CERTAIN EFFECTS OF SALTS ON THE PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA

The effect of various substances on living cells may be advantageously studied by exposing them to such substances and observing their subsequent behavior in solutions of a basic dye, brilliant cresyl blue. The rate of penetration of the basic dye, brilliant cresyl blue, is decreased when cells are exposed to salts with monovalent cations before they are placed in the dye solution (made up with borate buffer mixture). This inhibiting effect is assumed to be due to the effect of the salts on the protoplasm. This effect is not readily reversible when cells are transferred to distilled water, but it is removed by salts with bivalent or trivalent cations. In some cases it disappears in dye made up with phosphate buffer mixture, or with borate buffer mixture at the pH value in which the borax predominates, and in the case of NaCl it disappears in dye containing NaCl. No inhibiting effect is seen when cells are exposed to NaCl solution containing MgCl₂ before they are placed in the dye solution. The rate of penetration of dye is not decreased when cells are previously exposed to salts with bivalent and trivalent cations. The rate is slightly increased when cells are placed in the dye solution containing a salt with monovalent cation and probably with bivalent or trivalent cations. In the case of the bivalent and trivalent salts the increase is so slight that it may be negligible.     

STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : IV. PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA FLEXILIS

Glass electrode measurements of the pH value of the sap of Nitella show that cresyl blue in form of free base penetrates the vacuoles and raises pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, while the dye salt dissolved in the sap does not alter its pH value. It is proved conclusively that the increase in the pH value of the sap is due only to the presence of the dye and not to some other alkaline substance. Spectrophotometric measurements show that the dye which penetrates the vacuole is chiefly cresyl blue. When the protoplasm is squeezed into the sap, the pH value of the sap is higher than that of the pure sap. Such a mixture behaves very much like the sap in respect to the dye.     

THE PENETRATION OF BASIC DYE INTO NITELLA AND VALONIA IN THE PRESENCE OF CERTAIN ACIDS,BUFFER MIXTURES,AND SALTS

When living cells of Nitella are exposed to an acetate buffer solution until the pH value of the sap is decreased and subsequently placed in a solution of brilliant cresyl blue, the rate of penetration of dye into the vacuole is found to decrease in the majority of cases, and increase in other cases, as compared with the control cells which are transferred to the dye solution directly from tap water. This decrease in the rate is not due to the lowering of the pH value of the solution just outside the cell wall, as a result of diffusion of acetic acid from the cell when cells are removed from the buffer solution and placed in the dye solution, because the relative amount of decrease (as compared with the control) is the same whether the external solution is stirred or not. Such a decrease in the rate may be brought about without a change in the pH value of the sap if the cells are placed in the dye solution after exposure to a phosphate buffer solution in which the pH value of the sap remains normal. The rate of penetration of dye is then found to decrease. The extent of this decrease is the greater the lower the pH value of the solution. It is found that hydrochloric acid and boric acid have no effect while phosphoric acid has an inhibiting effect at pH 4.8 on stirring. Experiments with neutral salt solutions indicate that a direct effect on the cell (decreasing penetration) is due to monovalent base cations, while there is no such effect directly on the dye. It is assumed that the effect of the phosphate and acetate buffer solutions on the cell, decreasing the rate of penetration, is due (1) to the penetration of these acids into the protoplasm as undissociated molecules, which dissociate upon entrance and lower the pH value of the protoplasm or to their action on the surface of the protoplasm, (2) to the effect of base cations on the protoplasm (either at the surface or in the interior), and (3) possibly to the effect of certain anions. In this case the action of the buffer solution is not due to its hydrogen ions. In the case of living cells of Valonia under the same experimental conditions as Nitella it is found that the rate of penetration of dye decreases when the pH value of the sap increases in presence of NH₃, and also when the pH value of the sap is decreased in the presence of acetic acid. Such a decrease may be brought about even when the cells are previously exposed to sea water containing HCl, in which the pH value of the sap remains normal.     

SPECTROPHOTOMETRIC STUDIES OF PENETRATION : IV. PENETRATION OF TRIMETHYL THIONIN INTO NITELLA AND VALONIA FROM METHYLENE BLUE.

