ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.     

PRODUCTION OF MEMBRANE WHORLS IN RAT LIVER BY SOME INHIBITORS OF PROTEIN SYNTHESIS

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [³H]leucine and of [³H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [³H]leucine incorporation into cytoplasmic membranes was inhibited, while [³H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.     

ON THE MECHANISM OF ELECTROSHOCK-INDUCED INHIBITION OF PROTEIN SYNTHESIS IN RABBIT CEREBRAL CORTEX

Abstract— The mechanism of electroshock (ES)-induced inhibition of protein synthesis in rabbit cerebral cortex has been investigated by using a cell-free system. The protein biosynthetic activity of the post-mitochondrial supernatant (PMS) obtained from the cerebral cortex of ES-treated animals was found to be markedly lower than in controls (C). This inhibition was accompanied by a decrease of polysomes and an increase of monomers. In addition, a relative increase in light polysomes was evident at short intervals after ES treatment. No difference was found in the total soluble activity and in the activity of the elongation factors and ribonuclease present in the cell sap of C and ES animals. The biosynthetic activity of ES-total. free and membrane-bound ribosomes was approx 45% lower than that of the corresponding C fractions: polysome/monomer ratios were similarly reduced. The total content of cortical ribosomes was not affected by ES. Following ES treatment there was no change in the ribo-somal ability to elongate, terminate and release polypeptide chains, nor a decrease in the polysomal content of poly(A)-containing mRNA. These data strongly suggest that the ES-induced inhibition of protein synthesis results from a defect in the initiation process. The possible mechanisms mediating this defect have been discussed.     

THE EFFECTS OF INHIBITORS OF RNA AND PROTEIN SYNTHESIS ON CHLOROPLAST STRUCTURE AND FUNCTION IN WILD-TYPE CHLAMYDOMONAS REINHARDI

Wild-type cells of the unicellular green alga Chlamydomonas reinhardi have been grown for several generations in the presence of rifampicin, an inhibitor of chloroplast DNA-dependent RNA polymerase, spectinomycin and chloramphenicol, two inhibitors of protein synthesis on chloroplast ribosomes, and cycloheximide, an inhibitor of protein synthesis on cytoplasmic ribosomes. The effects of cycloheximide are complex, and it is concluded that this inhibitor cannot give meaningful information about the cytoplasmic control over the synthesis of chloroplast components in long-term experiments with C. reinhardi. In the presence of acetate and at the appropriate concentrations, the three inhibitors of chloroplast protein synthesis retard growth rates only slightly and do not affect the synthesis of chlorophyll; however, photosynthetic rates are reduced fourfold after several generations of growth. Each inhibitor produces a similar pattern of lesions in the organization of chloroplast membranes. Only rifampicin prevents the production of chloroplast ribosomes.     

THE PREVENTION BY INHIBITORS OF BRAIN PROTEIN SYNTHESIS OF THE HYPERACTIVITY AND HYPERPYREXIA PRODUCED IN RATS BY MONOAMINE OXIDASE INHIBITION AND THE ADMINISTRATION OF L-TRYPTOPHAN OR 5-METHOXY-N,N-DIMETHYLTRYPTAMINE

Abstract— In rats treated with a monoamine oxidase inhibitor, (tranylcypromine), L- tryptophan produces a stereotyped syndrome of hyperactivity and hyperpyrexia due to an increased rate of brain serotonin (5-hydroxytryptamine) synthesis. Pretreatment of rats with intraperitoneal injections of cycloheximide, acetoxycycloheximide, emetine and dehydroemetine and of mice with puromycin inhibited this syndrome. Cycloheximide also inhibited the hyperactivity caused by tranylcypromine and DL-15-hydroxtryptophan and did not affect the increased rate of brain serotonin ‘synthes’ is produced by tryptophan thus excluding a primary effect on tryptophan-5-hydroxylase. Inhibition of hyperactivity did not occur until brain protein synthesis was inhibited by greater than 65 per cent as measured by the incorporation of L-[U-¹⁴C]tyrosine into brain protein in vivo. Emetine, which has been shown to inhibit brain protein synthesis inhibited hyperactivity whereas isoemetine which did not inhibit brain protein synthesis, did not inhibit hyperactivity. Under conditions where cycloheximide inhibited hyperactivity produced by tranylcypromine and L-tryptophan, a large dose of 5-methoxy-N,N-dimethyltryptamine(5-MeODMT) still produced hyperactivity showing that the rats were still capable of the same pattern of hyperactivity. However, cycloheximide did inhibit hyperactivity due to 5-MeODMT, the degree of this inhibition being dependent upon a balance between the doses of cycloheximide and 5-MeODMT. 5-MeODMT probably acts directly within the brain to cause behavioural excitation and it seems likely that the inhibitors of brain protein synthesis interfere with the mechanism of action of brain 5HT and administered 5-MeODMT rather than upon any aspect of synthesis, storage or release of brain 5HT. It is suggested that the behaviourally excitant effects of brain 5HT and 5-MeODMT are mediated in some way by a brain protein with a short biological half-life. Such a protein may act either as a factor specifically mediating the central effects of brain 5HT or as a factor regulating the neuronal response to excitation by 5HT and 5-MeODMT.     

