anomaly. Over part of the potential range the light effects are dependent upon intensity. Increased CO₂ content of the sea water likewise depresses the P.D. in the dark, and light overcomes this depression if it is not carried too far. Recovery is probably due to photosynthetic consumption of CO₂, unless there is too much present. Again there are anomalous cusps during the first moments of illumination, and these may be the only effect if the P.D. is too low. The presence of ammonium salts in the sea water markedly sensitizes the cells to light. Subthreshold NH₄ concentrations in the dark become effective in the light, and the P.D. reverses to a negative sign on illumination, recovering again in the dark. This is due to increase of pH outside the cell as CO₂ is photosynthetically reduced, with increase of undissociated NH₃ which penetrates the cell. Anomalous cusps which first carry the P.D. in the opposite direction to the later drift are very marked in the presence of ammonia, and may represent an increased acidity which precedes the alkaline drift of photosynthesis. This acid gush seems to be primarily within the protoplasm, persisting when the sea water is buffered. Glass electrode measurements also indicate anomalies in the pH drift. There are contrary cusps on darkening which suggest temporarily increased alkalinity. Even more complex time courses are given by combining low O₂ and NH₄ exposures with light; these may have three or more cusps, with reversal, recovery, and new reversal. The ultimate cause of the light effects is to be found in an alteration of the surface properties by the treatments, which is overcome (low O₂, high CO₂), or aided (NH₄) by light. This alteration causes the surface to lose much of its ionic discrimination, and increases its electrical resistance. Tests with various anion substitutions indicate this, with recovery of normal response in the light. A theory of the P.D. in Halicystis is proposed, based on low mobility of the organic anions of the protoplasm, with differences in the two surfaces with respect to these, and the more mobile Na and K. ions.     

EFFECTS OF CYANIDE ON THE PROTOPLASM OF AMEBA

The experiments seem to indicate that the toxicity of HCN and KCN for amebæ is due to their effect on the cell membrane and not on the internal protoplasm. Concentrated solutions (N/10–N/300) of HCN or KCN produce an initial increase in viscosity of the protoplasm of amebæ (immersed) which is followed by liquefaction and disintegration of the cell. Dilute solutions of HCN or KCN decrease the viscosity of the protoplasm of amebæ. Injections of HCN or KCN into amebæ produce a reversible decrease in viscosity of the protoplasm.     

ON GUAIACOL SOLUTIONS : I. THE ELECTRICAL CONDUCTIVITY OF SODIUM AND POTASSIUM GUAIACOLATES IN GUAIACOL

1. Measurements on the densities, viscosities, dielectric constants, and specific conductances of pure anhydrous and water-saturated guaiacol at 25°C. are reported. 2. The solubility of water in guaiacol at 25°C., and its effect on the electrical conductivity of a sodium guaiacolate solution is given. 3. Electrical conductivity measurements are reported on solutions of sodium and potassium guaiacolates in water-saturated guaiacol at 25°C. 4. The decrease of electrical conductivity with increasing concentration for these salts is explained on the basis of an ionic equilibrium combined with the interionic attraction theory of Debye and Hückel. 5. The limiting equivalent conductances of sodium and potassium guaiacolates in water-saturated guaiacol at 25°C., the corresponding limiting ionic mobilities, and the dissociation constants are computed from the conductivity measurements. The salts are found to be weak electrolytes with dissociation constants of the order of 5 x 10^–6.     