Spectrophotometric measurements show that it is chiefly the trimethyl thionin that is present in the sap extracted from the vacuoles of uninjured cells of Nitella or Valonia which have been placed in methylene blue solution at a little above pH 9. Whether these measurements were made immediately or several hours later the same results were obtained. Methylene blue is detected in the sap (1) when the cells are injured or (2) when the contamination of the sap from the stained cell wall occurs at the time of extraction. The sap is found to be incapable of demethylating methylene blue dissolved in it even on standing for several hours. It is somewhat uncertain as to whether the trimethyl thionin penetrated as such from the external methylene blue solution which generally contains this dye as impurity (in too small concentration for detection by spectrophotometer but detectable by extraction with chloroform), or whether it has formed from methylene blue in the protoplasm. The evidences described in the text tend to favor the former explanation. Theory is discussed on basis of more rapid penetration of trimethyl thionin (in form of free base) than of methylene blue, or of trimethyl thionin in form of salt.     

THE ANTAGONISM BETWEEN ACETIC ACID AND THE CHLORIDES OF SODIUM,POTASSIUM, AND CALCIUM AS MANIFESTED IN DEVELOPING FUNDULUS EMBRYOS

1. Developing Fundulus embryos react in much the same way to mixtures of acetic acid in salt solutions whether their membranes are removed or not. It is therefore not necessary to assume any specific rôle for the membrane. 2. The primary toxic effect of acetic acid is to kill the surface of the embryo; heart stoppage is due to penetration of the acid after the surface has been injured. 3. The salt (NaCl, KCl, CaCl₂) antagonizes the acetic acid by slowing or preventing the killing of the surface of the embryo. 4. If embryos are immersed in certain KCl-acetic acid mixtures, the surface killing effect of the acid is antagonized but at the same time the KCl penetrates and stops the heart in the manner which is characteristic of a KCl solution alone.     

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."     

Studies on Foliar Penetration: III. THE EFFECTS OF CHLORINATION ON THE RATE OF PENETRATION OF PHENOXYACETIC ACID AND BENZOIC ACID INTO LEAVES OF PHASEOLUS VULGARIS

A comparative study has been made of the penetration into leafdisks of Phaseolus vulgaris of (a) phenoxyacetic acid and its2-, 4-, 2, 4-, 2, 6-, 3-5, 2, 4, 5-, and 2, 4, 6- chloro derivatives,and (b) benzoic acid and its 2-, 2, 4-, 2, 5-, 2, 3, 6-chloroand 3, 6-dichloro-2-methoxy derivatives. The methods of synthesisof each compound with ¹⁴C incorporated in the carboxyl groupare described. In the series of substituted phenoxyacetic acids it was establishedthat only for 2, 4, 5-T was there an appreciable loss of radioactivityfrom the system either in the light or darkness. In contrast,with the exception of 2, 4-DCBA, ¹⁴C is lost from each memberof the series of substituted benzoic acids. The level and pattern of penetration in the two series is differentiallyaffected by chlorination. In general progressive chlorinationof phenoxyacetic acid leads to an increase in the rate of penetrationin both light and darkness. There are, however, exceptions;for illuminated disks the rate of entry of 2,4,5-T is exceededby 2,4,6-T, 2, 4-D, and 2, 6-D whereas in the dark 2,4,5-T hasby far the highest rate of penetration. Progressive chlorinationof benzoic acid, however, depresses entry in both light anddarkness. Possible relationships between these findings and changes inselected physical properties of each series of compounds havebeen examined. Between members of the series of substitutedphenoxyacetic acids there is little variation in the dissociationconstant whereas for the substituted benzoic acids there isa marked lowering of the pK as the degree of chlorination increases.The rate of elution with chloroform of each compound from abuffered Silocel column gives a measure of the degree of partitioninto a lipid medium at a given pH. At pH 5.2 chlorination ofphenoxyacetic acid results in more rapid elution whereas chlorinationof benzoic acid causes a longer hold up. An apparatus was designed to enable measurements to be madeof the rate at which a compound moves from one aqueous phaseto another through a layer of lipid. Chlorination promotes therate of transfer of phenoxyacetic acid through octanol but retardsthe transfer of benzoic acid. The extent to which chlorinationdepresses the rate of transfer of benzoic acid matches the diminutionin the rate of leaf penetration. For the series of substitutedphenoxyacetic acids transfer rates and penetration rates followsimilar trends in the dark but in the light agreement is lessgood. These findings are discussed in relation to prior studies onthe rate of uptake and the metabolic fate of the ndividual compoundsin a number of plant tissues.     

