Limit Protein Synthesis Research Articles

Interferon-α and Interferon-β Induce Ia Expression in Limited Protein Synthesis Condition

We investigated the regulatory effect of interferons (IFNs) on macrophage (m phi) Ia antigen expression. Unlike type II IFN (IFN-gamma), type I IFNs (IFN-alpha and IFN-beta) were incapable of inducing the accumulation of Ia mRNA. However, when cultured with type I IFN plus cycloheximide (CHX), M phi accumulated significant amounts of Ia-specific mRNA, which was translated into Ia molecules expressed on the cell surface after the removal of CHX. Significant enhancement of Ia mRNA accumulation and the Ia expression were observed also in M phi stimulated with IFN-gamma plus CHX. Type I IFNs suppressed IFN-gamma-dependent Ia expression through the inhibition of Ia mRNA accumulation, but such suppression was not directed to the post-transcriptional step in M phi that had accumulated Ia mRNA in response to stimulation by type I or type II IFN plus CHX. These results seem to indicate that type I IFNs can induce Ia mRNA accumulation but that they also induce some inhibitory protein, the synthesis of which precedes it and is dominant, so that the Ia expression is masked.

Translational control in early sea urchin embryogenesis: initiation factor eIF4F stimulates protein synthesis in lysates from unfertilized eggs of Strongylcentrotus purpuratus

We have used cell-free translation systems from unfertilized eggs and embryos of the sea urchin Strongylocentrotus purpuratus to analyze the mechanisms limiting protein synthesis in early embryogenesis. Unfertilized egg lysates supplemented with nuclease-treated reticulocyte lysate were stimulated 2-4-fold in incorporation of radioactive amino acid into protein. Thirty-minute zygote lysates supplemented in this way were not stimulated. These results suggested that a component limiting translation in the unfertilized egg lysate was provided by the nuclease-treated lysate and that this component was no longer limiting protein synthesis following fertilization. In view of these results, partially fractionated lysates and individual purified translational components from mammalian cells were tested for stimulation of the unfertilized egg lysate. A 1000000g supernatant devoid of ribosomal subunits also stimulated the unfertilized egg lysate. Thus, the stimulation was not due to the addition of active ribosomal subunits but to soluble elements in the reticulocyte lysate. Of the soluble components tested, only the cap-binding protein complex eIF4F caused a dramatic stimulation of the unfertilized egg lysate (2-3.5-fold). The 30-min zygote lysate was not stimulated by eIF4F or by any of the other components tested, supporting the hypothesis that a block in the translational machinery is removed at fertilization. A rabbit reticulocyte shift assay was used to analyze whether mRNA is limiting in early development. When unfertilized egg lysate was added to the shift assay, there was no shift in radioactivity from 43S to 80S complexes, indicating the unfertilized egg mRNA is not available for translation.(ABSTRACT TRUNCATED AT 250 WORDS)

The sugar-specific adhesion/deadhesion apparatus of the marine bacterium [formula omitted] is a sensorium that continuously monitors nutrient levels in the environment

Our earlier studies on cell adhesion to immobilized carbohydrates are extended here to a marine bacterium, Vibriofurnissii. Apparently one lectin mediates the binding of these cells to glycosides of N-acetylglucosamine, mannose, and glucose covalently linked to Agarose beads. Kinetic studies show that protein synthesis is required for initiating and for maintaining adhesion to the glycosides. Furthermore, a pro− mutant binds to GlcNAc-beads at Pro concentrations insufficient to support cell growth. Expression of the functional lectin therefore predominates under conditions of limiting protein synthesis. Thus, cells adhere to the sugars in an environment compatible with protein synthesis, and deadhere when depleted of any required nutrient, presumably to migrate to a more favorable locale. The adhesion-deadhesion apparatus thereby permits constant monitoring of the surrounding environment, comprising a “nutrient sensorium”.

Translational control and the cytoskeleton in Physarum polycephalum.

