Energy cost of whole-body protein synthesis measured in vivo in chicks

1. Energy cost of whole-body protein synthesis was measured in vivo in chicks by comparing the changes in protein synthesis and heat production after the administration of cycloheximide, an inhibitor of protein synthesis. 2. Incorporation of phenylalanine into whole-body protein fraction was promptly inhibited after the intravenous injection of cycloheximide, and the effect was sustained for at least 3 hr. 3. Both whole-body protein synthesis and total heat production were significantly reduced by the cycloheximide administration. 4. The energy cost of whole-body protein synthesis was calculated to be 5.35 kJ per g protein synthesis, and hence on a molar basis 7.52 ATPs are required per peptide bond synthesis.     

Role of precursor translocation in coordination of murein and phospholipid synthesis in Escherichia coli.

Inhibition of phospholipid synthesis in Escherichia coli by either cerulenin treatment or glycerol starvation of a glycerol-auxotrophic mutant resulted in a concomitant block of murein synthesis. The intracellular pool of cytoplasmic and lipid-linked murein precursors was not affected by an inhibition of phospholipid synthesis, nor was the activity of the penicillin-binding proteins. In addition, a decrease in the activity of the two lipoprotein murein hydrolases, the lytic transglycosylases A and B, could not be demonstrated. The indirect inhibition of murein synthesis by cerulenin resulted in a 68% decrease of trimeric muropeptide structures, proposed to represent the attachment points of newly added murein. Importantly, inhibition of phospholipid synthesis also inhibited O-antigen synthesis with a sensitivity and kinetics similar to those of murein synthesis. It is concluded that the step common for murein and O-antigen synthesis, the translocation of the respective bactoprenolphosphate-linked precursor molecules, is affected by an inhibition of phospholipid synthesis. Consistent with this assumption, it was shown that murein synthesis no longer depends on ongoing phospholipid synthesis in ether-permeabilized cells. We propose that the assembly of a murein-synthesizing machinery, a multienzyme complex consisting of murein hydrolases and synthases, at specific sites of the membrane, where integral membrane proteins such as RodA and FtsW facilitate the translocation of the lipid-linked murein precursors to the periplasm, depends on ongoing phospholipid synthesis. This would explain the well-known phenomenon that both murein synthesis and antibiotic-induced autolysis depend on phospholipid synthesis and thereby indirectly on the stringent control.     

Role of light in the synthesis of nitrate reductase and nitrite reductase in rice seedlings

1. In rice seedlings synthesis of methyl viologen-nitrite reductase was stimulated by light, as was that of NADH-nitrate oxidoreductase (EC 1.6.6.1). A small residual effect of light on the synthesis of the enzymes persisted in the dark for a short time. 2. In etiolated seedlings exposed to light and nitrate, a lag period of 3h was necessary before enzyme synthesis commenced, whereas in green seedlings kept in the dark for 36h, synthesis of both the enzymes started as soon as light and nitrate were provided. 3. Experiments with cycloheximide suggested that fresh protein synthesis in light was necessary for formation of active enzymes. Mere activation by light of inactive enzymes or their precursors, was not involved. 4. In green seedlings synthesis of nitrite reductase was more sensitive to chloramphenicol than that of nitrate reductase. In chloramphenicol-treated etiolated seedlings, however, synthesis of both the enzymes was inhibited to the same extent on subsequent light-treatment. 5. A close correlation was observed between inhibition of the Hill reaction by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and simazin [2-chloro-4,6-bis(ethylamino)-s-triazine] (at high concentration) and the inhibition of enzyme synthesis. At lower concentrations, however, simazin stimulated nitrate reductase. 6. In a single leaf synthesis of enzymes was observed only in portions exposed to light, whereas little activity was present in the dark covered part. 7. CO(2) deprivation severely inhibited the synthesis of enzymes in the light. Sucrose could not reverse this effect. 8. In excised embryos cultured in synthetic media containing sucrose, light was also essential for enzyme formation. 9. It is suggested that redox changes taking place in the green tissues as a result of the Hill reaction create conditions favourable for the induced synthesis of nitrate reductase and nitrite reductase.     

Lead inhibition of enzyme synthesis in soil.

