Brief fasting decreases protein synthesis in the brain of adult rats

The influence of starvation on protein synthesis in the adult rat brain was studied in vivo by an intravenous injection of a flooding dose of unlabeled valine including a tracer dose ofL-[3,4(n)-³H]valine. Brief starvation (24 hours) induced a 20% decline in fractional and absolute rates of brain protein synthesis. This decline resulted from a 20% decrease in the efficiency of protein synthesis (g protein synthesized per day per g RNA) whereas the capacity for protein synthesis (g RNA per mg protein) was maintained. Prolonged starvation (5 days) was marked by no further significant changes in the fractional rate, absolute rate and efficiency of protein synthesis, whereas the capacity for protein synthesis cecreased slightly. The relative contribution of brain to wholebody body protein synthesis increased during fasting, and neither the protein nor the RNA brain content did change during the experiment. These results clearly indicate that brain proteins are spared in response to brief and prolonged food deprivation, and that brain protein synthesis is very sensitive to short-term fasting.     

Dynamic visualization of local protein synthesis in hippocampal neurons

Using pharmacological approaches, several recent studies suggest that local protein synthesis is required for synaptic plasticity. Convincing demonstrations of bona fide dendritic protein synthesis in mammalian neurons are rare, however. We developed a protein synthesis reporter in which the coding sequence of green fluorescent protein is flanked by the 5' and 3' untranslated regions from CAMKII-alpha, conferring both dendritic mRNA localization and translational regulation. In cultured hippocampal neurons, we show that BDNF, a growth factor involved in synaptic plasticity, stimulates protein synthesis of the reporter in intact, mechanically, or "optically" isolated dendrites. The stimulation of protein synthesis is blocked by anisomycin and not observed in untreated neurons. In addition, dendrites appear to possess translational hot spots, regions near synapses where protein synthesis consistently occurs over time.     

Effect of cytosine arabinoside on viral-specific protein synthesis in cells infected with herpes simplex virus.

The relationship between viral DNA and protein synthesis during herpes simplex virus type 1 (HSV-1) replication in HeLa cells was examined. Treatment of infected cells with cytosine arabinoside (ara-C), which inhibited the synthesis of HSV-1 DNA beyond the level of detection, markedly affected the types and amounts of viral proteins made in the infected cell. Although early HSV-1 proteins were synthesized normally, there was a rapid decline in total viral protein synthesis beginning 3 to 4 h after infection. This is the time that viral DNA synthesis would normally have been initiated. ara-C also prevented the normal shift from early to late viral protein synthesis. Finally, it was shown that the effect of ara-C on late protein synthesis was dependent upon the time after infection that the drug was added. These results suggest that inhibition of progeny viral DNA synthesis by ara-C prevents the "turning on" of late HSV-1 protein synthesis but allows early translation to be "switched off."     

The energetic cost of protein synthesis in isolated hepatocytes of rainbow trout (Oncorhynchus mykiss)

Summary To establish the energetic cost of protein synthesis, isolated trout hepatocytes were used to measure protein synthesis and respiration simultaneously at a variety of temperatures. The presence of bovine serum albumin was essential for the viability of isolated hepatocytes during isolation, but, in order to measure protein synthesis rates, oxygen consumption rates and RNA-to-protein ratios, BSA had to be washed from the cells. Isolated hepatocytes were found to be capable of protein synthesis and oxygen consumption at constant rates over a wide range of oxygen tension. Cycloheximide was used to inhibit protein synthesis. Isolated hepatocytes used on average 79.7±9.5% of their total oxygen consumption on cycloheximide-sensitive protein synthesis and 2.8±2.8% on maintaining ouabain-sensitive Na⁺/K⁺-ATPase activity. The energetic cost of protein synthesis in terms of moles of adenosine triphosphate per gram of protein synthesis decreased with increasing rates of protein synthesis at higher temperatures. It is suggested that the energetic cost consists of a fixed (independent of synthesis rate) and a variable component (dependent on synthesis rate).Abbreviations BSA bovine serum albumin - dpm disintegrations per min - k _s fractional rate of protein synthesis - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid - PHE phenylalanine; PO₂ oxygen tension - PCA perchloric acid     