Translationally active plasmodia of the syncytial slime mold Physarum polycephalum develop into translationally dormant sclerotia during starvation. Although functional mRNA and ribosomes exist in sclerotia, protein synthesis is suppressed at the level of initiation. To test the possibility that alterations in the cytoskeleton may limit protein synthesis, we have examined the distribution of polysomes and actin mRNA in the cytoskeletal (CSK) and soluble (SOL) fractions of Triton X-100-extracted plasmodia and sclerotia. Most of the polysomes and actin mRNA were located in the CSK of plasmodia, while most of the ribosomes and actin mRNA were located in the SOL of sclerotia. The results suggest that ribosomes and mRNA shift from the CSK to the SOL as protein synthesis is suppressed during starvation. Plasmodia and sclerotia can be induced to accumulate excess polysomes by treatment with low levels of the elongation inhibitor cycloheximide. Treatment of plasmodia with cycloheximide caused excess polysomes to accumulate in the SOL, suggesting that the CSK contains a limited capacity for binding translational components and that the association of polysomes with the cytoskeleton is not required for protein synthesis. Treatment of sclerotia with cycloheximide, however, caused polysomes and actin mRNA to accumulate in the CSK, suggesting that the sclerotial cytoskeleton, although depleted in ribosomes and mRNA, is capable of binding translational components. It is concluded that alterations in the sclerotial cytoskeleton are not involved in translational control.

Oligodendroglial pathology in canine distemper virus infection in vitro.

Dog brain cell cultures were infected with different canine distemper virus (CDV) strains to study the oligodendrocytes, which were characterized with eight different antibodies to cover the whole oligodendroglial population in the culture. A few weeks after infection all oligodendroglial cell types started to degenerate and disappeared from the culture. However, since no CDV protein could be demonstrated in the degenerating oligodendrocytes with extensive double-labelling studies, this lesion can not be explained as being a result of cytolytic infection. This conclusion was further supported in experiments with plaque-forming CDV, in which viral replication is restricted to the cytolytic areas only; oligodendrocytes also degenerated in virus-free areas between the plaques. The hypothesis of toxic factors released by other infected cell types in the culture leading to secondary damage of the oligodendrocyte could not be confirmed by transferring supernatants from infected to normal cultures. Whereas the presence of toxic factors can not be completely excluded, the possibility of an abortive infection of the oligodendrocytes with no or very limited viral protein synthesis is discussed.

Functional aspects of bacterial polysomes during limited protein synthesis

The effects of amino acid starvation on the metabolic behavior of polysomes and the size distribution of proteins have been studied in an otherwise isogenic pair of stringent ( relA +) and relaxed ( relA) strains of Escherichia coli. The stability of polysomes has been analyzed by using two different approaches. First, the process of their degradation has been followed after treating the cells with rifampicin, an inhibitor of the synthesis of all classes of RNA including messenger RNA. Secondly, the process of their assembly has been studied after their previous conversion to monosomes, as induced by glucose deprivation of cells. It is shown that, in either type of bacterial strain, polysomes are continually broken down and re-synthesized during amino acid starvation. However, such polysome turnover is then less rapid than in normally growing bacteria, and, moreover, it seems amino acid specific since it occurs at a lower rate during arginine starvation than during histidine starvation, namely, in the relaxed strain. The molecular weight distribution of proteins has been determined after labeling of cells with radioactive methionine and separation of polypeptides by one-dimensional polyacrylamide gel electrophoresis. The average size of polypeptides synthesized in the stringent strain during starvation is quite similar to that measured during normal growth. By contrast, a significant shift towards smaller molecules is observed in the relaxed strain deprived of an essential amino acid. Here again, this reduction of the size of polypeptides seems amino acid specific since it is especially marked during arginine starvation. These results are discussed in terms of ribosomes translocation and premature peptide chain termination in connection with the accuracy of the translational process.

Response to Abomasal Infusion of Methionine of Weaned Dairy Calves Fed a Complete Pelleted Starter Ration Based on By-Product Feeds1,2

Three abomasal infusion trials were to determine the first-limiting amino acid for nitrogen retention of postweaned Holstein bull calves fed a starter ration based on cereal by-product feeds. Ration ingredients contributed the following percentages of total amino acids: wheat middlings 38, brewer's grains 24, hominy feed 21, oat hulls 10, distiller's grains 6, and molasses 1. Dry matter of the pelleted ration contained 13 to 14% crude protein and .23 to .26% sulfur. Each trial was a replicated 4 × 4 Latin square with 9-day periods. Calves were weaned at 4 wk and infusions initiated at 5 to 6 wk of age. Abomasal infusion of the 10 essential amino acids, an isonitrogenous quantity of an equimolar mixture of alanine, asparagine, glutamate, glycine, and serine, or an isocaloric quantity of glucose indicated that essential amino acid(s) rather than nonspecific nitrogen or energy was limiting protein synthesis. Quadratic responses in urine nitrogen excretion and retained nitrogen occurred when 0, .3, .6, and 1.2g L-methionine/kg starter ration were infused. Greatest responses occurred with .6g methionine, which increased retained nitrogen 11 and 19% and decreased urine nitrogen 7 and 10%. The relationship between infused methionine and plasma methionine was linear. Infusions in all trials had no effect on digestibility of energy and nitrogen.