Addition of 2 mg of Pb2+/g of soil concident with or after amendment with starch or maltose resulted in 75 and 50% decreases in net synthesis of amylase and alpha-glucosidase, respectively. Invertase synthesis in sucrose-amended soil was transiently reduced after Pb2+ addition. Amylase activity was several times less sensitive to Pb2+ inhibition than was enzyme synthesis. In most cases, the rate of enzyme synthesis returned to control (Pb2+) values 24 to 48 h after the addition of Pb. The decrease in amylase synthesis was paralleled by a decrease in the number of Pb-sensitive, amylase-producing bacteria, whereas recovery of synthesis was associated with an increase in the number of amylase-producing bacteria. The degree of inhibition of enzyme synthesis was related to the quantity of Pb added and to the specific form of lead. PbSO4 decreased amylase synthesis at concentrations of 10.2 mg of Pb2+/g of soil or more, whereas PbO did not inhibit amylase synthesis at 13 mg of Pb2+/g of soil. Lead acetate, PbCl2, and PbS reduced amylase synthesis at total Pb2+ concentrations of 0.45 mg of Pb2+/g of soil or higher. The results indicated that lead is a potent but somewhat selective inhibitor of enzyme synthesis in soil, and that highly insoluble lead compounds, such as PbS, may be potent modifiers of soil biological activity.     

Role of RNA synthesis in DNA replication of synchronized populations of cultured mammalian cells

Using the harvesting method of synchronizing L cells, the relationship of RNA synthesis of DNA replication was studied by the use of selective inhibitors of RNA synthesis such as actinomycin D and chromomycin succinate. The synthesis of the early replicating DNA fraction is a process sensitive to the inhibition of RNA synthesis during the G1 period. The synthesis of early replicating DNA was inhibited by chromomycin succinate without affecting the initation of DNA synthesis. However, actinomycin D inhibited the synthesis of early replicating DNA and prevented the initiation of DNA synthesis in 50% of the synchronized cells. However, it was found that the continued synthesis of RNA during the S period is not essential for the synthesis of late replicating DNA. In addition to this specific response of DNA synthesis to the inhibitors of RNA synthesis, another function of early and late replicating DNA was determined relative to the cell viability. Cells synthesizing early replicating DNA were killed more efficiently by chromomycin than at other stages of the cell cycle. This indicates that the early replicating DNA unit plays a more important role in cell reproduction than the late replicating DNA unit.     

Intensity of RNA and protein synthesis in fibroblasts of mice in wound healing]

The RNA and protein synthesis in mouse fibroblasts was studied on the 3d and 7th day of wound healing by electron-microscopic autoradiography with double-labeling technique. It was shown that the ratio between the protein synthesis in cytoplasm and the RNA synthesis in the nucleus of some cells significantly differed resulting in relative predominance of one of the two processes studied in the fibroblasts during both terms of wound healing. On an average the fibroblasts were characterized by a higher level of the protein synthesis on the 3d day of wound healing. This average high level was provided by an increase in the fibroblasts, in which the protein synthesis prevailed over the RNA one.     

The Activation by Glucose of Liver Membrane Nitric Oxide Synthase in the Synthesis and Translocation of Glucose transporter-4 in the Production of Insulin in the Mice Hepatocytes

Introduction

Glucose has been reported to have an essential role in the synthesis and secretion of insulin in hepatocytes. As the efflux of glucose is facilitated from the liver cells into the circulation, the mechanism of transportation of glucose into the hepatocytes for the synthesis of insulin was investigated.

Methods

Grated liver suspension (GLS) was prepared by grating intact liver from adult mice by using a grater. Nitric oxide (NO) was measured by methemoglobin method. Glucose transporter-4 (Glut-4) was measured by immunoblot technique using Glut-4 antibody.

Results

Incubation of GLS with different amounts of glucose resulted in the uptake of glucose by the suspension with increased NO synthesis due to the stimulation of a glucose activated nitric oxide synthase that was present in the liver membrane. The inhibition of glucose induced NO synthesis resulted in the inhibition of glucose uptake. Glucose at 0.02M that maximally increased NO synthesis in the hepatocytes led to the translocation and increased synthesis of Glut-4 by 3.3 fold over the control that was inhibited by the inhibition of NO synthesis. The glucose induced NO synthesis was also found to result in the synthesis of insulin, in the presence of glucose due to the expression of both proinsulin genes I and II in the liver cells.