THE KINETICS OF SAPONIFICATION OF IODOACETIC ACID BY SODIUM HYDROXIDE AND BY CERTAIN ALKALINE BUFFER SOLUTIONS

1. The rate of the saponification of iodoacetic acid in sodium hydroxide and alkaline buffer solutions yielding glycollic acid was measured by means of Heyrovský''s polarographic method. 2. From the bimolecular velocity constants, increasing with the ionic strength of the solution, the Brönsted factor, F, which characterizes the primary salt effect, was calculated. 3. In the borate buffer solutions the monomolecular constants of the saponification were determined which, at values above the pH of neutralization of boric acid, show a proportionality to the concentration of hydroxyl anions. Below the pH of neutralization of boric acid, they are proportional to the concentration of borate anions.     

萘乙酸、激动素和伤害对绿豆子叶愈伤组织形成的作用以及和色氨酸、吲(口朶)乙酸生物合成的关系

本文以绿豆子叶为材料研究了切伤、外源萘乙酸及激动素诱导形成愈伤组织的作用及其与内源色氨酸和吲哚乙酸生物合成的关系。实验结果表明,切伤对于愈伤组织的形成具有重要作用,切伤面积的大小与愈伤组织的增殖成正比。在绿豆子叶愈伤组织形成的初期,游离色氨酸和内源吲哚乙酸的水平均降低,而在后期,组织内部游离色氨酸和吲哚乙酸的含量都有增加。在培养基中加入外源色氨酸可以部分代替萘乙酸促进愈伤组织的形成。可以认为,外源激素诱导愈伤组织的形成是通过促进内源色氨酸和内源吲哚乙酸的生物合成而实现的。受伤对愈伤组织的形成也起了重要的协同作用。     

THE EFFECT OF METHIONINE SULPHOXIMINE ON THE INCORPORATION OF LABELLED GLUCOSE, ACETATE, PHENYLALANINE AND PROLINE INTO GLUTAMATE AND RELATED AMINO ACIDS IN THE BRAINS OF MICE

—The incorporation of radioactivity from labelled glucose, acetate, phenylalanine and proline into glutamate, aspartate and glutamine was measured in mice treated with methionine sulphoximine and in the control animals. The labelled precursors were injected and their incorporation determined before the onset of convulsions. The incorporation of radioactivity from labelled glucose into the dicarboxylic amino acids was reduced, in particular the incorporation into glutamine. The incorporation of radioactivity from labelled acetate and phenylalanine into glutamate and aspartate was increased by methionine sulphoximine, while the incorporation into glutamine was not changed very much. The labelling of glutamine, relative to glutamate, was reduced with all precursors, indicating that glutamine synthetase was inhibited in vivo by methionine sulphoximine. It is very likely that methionine sulphoximine affects many aspects of energy metabolism in brain; in particular the metabolism of glucose seems to be inhibited, while the rate of conversion of substrates other than glucose seems to be increased.     

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.     

植酸钠对黑曲霉柠檬酸发酵产酸的促进效应

研究了植酸钠对黑曲霉柠檬酸发酵产酸的促进效应。在葡萄糖全合成培养基中添加１％的植酸钠,可使产酸比对照提高２．４倍;在薯粉、玉米粉等天然培养基中添加１％植酸钠,柠檬酸产量分别提高１．７倍和１．３倍。酶活性测定分析表明,植酸钠对柠檬酸代谢途径中的几种关键酶的活性有影响。     

EFFECT OF INCREASED RAT BRAIN TRYPTOPHAN ON 5-HYDROXYTRYPTAMINE AND 5-HYDROXYINDOLYL ACETIC ACID IN THE HYPOTHALAMUS AND OTHER BRAIN REGIONS

—Tryptophan was found at higher concentration in the rat hypothalamus than in other brain regions. This difference was explicable neither by regional differences in blood content nor by differences in tryptophan recovery from different weights of tissue. It was not due to interference by other known brain indoles. After food deprivation or tryptophan injection the tryptophan concentration rose in all regions. Total 5-hydroxyindole increases showed regional differences but relative changes were similar after both procedures. Increases in 5-hydroxytryptamine were clearest in midbrain + hippocampus. In general, 5-hydroxyindolylacetic acid increased more markedly than 5-hydroxytryptamine. The hypothalamus appeared refractory with negligible increases of both 5-hydroxyindoles upon either food deprivation or tryptophan administration even though hypothalamic tryptophan concentration rose considerably. Results are discussed in relation to other evidence suggesting special characteristics of 5-HT regulation in the hypothalamus.