Inhibition by Chloramphenicol of Chlorophyll and Protein Synthesis and Growth in Euglena gracilis

SYNOPSIS. Chloramphenicol inhibits growth of Euglena gracilis. It also interferes in the "greening" process by inhibiting protein synthesis (measured by leucine uptake), but RNA synthesis is essentially unafiected. The antibiotic acts on all cell fractions, but prefermtially inhibits the synthesis of plastid protein. Inhibition of chlorophyll synthesis is transitory and does not affect the self-reproduction system for chloroplast formation.     

Site of synthesis of the proteins of mammalian mitochondrial ribosomes. Evidence from cultured bovine cells

In order to determine the sites of synthesis of the proteins of the mammalian mitochondrial ribosome (mitoribosome), bovine (MDBK) cells were labeled with [35S]methionine in the presence of inhibitors of mitochondrial and cytoplasmic protein synthesis. Labeling in the absence of cytoplasmic protein synthesis produced a "blank" fluorogram, indicating that there is no mitochondrial product. Additionally, incorporation of [35S]methionine into the enumerated mitoribosomal proteins continued in the absence of mitochondrial protein synthesis. Finally, it was demonstrated that mitoribosomal proteins can be both translated and assembled into complete mitoribosomes in the absence of mitochondrial protein synthesis. These results indicate that in mammals, as opposed to lower eukaryotes, all of the mitoribosomal proteins are products of cytoplasmic protein synthesis.     

Relationship Between Protein Synthesis and Viral Deoxyribonucleic Acid Synthesis

Evidence is presented that poxvirus deoxyribonucleic acid (DNA) synthesis required concurrent protein synthesis. The protein requirement in question can be distinguished from viral-induced thymidine kinase and DNA polymerase by virture of the instability of its messenger ribonucleic acid and its stoichiometric rather than catalytic relation to DNA synthesis. The protein(s) required did accumulate in the presence of fluorodeoxyuridine, an inhibitor of DNA synthesis, and, thus, appeared to be an "early" poxvirus function. The protein(s) was stable since it did function several hours after its synthesis had been terminated by puromycin. Two possible roles for such a protein requirement are discussed.     

The regulation of RNA synthesis in yeast I: Starvation experiments

Summary The synthesis of tRNA in yeast is shown to be under separate control to that of rRNA during amino acid and nitrogen starvation. Inhibitors of the elongation and termination steps of protein synthesis were found to stimulate the synthesis of tRNA in starved yeast cells. This effect appeared to be due to the trickle-charging of tRNA. Two inhibitors of early steps in the initiation of protein synthesis were found to be unable to stimulate RNA synthesis in starved cells. It is proposed that yeast tRNA synthesis is under autoregulatory control and that the level of tRNA charging and the mRNA-ribosome complex are important components of this control system.     

Cycloheximide insensitive nuclear protein synthesis partially suppressed by chloramphenicol: Some possible implications in the evolution of eukaryotes

• 1.1. Cycloheximide-resistant nuclear protein synthesis that is almost completely suppressed by puromycin and partially inhibited by chloramphenicol can be demonstrated with the use of a mutant Chinese hamster ovary cell line exhibiting a temperature-sensitive defect in cytoplasmic non-mitochondrial protein synthesis.
• 2.2. For a number of reasons, mitochondria are unlikely to be the source of chloramphenicol-sensitive cycloheximide-resistant nuclear protein synthesis.
• 3.3. The existence of nuclear protein synthesis that is unaffected by cycloheximide and partially inhibited by chloramphenicol raises a number of questions about the evolution of eukaryotic cell nuclei.

     

Tropic effect of dexamethasone on goldfish melanocytoma cells: induction of calcium-dependent but protein synthesis-independent morphological changes

Treatment of melanocytoma cells of goldfish origin with dexamethasone leads to rapid morphological changes, flattening of cell body and extension of dendrites. This effect is independent of protein synthesis but requires the presence of extracellular calcium, indicating that it is a "tropic effect" distinct from the typical "trophic effects" of steroid hormones that involve de novo protein synthesis.     