Conclusion

It was concluded that glucose itself facilitated its own transportation in the liver cells both via Glut-4 and by the synthesis of NO which had an essential role for insulin synthesis in the presence of glucose in these cells.     

Changes in ovalbumin and protein synthesis in vivo in the magnum of laying hens during the egg formation cycle.

1. The present study was carried out to investigate whether or not the rate of synthesis of total protein in various oviducal segments and ovalbumin, a major egg white protein, in the magnum fluctuated during the egg formation cycle in laying hens. 2. Synthesis of total protein and ovalbumin was measured in vivo by the incorporation of [15N]methionine after a primed continuous infusion of tracer for 3 hr. 3. Protein and ovalbumin contents in the magnum and the entire oviduct decreased sharply when an ovum moved down from the magnum to the isthmus, probably due to the secretion of egg white proteins. 4. In contrast, total protein and ovalbumin synthesis in the magnum was significantly higher when an ovum was in there than when it was in any other segments. Fluctuations of ovalbumin synthesis and total protein synthesis in the magnum were roughly parallel to those of total protein synthesis in the entire oviduct. 5. It was concluded, therefore, that the changes seen in total protein synthesis in the whole oviduct during the egg formation cycle were mainly attributable to those in magnum protein synthesis, of which a significant portion was accounted for by the synthesis of ovalbumin.     

Polyamine depletion results in impairment of polyribosome formation and protein synthesis before onset of DNA synthesis in mitogen-activated human lymphocytes

Lymphocytes, exposed to mitogens in culture, show enhanced protein and RNA synthesis before the onset of DNA synthesis. Inhibition by DL-alpha-difluoromethylornithine of polyamine synthesis in phytohaemagglutinin-activated human lymphocytes resulted in a suppression of protein synthesis, which was evident before the initiation of DNA synthesis. The mitogen-induced increase in the incorporation of [3H]thymidine into DNA was subsequently inhibited in parallel with the activity of thymidine kinase in the polyamine-depleted cells. Ultraviolet absorbance measurement of the ribosomes after sucrose gradient centrifugation revealed a suppression of polyribosome formation that coincided with the decrease in the rate of protein synthesis. The disturbance in the polysomal profiles did not appear to be due to a shortage of mRNA, since the synthesis of poly(A)-rich mRNA was reduced less than that of rRNA after inhibiting polyamine synthesis. Entry of both the pre-existing and newly synthesized ribosomal subunits into polysomal structures was found to be impaired. These results thus suggest an important role for polyamines in the initiation step of protein synthesis.     

Activation and inactivation of synthesis of secondary wall polymers in Bacillus subtilis W23

The progress of activation and inactivation of synthesis of the wall polymers, teichoic acid and teichuronic acid, in response to changes in the phosphate content of the growth medium has been examined using toluenised cells of B. subtilis W23. Activation of teichoic acid synthesis from nucleotide precursors was independent of protein synthesis, but chloramphenicol prevented activation when DL-glycerol 3-phosphate and CTP replaced CDP-glycerol as one of the substrates of the reaction. Activation of teichuronic acid synthesis was dependent on synthesis of protein. Inactivation of synthesis of both polymers was slowed, but not prevented, by inhibition of protein synthesis. Evidence was obtained that a protein synthesised during phosphate starvation retards the activation of teichoic acid synthesis.     

Translational control of alpha-amylase gene expression in Aspergillus awamori

Regulation of alpha-amylase gene expression in Aspergillus awamori was studied by analyzing the enzyme activity levels, rate of protein synthesis, and alpha-amylase-specific mRNA levels under various conditions of growth. alpha-Amylase synthesis was sensitive to catabolite repression as glucose repressed its synthesis by about fourfold. The stimulation of alpha-amylase synthesis in the presence of its substrate starch was shown to be due to derepression rather than induction as the enzyme was synthesized at similar rates in both starch and starvation media. Repression and derepression of enzyme synthesis was found to be mediated at the translational level. The cellular levels of alpha-amylase-specific mRNA as measured by an in vitro translation assay system, were almost identical under all conditions of enzyme synthesis. Relative in vivo and in vitro alpha-amylase mRNA template activities suggest that alpha-amylase mRNA is translated much more efficiently during the derepression than under the conditions of repressed synthesis.     