锌诱导的蛋白合成增加可抵抗链佐霉素所致的胰岛细胞损伤

本工作在离体细胞水平观察ZnGl_2对链佐霉素(STZ)诱发的胰岛β细胞损伤的保护作用,并分析其可能的作用机制。结果如下:向培养的胰岛细胞中加入生理盐水和STZ(3mmol/L),孵育12h后,活细胞数由实验前的70万个/ml降至43.93±1.16万个/ml;将ZnCl_2(0.25、0.5、1.0mmol/L)和相同剂量的STZ一同加入细胞,可不同程度地缓解STZ对胰岛的破坏作用,在含不同浓度ZnCl_2的培养液中活细胞数分别恢复至47.39±0.88,58.06±2.29,67.72±1.48万个/ml,与STZ破坏组相比,分别具有显著差异,并呈量效关系。在给予ZnCl_2(1.0mmol/L)的同时,向细胞中加入蛋白合成抑制剂亚胺环已酮(100μg/ml),可翻转ZnCl_2的作用,活细胞数由63.17±2.15万个/ml,又重新减至45.77±0.76万个/ml。单独加亚胺环己酮对活细胞数目无明显影响。用~3H-亮氨酸掺入实验观察ZnCl_2对胰岛细胞蛋白合成的影响发现,单独给予ZnCl_2(1.0mmol/L)仅使蛋白合成轻微增加,与盐水对照组无显著差异;在给ZnCl_2的同时加入STZ,则蛋白合成明显增多,保护组与STZ破坏组比较,差异显著。上述结果表明,增加细胞内蛋白合成,以加强细胞自身对外来损伤的修复力,可能是ZnCl_2保护胰岛细胞的机制之一。     

FETAL DEVELOPMENT: THE EFFECTS OF MATURATION ON IN VITRO PROTEIN SYNTHESIS BY MOUSE BRAIN TISSUE

—The elucidation of the translational regulatory events which function during the critical fetal and neonatal period is an important prerequisite to our understanding of normal, as well as abnormal, brain growth and differentiation. Brain cell suspensions and cell-free homogenates were employed to study the protein synthetic activity during the maturation of fetal- neural tissue. The results clearly demonstrated that while neural tissue from 1-day postnatal mice was 10 times more active in protein synthesis than brain tissue from adult mice, the former was many fold less active in translational events than fetal neural tissue from 13-day post-zygotic mice. Fetal polypeptide synthetic activity was found to decrease from the 13th day to the 19th day post-zygotic. This decrement in the translational activity was not due to amino acid availability or pools, or to differences, quantitatively or qualitatively, in polysome concentrations. The enhanced rate of protein synthetic activity measured with neural tissue from 13-day post-zygotic mice was shown to be due to an increase in rate of protein synthesis and not to an enhanced rate of protein degradation.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO THIRAM, ZIRAM, FERBAM, NABAM AND ZINEB

The concentrations of thiram preventing germination of spores of Botrytis cinerea in drops of a 1% solution of sucrose, and on the surface of a sucrose-nitrate agar have been determined. Thiram had much less effect on germination in the agar medium, even when a purified agar was used. There was no growth on sucrose-nitrate agar if the concentration of thiram exceeded 31 p.p.m. Attempts to obtain strains able to grow at higher concentrations were unsuccessful.
Similar results were obtained with ziram, nabam and zineb.
Ferbam also was more effective in preventing spore germination in spore drops than on agar media; this effect was obtained with ordinary and with purified agar.
On a sucrose-nitrate agar generally there was no growth if the concentration of ferbam exceeded 125 p.p.m., but in one of forty-eight plates containing 250 p.p.m. ferbam, five slowly growing colonies were produced, and from these colonies arose mycelium which grew and sporulated rapidly on 500 p.p.m. ferbam agar. Agar disk inocula were transferred from these cultures to agar containing higher concentrations of ferbam and in this way, and by repeating the process, a strain was obtained which grew slowly but continuously, and sporulated on agar containing 5000 p.p.m. ferbam. However, the poor solubility of this fungicide made it difficult to assess quantitatively the degree of adaptation.
A proportion of the spores from this strain germinated in drops containing about twice the concentration of ferbam which prevented germination of parent spores.
The resistance of the mycelium of the resistant strain was not lost after repeated subculture on fungicide-free agar. The resistant strain was as susceptible as the parent strain to thiram, ziram, nabam and zineb.
Attempts to obtain strains of Venturia inaequalis resistant to thiram, ferbam, ziram and zineb were unsuccessful.     