Pleiotropic effect of his gene mutations on nitrogen fixation in Klebsiella pneumoniae

Several his mutations were found to influence nitrogen fixation in Klebsiella pneumoniae: hisB, hisC, and hisD mutants had 50% of wild-type levels of nitrogenase activity when supplied with 30 mug or less histidine/ml although this concentration did not limit protein synthesis and the mutants retained a Nif plate phenotype. A hisA mutation had a similar but more dramatic effect. At low concentrations of histidine the hisA mutant strain had only 5% of the nitrogenase activity found at high histidine concentration or in a his strain, and was also Nif on low histidine agar plates. Addition of adenine restored nitrogenase activity in the hisA but not the hisB, hisC, or hisD mutants. Low levels of intracellular ATP, a consequence of hisG enzyme activity, correlated with loss of nitrogen-fixing ability in the hisA mutant which failed to sustain nif gene expression under these conditions. Synthesis of other major cell proteins was relatively unaffected indicating that nif gene expression is selectively regulated by the energy status of the organism.

Messenger RNA Competition in Living Xenopus Oocytes

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein-synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs this become endogenous mRNAs. These results, together with previous estmates of the translational efficiency of injected heterologous mRNA species, are compatible with the assumption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in the temporarily inactivated form.

Regulation of K+ Influx in Barley Roots: Evidence for Direct Control by Internal K+

Values for plasmalemma influx of K+ into excised barley roots, from solutions containing 0.05 mM KCl plus 0.5 mM CaSO4, were reduced by 50-60% following a 6-h pretreatment period in 50 mM KCl plus 0.05 mM CaSO4 solution. This reduction of influx, associated with increased internal K+ concentration, was independent of DNA, RNA and protein synthesis during the pretreatment period as indicated by its insensitivity to the presence of 5-fluorodeoxyuridine, actinomycin D, cycloheximide, p-fluorophenylalanine or anisomycin in the pretreatment solutions. Roots of plantlets grown from gamma-irradiated barley seeds, which were incapable of under-going cell division and capable of only limited protein synthesis, were nevertheless able to reduce K+ influx values in response to increased internal K+ concentration. The measurement of K+ influx from 0.05 mM KCl solutions following pretreatment periods as short as 15 min in 50 mM KCl gave no evidence of any lag period in the development of reduced influx associated with increased internal K+ status. The above experiments are discussed in terms of a model for the regulation of K+ influx which ascribes a direct 'allosteric' role to internal K+ in controlling influx.

Spores of microorganisms. XXVI. Synthetic activities of germinating spores of Bacillus cereus prevented from outgrowth.

Spores of Bacillus cereus were germinated in a germination limited medium (GL-medium) which facilitates only germination but not the postgerminative development of spores. Under these conditions a limited protein synthesis occurs. However, this protein synthesis is stopped after a short time interval. The rate of synthesis of new proteins, as well as their total amount, is influenced by the length of the activation heat shock. Synthesis of the wall material continues for several hours and thick-walled cells with a changed ultrastructure are formed. Synthesis of the diaminopimelic acid (dap) containing material of the cell wall is sensitive to actinomycin D and relatively resistant to chloramphenicol. Similarly, protein synthesis is relatively chlorapmhenicol-resistant but is fully inhibited by azauracil or spiramycin. Whereas RNA formed in the control culture is partially decomposed after 30 min of incubation, chloramphenicol accelerates its synthesis and prevents its decay. Exudate components apparently stimulate synthesis of ribonucleic acid, proteins and the wall material. The 14-C-dap containing material released by prelabelled spores in the form of the exudate during the germination is not re-utilized by the spores germinated in the GL-medium. The results are discussed with respect to the atypical primary synthetic activities of spores under conditions when the postgerminative development is prevented and from the point of view of participation of the germination exudate during these syntheses.

Protein synthesis by synaptosomes from rat brain. Contribution by the intraterminal mitochondria.