Extracellular matrix metabolism by chondrocytes. III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. Benzyl-beta-D-xyloside, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.     

Control of Replication in RNA Bacteriophages

The rates of viral RNA and protein syntheses for wild-type RNA bacteriophages and their nonpolar, coat protein amber mutants were determined in amber suppressor (S26R1E, Su-1 and H12R8a, Su-3) and nonsuppressor (AB259, S26, and Q13) strains of Escherichia coli in the presence of rifamycin. It was demonstrated that the rates of synthesis of phage-specific replicase and RNA minus strands drop off concurrently in both wild-type and coat protein mutant-infected Su(-) and Su(+) cells after 10 and 15 min postinfection, respectively. The rate of synthesis of RNA plus strands started to decline 5 to 10 min later in both cases. Excessive synthesis of replicase in the coat protein mutant-infected cells was accompanied by a similar overproduction of RNA minus strands, but not of plus strands. Partial suppression of protein synthesis in wild-type phage-infected cells abolishing coat protein control over replicase accumulation led to prolongation of replicase synthesis. Such an effect was observed also in coat protein mutant-infected cells, indicating that the excess of replicase itself may be capable of suppression of replicase synthesis in the absence of coat protein. The prolongation of replicase synthesis was followed by the prolonged synthesis of RNA minus strands in both cases. Moreover, replicase and minus strands were formed in nearly equal amounts when protein synthesis was partially inhibited. Assuming functional instability of phage RNAs, the observed coupling of replicase and minus-strand RNA synthesis offers a possibility for control of viral RNA replication by means of control of replicase synthesis on the translational level. A hypothesis is put forward to explain the molecular mechanism of such coupling between the syntheses of replicase and RNA minus strands.     

Temperature sensitivity of DNA synthesis in mouse testicular germ cells in vitro

The effect of temperature on testicular DNA synthesis in mice was studied in vitro. By using cultures of cryptorchid testis, DNA synthesis of differentiated germ cells, such as intermediate and type B spermatogonia and resting primary spermatocytes, was shown to be temperature-sensitive, while that of undifferentiated type A spermatogonia was not. DNA synthesis of non-germ cells was not temperature-sensitive. This temperature sensitivity of germ cells in DNA synthesis may be one cause of the thermal inhibition of germ cell differentiation.     

Effect of diabetes on the rates of synthesis and degradation of ribosomes in rat muscle and liver in vivo

An important component of the decrease in protein synthesis in muscle of diabetic animals is a fall in the ribosome content. Therefore, we have investigated the turnover of ribosomes in skeletal muscle, heart, and liver of rats during the onset of diabetes. Synthesis rates were measured by incorporation of label into the protein moieties of the ribosomes, and a dual isotope technique was used to relate ribosome synthesis to that of total tissue protein. Degradation rates were calculated as the difference between the rates of synthesis and accumulation. The loss of ribosomes from gastrocnemius muscle and heart took place mainly between the 2nd and 4th days of insulin deficiency and was brought about largely by a very pronounced increase in the degradation rate, though synthesis also fell by a substantial amount. Rates of total tissue protein synthesis decreased markedly, but the degradation rates were only slightly elevated, if at all. Thus, the effect of diabetes on muscle ribosome breakdown was quite distinct from that on degradation of total tissue protein. In liver the response of protein synthesis to diabetes was much less pronounced than in muscle, and ribosome synthesis was not affected.     