Study of the ultrastructural and functional expression of the hepatocyte genome under conditions of prolonged depression of protein biosynthesis by cycloheximide. III]

Dynamics of changes in mtRNA synthesis and mitochondria ultrastructure is strictly dependent on the level of inhibition of biosynthesis of cytoplasm proteins and "soluble" proteins of mitochondria by cycloheximide in hepatocytes: 1-6 hrs later a progressive weakening of protein synthesis is accompanied by a drop in mtRNA synthesis and essential destruction of mitochondria; from 12 to 24 hrs a partial restoration of protein biosynthesis induces the processes of the above-mentioned indexes normalization.     

Aging Is Accompanied by a Blunted Muscle Protein Synthetic Response to Protein Ingestion

Purpose

Progressive loss of skeletal muscle mass with aging (sarcopenia) forms a global health concern. It has been suggested that an impaired capacity to increase muscle protein synthesis rates in response to protein intake is a key contributor to sarcopenia. We assessed whether differences in post-absorptive and/or post-prandial muscle protein synthesis rates exist between large cohorts of healthy young and older men.

Procedures

We performed a cross-sectional, retrospective study comparing in vivo post-absorptive muscle protein synthesis rates determined with stable isotope methodologies between 34 healthy young (22±1 y) and 72 older (75±1 y) men, and post-prandial muscle protein synthesis rates between 35 healthy young (22±1 y) and 40 older (74±1 y) men.

Findings

Post-absorptive muscle protein synthesis rates did not differ significantly between the young and older group. Post-prandial muscle protein synthesis rates were 16% lower in the older subjects when compared with the young. Muscle protein synthesis rates were >3 fold more responsive to dietary protein ingestion in the young. Irrespective of age, there was a strong negative correlation between post-absorptive muscle protein synthesis rates and the increase in muscle protein synthesis rate following protein ingestion.

Conclusions

Aging is associated with the development of muscle anabolic inflexibility which represents a key physiological mechanism underpinning sarcopenia.     

Rats that consume caffeine show decreased brain protein synthesis

The effect of caffeine on protein synthesis in brain and liver was studied. When caffeine was added to a post-mitochondrial supernatant from rat brain protein synthesis was inhibited, i.e. 1 mM caffeine about 20%. The effect on protein synthesis of two weeks administration of large doses of caffeine in the drinking fluid of rats was also measured. Caffeine decreased protein synthesis in rat brain by about 32% and 20% compared with ad libitum and pair-fed controls. Protein synthesis was calculated taking into account the levels of free leucine determined by HPLC: 0.10 mol/g brain of ad libitum; 0.11 for pair-fed and 0.07 for caffeine. The pattern of proteins synthesized was not significantly altered by caffeine as shown by gel-electrophoresis and fluorography. There was no effect on protein synthesis of liver. The possible significance of these results is briefly discussed.     

Effect of protein synthesis inhibition on recovery of UV- and γ-irradiated Schizosaccharomyces pombe from repair inhibition by caffeine

Summary The progress of repair in Schizosaccharomyces pombe may be followed during post-irradiation incubation by measuring, after various intervals, the ability of UV- or -irradiated cells to avoid enhanced lethality when exposed to the repair inhibitor caffeine (Gentner and Werner, 1975). This technique has now been used to investigate the effect of inhibition of protein synthesis on repair of UV- and -irradiation-induced damage in this organism.When protein synthesis was inhibited with cycloheximide in UV-irradiated wild-type cells, only a small amount of recovery from caffeine inhibition occurred; this indicated that post-irradiation protein synthesis was required for repair, and in particular for the recombinational repair pathway, which is a major mechanism for repair of UV damage in this organism.In -irradiated wild-type cells, inhibition of post-irradiation protein synthesis reduced the rate of recovery from repair inhibition by caffeine, but full recovery from caffeine-sensitive damage did occur at longer incubation times. We attribute the reduction in rate to the effect of protein synthesis inhibition on the recombinational repair pathway, because this pathway is known to be involved in the repair of both -ray and UV damage. The recovery that took place at the slower rate must reflect a caffeine-sensitive pathway which is involved only in repair of -ray damage and which does not require post-irradiation protein synthesis for activity.AECL Reference No. 5402     