(1) The characteristics of protein synthesis in microsomal and synaptosomal fractions from rat brain were examined. A high sensitivity to ribonuclease and to cycloheximide, and the need for the presence of pH5 enzymes distinguished protein synthesis in microsomal fractions from protein synthesis in synaptosomes. (2) Under various conditions of incubation synaptosomal fractions prepared in sucrose showed limited protein synthesis compared with synaptosomal fractions prepared by using Ficoll. Such discrepancies could not be attributed to: (i) animal age, (ii) the metabolic state of the synaptosomal fraction, (iii) the absence of bivalent cations in the incubation medium or (iv) the temperature. (3) Protein synthesis in synaptosomal fractions was inhibited 50-65% by cycloheximide, 38-50% by chloramphenicol, 95% by puromycin, 70% by azide and 40% by deoxyglucose; ribonuclease had only a negligible inhibitory effect. (4) As a first approximation to the localization of the protein-synthetic machinery present in the synaptosomal fraction, the distribution of enzymes and radioactivity in subfractions of prelabelled synaptosomes was determined after osmotic shock with water. Approximately 60% of the total protein synthesis in the synaptosomal fraction occurred in the intraterminal mitochondria. (5) Protein synthesis in the intraterminal mitochondria did not show any fundamental difference from synthesis in somatic mitochondria, with respect to inhibition by cycloheximide and chloramphenicol. (6) It was concluded that if extramitochondrial protein synthesis occurs in synaptosomes, it must be very low.

Incorporation of [Me-3H]methionine into wool follicle proteins: a biochemical and ultrastructural study.

ABSTRACT The formation of the ‘low-sulphur’ group of proteins which constitutes more than half the cortical cell content of wool was investigated. Of the amino acids present in the low- and high-sulphur proteins isolated from wool, methionine is the only one which is exclusive to the low-S group. The incorporation of [Me-3H]methionine, administered intradermally to a sheep, was examined using both biochemical and autoradiographic procedures. During the first hour after injection, most of the [3H]methionine appeared in the blood, but some was incorporated into the wool follicle proteins at the injection sites. Most of the radio-activity incorporated by the fibre during this period was subsequently retained. Initially, more than 90 % of the activity was present in the prekeratin (8 M urea-soluble) portion of the fibre; 50 h elapsed before half of the radioactive material appeared in the keratinized (8 M urea-insoluble) part of the fibre. Methionine and its oxidation products accounted for more than 80 % of the radioactivity present in hydrolysates of wool removed from injection sites after 4 h or 22 days. Analysis of labelled wool showed that the specific radioactivities of those fractions low in sulphur (α- and β-keratose) were 7 and 14 times greater than that of the high-S (γ-keratose) fraction. Autoradiographic studies showed that the main site of formation of the low-S proteins was in the bulb region of the follicle. Here there was no specific intracellular site of low-S protein synthesis, the [3H]methionine being incorporated into the nucleus, cytoplasm, the various cell organelles and intercellular membranes. The amount of radioactivity incorporated by the cortical cells diminished at progressively higher levels in the fibre. In the keratinization zone, the various cell components showed limited protein synthesis, but 1–8 h after injection there was a localization of tritium over the keratin fibrils. It is suggested that a large proportion of the low-S proteins formed within the developing cortical cell ultimately contribute to the keratin microfibrils. The results provide further evidence for the formation of the low-S and high-S proteins at different sites in the follicle.

Protein synthesis during fungal spore germination. IV. Transfer ribonucleic acid from germinated and ungerminated spores.

Transfer ribonucleic acid (tRNA) fractions isolated from germinated and ungerminated spores of Botryodiplodia theobromae and Rhizopus stolonifer had acceptor activity for all 20 amino acids commonly found in protein, when tested with an enzyme fraction from germinated spores. Accordingly, it is unlikely that the absence of tRNA for a particular amino acid limits protein synthesis in fungal spores.

Synthesis of protein and ribonucleic acid by starved Streptococcus lactis in relation to survival.

SUMMARY Protein synthesis, as estimated from incorporation studies with [14C]valine, was barely detectable when organisms were starved in phosphate buffer containing Mg2+. The addition of an energy source promoted limited protein synthesis. In this respect glucose produced a much higher rate of [14C]valine uptake and incorporation than arginine. Although arginine prolonged survival, experiments with inhibitors showed that this was unlikely to be due to the protein synthesis which had been promoted. The ability of starved organisms to assimilate [14C]valine and to incorporate the isotope into protein in the presence of glucose appeared to be correlated with survival. Synthesis of RNA in starved organisms, as determined from [14C|uracil incorporation experiments, depended on exogenous glucose. During brief exposures to [14C]uracil plus glucose at intervals during starvation, organisms incorporated decreasing amounts of isotope into RNA although incorporation by non-viable organisms occurred at approximately half the initial rate. Although resynthesis of both protein and RNA was demonstrated with added energy sources, no evidence was obtained to suggest that this favoured survival.