Induction of deoxyribonucleic Acid synthesis in potato tuber slices: role of protein synthesis

Timing of protein synthesis which is a prerequisite to DNA synthesis induced in potato tuber tissue (Solanum tuberosum L.) by cut injury has been studied using cycloheximide. The induction of DNA synthesis which was measured by incorporation of ³H-thymidine was completely inhibited when the inhibitor was applied to the tuber discs immediately after slicing. When the application of cycloheximide was delayed for 6 hours or more after slicing, DNA synthesis was observed but its rate was reduced to 20% of control. The inhibitory effect of cycloheximide, however, rapidly decreased when the inhibitor was applied at 6 or less hours immediately prior to determination of DNA synthesis. The effect of cycloheximide on the incorporation of ¹⁴C-leucine suggests that the change in the effect of cycloheximide on the induction of DNA synthesis is not due to incomplete inhibition of protein synthesis. Cycloheximide did not have significant effects on either uptake or phosphorylation of ³H-thymidine in the discs. Inhibition of both protein and DNA synthesis by cycloheximide was reversed by washing and further incubation of the discs. Almost no qualitative difference was detected by buoyant density analysis between DNA formed under inhibition of protein synthesis of the later stage and DNA synthesized under normal conditions. These results suggest that DNA synthesis induced in potato tuber tissue by cut injury requires continuous synthesis of new protein molecules in a characteristically programmed sequence.     

Vagotomy effect on the content and synthesis of acetylcholine in autonomic ganglia in dog stomach

The effect of total, selective and highly selective vagotomy was studied on the content and synthesis of acetylcholine in the tissues of the gastric fundus and pyloric part of the stomach in mongrel dogs of either sex aged 3 to 6 years. Total vagotomy produced after 3 weeks a non-significant rise in the amount and a highly significant increase of the synthesis of ACh in the fundus, and a significant increase in the amount and synthesis of ACh in the pyloric part. Six weeks after the procedure the Ch content of the fundus and pyloric part decreased statistically significantly, while the synthesis rate was not different from the initial one. Three weeks after selective vagotomy the ACh content of the fundus and pyloric part was significantly increased. The rate of ACh synthesis was normal. Six weeks after vagotomy the content of ACh and its synthesis in the fundus were similar to those before vagotomy. In the same time the ACh synthesis rate was significantly decreased in the pyloric part, while the ACh content there was similar to the control value. Highly selective vagotomy with denervation of the fundus and corpus of the stomach but with maintenance of the innervation of the pyloric part caused after 3 weeks a rise of the ACh content of the fundus tissues. The ACh synthesis rate was not different from the normal one. In the pyloric part the ACh content was significantly decreased and the synthesis of ACh was somewhat inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of synthesis of alkaline phosphatase by deoxyribonucleic acid synthesis in a constitutive mutant of Bacillus subtilis

Hiraga, Sota (Osaka University, Osaka, Japan). Regulation of synthesis of alkaline phosphatase by deoxyribonucleic acid synthesis in a constitutive mutant of Bacillus subtilis. J. Bacteriol. 91:2192-2199. 1966.-It was found that synthesis of alkaline phosphatase (APase) correlated with deoxyribonucleic acid (DNA) synthesis in a partially constitutive mutant of Bacillus subtilis. When cultures of the mutant were made to undergo synchronous growth by germination of spores in an excess-phosphate medium, synthesis of APase was repressed at the beginning of DNA synthesis. If the initiation of DNA synthesis was inhibited by thymine starvation, the repression of APase was not observed. When DNA synthesis, previously initiated, was inhibited by thymine or uracil starvation, or by addition of mitomycin C, the repression was partially released at a later stage. In contrast, this correlation between repression and DNA synthesis was not observed in a repressible strain.     

Inhibition of macromolecular synthesis in cultured macrophages by Pseudomonas pseudomallei exotoxin

Pseudomonas pseudomallei exotoxin was found to be a potent inhibitor of protein and DNA synthesis in cultured macrophages. Inhibition of DNA synthesis occurred at toxin concentrations as low as 1-2 micrograms/ml and inhibition of 3H-thymidine uptake was almost complete at concentrations of 8 micrograms/ml or more. A close correlation between cell damage and inhibition by DNA synthesis was observed. For protein synthesis, inhibition was obtained at much lower doses (0.06-2.0 micrograms/ml) of the toxin. At similar toxin concentrations, DNA synthesis was marginally affected. Further, it was shown that protein synthesis inhibition occurred almost immediately after incubation, reaching its maximal inhibitory effect of 70% after 6 hr. DNA synthesis, however, was minimally affected by a similar toxin concentration even after 10 hr of incubation. The inhibition of macromolecular synthesis in macrophages by P. pseudomallei exotoxin may be relevant to its modulatory effect on the host defense mechanism.