Transport of cholesterol into mitochondria is rate-limiting for bile acid synthesis via the alternative pathway in primary rat hepatocytes

Bile acid synthesis occurs mainly via two pathways: the "classic" pathway, initiated by microsomal cholesterol 7alpha-hydroxylase (CYP7A1), and an "alternative" (acidic) pathway, initiated by sterol 27-hydroxylase (CYP27). CYP27 is located in the inner mitochondrial membrane, where cholesterol content is very low. We hypothesized that cholesterol transport into mitochondria may be rate-limiting for bile acid synthesis via the "alternative" pathway. Overexpression of the gene encoding steroidogenic acute regulatory (StAR) protein, a known mitochondrial cholesterol transport protein, led to a 5-fold increase in bile acid synthesis. An increase in StAR protein coincided with an increase in bile acid synthesis. CYP27 overexpression increased bile acid synthesis by <2-fold. The rates of bile acid synthesis following a combination of StAR plus CYP27 overexpression were similar to those obtained with StAR alone. TLC analysis of (14)C-labeled bile acids synthesized in cells overexpressing StAR showed a 5-fold increase in muricholic acid; in chloroform-extractable products, a dramatic increase was seen in bile acid biosynthesis intermediates (27- and 7,27-hydroxycholesterol). High-performance liquid chromatography analysis showed that 27-hydroxycholesterol accumulated in the mitochondria of StAR-overexpressing cells only. These findings suggest that cholesterol delivery to the inner mitochondrial membrane is the predominant rate-determining step for bile acid synthesis via the alternative pathway.     

Inhibition of chitin biosynthesis in cultured imaginal discs: Effects of alpha-amanitin,actinomycin-D,cycloheximide, and puromycin

Summary Wing imaginal discs isolated from last instar larvae of the Indian meal moth,Plodia interpunctella, produced chitin when incubated in vitro with 2×10^–7 M 20-hydroxyecdysone. Chitin biosynthesis was initiated 8 h after the conclusion of a 24-h treatment with hormone. Simulataneous incubation of wing discs with 20-hydroxyecdysone and either inhibitors of RNA synthesis (alpha-amanitin, actinomycin-D) or inhibitors of protein systhesis (cycloheximide, puromycin) prevented chitin biosynthesis. We conclude from our results that RNA and protein synthesis must continue undiminished during the hormone-contact period, and that synthesis of protein, but not of new RNA is required during the posthormone culture period. Our findings are consistent with the hypothesis that ecdysteroids stimulate insect metamorphosis by promoting the synthesis of new RNA and protein during a hormone-dependent phase followed by hormone-independent protein synthesis.     

Heat-shock inhibits protein synthesis and eIF-2 activity in cultured cortical neurons

Stress, such as heat-shock, hypoxia and hypoglycemia, inhibits the initiation of protein synthesis. The effects of heat-shock on protein synthesis, eucaryotic initiation factor 2 (eIF-2) activity, protein kinase C (PKC), and casein kinase II (CKII) activities were studied in primary cortical neuronal cultures. In neurons exposed to heat-shock at 44°C for 20 min, protein synthesis is inhibited by more than 80%, and is accompanied by a 60% decrease in eIF-2 activity. Steady state PKC and CK II activities were not affected by heat-shock. Vanadate (200 M), a protein phosphotyrosine phosphatase inhibitor, partially prevented the depression of eIF-2 activity during heat-shock, and increased CKII activity by 90%. In contrast, staurosporine (62nM), a protein kinase C inhibitor, did not affect eIF-2 activity. We conclude that heat-shock causes a change in the phosphorylation/ dephosphorylation of regulatory proteins leading to a depressed eIF-2 activity and protein synthesis in neurons.