The roles of inducer and catabolite repressor in the synthesis of β-galactosidase by Escherichia coli

The mechanism of the induction of β-galactosidase in Escherichia coli at 30°C was investigated by separating the phase of enzyme induction from the phase of enzyme production. This separation was accomplished by removing the inducer by filtration after 3 to 4 min of contact with the cells, that is before enzyme had been formed; the cells then produced the enzyme in inducer-free medium at an exponentially declining rate for approximately 10 min. It was found that interference with the synthesis of normal RNA by 5-fluorouracil during the phase of enzyme induction largely prevents the subsequent production of normal enzyme and causes the formation of an altered protein serologically related to β-galactosidase; on the other hand, such an interference with RNA synthesis during the phase of enzyme production does not affect the formation of the enzyme by previously induced cells. Conversely, interference with protein synthesis by starvation for an amino acid or by chloramphenicol during the phase of induction does not prevent the subsequent production of normal enzyme; but such an interference with protein synthesis during the phase of enzyme production inhibits the formation of the enzyme by previously induced cells to the same extent as that of other protein. These results indicate that during the induction phase a messenger RNA specific for β-galactosidase is produced which directs the subsequent synthesis of the enzyme in the inducer-free medium. No additional β-galactosidase messenger RNA is formed after removal of the inducer and that present in the cell decays exponentially with a half-life of approximately 2·5 min. The rate of this decay is independent of the rate of protein synthesis or the rate of energy metabolism. An excess of intracellular catabolites, produced either by the rapid catabolism of glucose or by the restriction of protein synthesis in the presence of a utilizable energy source, inhibits the induction of the enzyme but does not affect the production of the enzyme by previously induced cells. Apparently, the catabolite repressor inhibits and the inducer stimulates the synthesis of the unstable messenger RNA specific for β-galactosidase.

Factors limiting protein synthesis by isolated ribosomes

The “active” and “inactive” ribosomal systems from peas have been examined with respect to differences between the two systems. Although the two systems differ in their response to varied concentrations of the components of the reaction system, simple adjustment of these concentrations to optimal levels does not bring amino acid incorporation by the “inactive” system up to a level which even approaches that of the “active” system. The ribosomes from the two systems do not seem to differ markedly in stability, and both systems have essentially equal ability to form amini acyl-RNA. However, the two systems differ greatly in their ability to bind amino acyl-RNA to ribosomes, and to transfer amino acid from amino acyl-RNA to ribosomal protein. Experiments with polyuridylate indicate that a lesion in the “inactive” system may be related to the binding of messenger RNA to the ribosome.

Factors limiting protein synthesis by isolated ribosomes

Factors limiting protein synthesis by isolated ribosomes

Factors limiting protein synthesis by isolated ribosomes

The “active” and “inactive” ribosomal systems from peas have been examined with respect to differences between the two systems. Although the two systems differ in their response to varied concentrations of the components of the reaction system, simple adjustment of these concentrations to optimal levels does not bring amino acid incorporation by the “inactive” system up to a level which even approaches that of the “active” system. The ribosomes from the two systems do not seem to differ markedly in stability, and both systems have essentially equal ability to form amini acyl-RNA. However, the two systems differ greatly in their ability to bind amino acyl-RNA to ribosomes, and to transfer amino acid from amino acyl-RNA to ribosomal protein. Experiments with polyuridylate indicate that a lesion in the “inactive” system may be related to the binding of messenger RNA to the ribosome.

Intracellular protein and nucleic acid turnover in resting yeast cells

In the absence of exogenous energy and nitrogen, resting yeast cells are capable of replenishing both their nucleotide and free amino acid pools. This replenishment phenomenon is the result of intracellular protein and nucleic acid breakdown rather than of cell lysis. Isotopically labeled cells showed turnover rates of 6.6 · 10 −3 h −1 and 1.5 · 10 −3 h −1 for protein and nucleic acid respectively. Protein and nucleic acid degradation, as well as their synthesis, are energy-requiring reactions. When exogenous energy is available, the degradation products are reutilized for protein and nucleic acid synthesis. Employing 15N-purine-grown cells, it was found that the amino groups of nucleic acid purines can serve as nitrogen reservoirs for limited protein synthesis.