THE ADAPTATION OF FUNGI TO FUNGICIDES: ADAPTATION TO COPPER AND MERCURY SALTS

The susceptibility of Botrytis cinerea to copper sulphate in liquid media increased when the volume, and therefore the depth, of the medium in culture bottles exceeded certain values; when the volume was 40 ml. the maximum concentration allowing growth was 300 p.p.m.
By growing mycelium in media containing progressively higher concentrations of copper sulphate a strain was produced which grew at a concentration of 750 p.p.m.
In high concentrations of copper sulphate growth always started at the edge of the liquid, and inocula grew only if they were placed in this position.
In germination tests spores from the resistant strain were more resistant to copper sulphate than were spores of the parent strain.
The resistance of mycelium, and to a lesser extent of spores, was retained after growth of the resistant strain for six months in fungicide-free media.
Spore and mycelial inocula grew in much higher concentrations of copper sulphate when nutrient media were solidified with agar.
The strain resistant in liquid media was no more resistant than the parent strain on agar media.
The resistance of the fungus was not increased after growth for long periods on agar containing high concentrations of copper sulphate. The resistance of the strain resistant in liquid media was not lost after growth on agar media for 3 months.
Attempts to produce strains more resistant than the parent to mercuric chloride were unsuccessful.
The results obtained with phenyl-mercuric acetate were essentially similar to those obtained with copper sulphate, but relatively much more resistant strains were produced.     

EFFECTS OF SOME PROTEIN AND NUCLEIC ACID SYNTHESIS INHIBITORS ON FERTILIZATION IN VOLVOX CARTERI1

The fertilization process in Volvox involves a series of events described as follows. A sperm bundle produced by the male colony attaches to the female colony. The bundle then dissociates to a cluster of individual sperm prior to formation of a fertilization pore in the sheath of the female colony. Sperm subsequently enter the matrix of the female colony and presumably fertilize the eggs. The protein synthesis inhibitors cycloheximide, chloramphenicol, and puromycin each, blocked fertilization pore formation. The nucleic acid synthesis inhibitors actinomycin D and 5-fluorouracil were also tested. Actinomycin D completely blocked fertilization pore formation, whereas 5-fluorouracil appeared to have no effect. A proposed mechanism of fertilization pore formation is discussed.     

THE DARK ADAPTATION OF THE EYE OF LIMULUS,AS MANIFESTED BY ITS ELECTRIC RESPONSE TO ILLUMINATION

On page 387, Vol. 13, No. 3, January 20, 1930, the numbers in the last column of Table 1 should be divided by 10 to give the true values of k''. In the next to the last line, under Table 1, for value of C = 0.337 read value of C = –0.337.     

EXPERIMENTS ON THE ADAPTATION OF ESCHERICHIA COLI TO SODIUM CHLORIDE

1. It has been shown that a fairly constant fraction of the total number of bacteria in a fresh-water culture of E. coli can reproduce on direct transfer to a saline medium with a definite NaCl concentration, as judged from the viable count determinations in such a medium. 2. The absolute value of this fraction depends on a number of factors other than the salt content of the test medium, such as the hydrogen ion and yeast autolysate concentrations, aeration, and the physiological condition of the bacteria. 3. A method for testing the degree and rate of adaptation of the bacteria to saline environment, depending on the analysis of changes in the value of the salt-viable fraction, was developed. 4. Maximum adaptability to saline environments was found during the early stationary phase of NaCl-free cultures. Low adaptability accompanied the logarithmic phase and the senescence of the cultures. 5. The limits of variation could be extended by treatment of non-dividing cells with gradually increasing concentrations of salt or by subjecting them to a single intermediate NaCl concentration. This acclimatization was independent of reproduction. The number of bacteria becoming capable of reproducing in a hitherto unfavorable environment increased with the period of exposure to intermediate salt concentrations until a maximum value was reached. 6. This maximum value was shown to depend on the salinity of the test medium, the age of the bacterial culture, and the method of preliminary treatment. "Optimal acclimatization" could be effected by subjecting the organisms to a single fairly low intermediate NaCl concentration. 7. The rate of the individual acclimatization process was shown to be greater at higher than at lower temperatures. 8. Acclimatized bacteria rapidly lost their increased ability to reproduce in saline media upon return to a salt-free environment, although no reproduction of the cells could be detected. This was interpreted as an indication that the processes involved are readily reversible. 9. Studies on the reproduction of E. coli in strongly saline broth indicated that only those cells originally acclimatized to the salt concentration of the medium could divide. All cells produced in such a medium could continue to reproduce. The propagation in the altered medium was not accompanied by any further acclimatization throughout five subcultures. 10. Both the division rate and the maximum crop of cultures in saline broth were considerably lower than of those in a fresh-water medium. No change in either occurred throughout five successive subcultures. The morphology of the organisms was also altered by the presence of salt. 11. The division rate, maximum crop, morphology, and adaptive power returned immediately to normal on re-transfer of bacteria grown in an NaCl-containing medium to "salt-free" broth. 12. The entire adaptive response of the bacteria to a considerable increase in the salinity of the environment could thus be separated into two components: an acclimatization, independent of reproduction, and a selection of those cells with the widest range of potentialities.