THE EFFECTS OF CURRENT FLOW ON BIOELECTRIC POTENTIAL : II. HALICYSTIS

The effect of direct current, of controlled direction and density, across the protoplasm of impaled cells of Halicystis, is described. Inward currents slightly increase the already positive P.D. (70 to 80 mv.) in a regular polarization curve, which depolarizes equally smoothly when the current is stopped. Outward currents of low density produce similar curves in the opposite direction, decreasing the positive P.D. by some 10 or 20 mv. with recovery on cessation of flow. Above a critical density of outward current, however, a new effect becomes superimposed; an abrupt reversal of the P.D. which now becomes 30 to 60 mv. negative. The reversal curve has a characteristic shape: the original polarization passes into a sigmoid reversal curve, with an abrupt cusp usually following reversal, and an irregular negative value remaining as long as the current flows. Further increases of outward current each produce a small initial cusp, but do not greatly increase the negative P.D. If the current is decreased, there occurs a threshold current density at which the positive P.D. is again recovered, although the outward current continues to flow. This current density (giving positivity) is characteristically less than that required to produce reversal originally, giving the process a hysteretic character. The recovery is more rapid the smaller the current, and takes only a few seconds in the absence of current flow, its course being in a smooth curve, usually without an inflection, thus differing from the S-shaped reversal curve. The reversal produced by outward current flow is compared with that produced by treatment with ammonia. Many formal resemblances suggest that the same mechanism may be involved. Current flow was therefore studied in conjunction with ammonia treatment. Ammonia concentrations below the threshold for reversal were found to lower the threshold for outward currents. Subthreshold ammonia concentrations, just too low to produce reversal alone, produced permanent reversal when assisted by a short flow of very small outward currents, the P.D. remaining reversed when the current was stopped. Further increases of outward current, when the P.D. had been already reversed by ammonia, produced only small further increases of negativity. This shows that the two treatments are of equivalent effect, and mutually assist in producing a given effect, but are not additive in the sense of being superimposable to produce a greater effect than either could produce by itself. Since ammonia increases the alkalinity of the sap, and presumably of the protoplasm, when it penetrates, it is possible that the reversal of P.D. by current flow is also due to change of pH. The evidence for increased alkalinity or acidity due to current flow across phase boundaries or membranes is discussed. While an attractive hypothesis, it meets difficulties in H. ovalis where such pH changes are both theoretically questionable and practically ineffective in reversing the P.D. It seems best at the present time to assign the reversal of P.D. to the alteration or destruction of one surface layer of the protoplasm, with reduction or loss of its potential, leaving that at the other surface still intact and manifesting its oppositely directed potential more or less completely. The location of these surfaces is only conjectural, but some evidence indicates that it is the outer surface which is so altered, and reconstructed on recovery of positive P.D. This agrees with the essentially all-or-none character of the reversal. The various treatments which cause reversal may act in quite different ways upon the surface.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : II. THE EFFECTS OF POTASSIUM ON TWO SPECIES WITH DIFFERENT SAPS

The potential difference across the protoplasm of impaled cells of two American species of Halicystis is compared. The mean value for H. Osterhoutii is 68.4 mv.; that for H. ovalis is 79.7 mv., the sea water being positive to the sap in both. The higher potential of H. ovalis is apparently due to the higher concentration of KCl (0.3 M) in its vacuolar sap. When the KCl content of H. Osterhoutii sap (normally 0.01 M or less) is experimentally raised to 0.3 M, the potential rises to values about equal to those in H. ovalis. The external application of solutions high in potassium temporarily lowers the potential of both, probably by the high mobility of K⁺ ions. But a large potential is soon regained, representing the characteristic potential of the protoplasm. This is about 20 mv. lower than in sea water. The accumulation of KCl in the sap of H. ovalis is apparently not due to the higher mobility of K⁺ ion in its protoplasm, since the electrical effects of potassium are practically identical in H. Osterhoutii, where KCl is not accumulated.     

APPARENT VIOLATIONS OF THE ALL-OR-NONE LAW IN RELATION TO POTASSIUM IN THE PROTOPLASM

In Vol. 37, No. 6, July 20, 1954, page 814, in the third paragraph, delete the first sentence and insert the following: This equation gives excellent results with Nitella for example with 0.001 M KCl outside and 0.05 M KCl in the sap we may use concentrations in place of activities in the equation and put a_s = 0.05 and a_o = 0.001. No allowance is made for any change in the concentration of incoming ions due to their combination with carrier molecules since this change would be small and difficult to estimate. If we put U = 73 and V = 1 we obtain P = 97 mv. which is close to the usual observed value. The expression (73 –1) ÷ (73 + 1) = 0.973.     

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.     

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.     

松树受害后一些化学物质含量的变化及其对马尾松毛虫种群参数的影响

通过探讨松树受害后松针的一些化学物质变化与马尾松毛虫Dendrolimus punctatus 种群参数的相互作用关系。结果表明：松针被马尾松毛虫为害后,针叶的氨基酸含量减少、单宁和总酚含量增加;马尾松毛虫取食被害松针后,体内的2种解毒酶(过氧化氢酶、羧酸酯酶)活性增加,其种群参数表现出发育历期延长、体重减轻、死亡率增加、生殖力下降的变 化。