Inhibition of protein synthesis by (aminooxy)acetate in rat liver

(Aminooxy)acetate and D-cycloserine, two inhibitors of hepatic transamination reactions, inhibited also protein synthesis in isolated cells and postmitochondrial supernatants from rat liver. Both inhibitors acted in extracts only in concentrations higher than 1 mM. However, while D-cycloserine acted in isolated cells, as in extracts, (aminooxy)acetate inhibits protein synthesis in isolated cells by 50% of the control in the range 0.03-3 mM. NH+4 and H2O2, two by-products of (aminooxy)acetate degradation, inhibited protein synthesis in isolated liver cells, but at such a high concentration that the inhibition of protein synthesis carried out by (aminooxy)acetate cannot be explained by generation of these species. The results point out that the inhibitory action of (aminooxy)acetate on protein synthesis appears to require the integrity of the molecule.     

DNA polymerase delta, RFC and PCNA are required for repair synthesis of large looped heteroduplexes in Saccharomyces cerevisiae

Small looped mispairs are corrected by DNA mismatch repair (MMR). In addition, a distinct process called large loop repair (LLR) corrects loops up to several hundred nucleotides in extracts of bacteria, yeast or human cells. Although LLR activity can be readily demonstrated, there has been little progress in identifying its protein components. This study identified some of the yeast proteins responsible for DNA repair synthesis during LLR. Polyclonal antisera to either Pol31 or Pol32 subunits of polymerase δ efficiently inhibited LLR in extracts by blocking repair just prior to gap filling. Gap filling was inhibited regardless of whether the loop was retained or removed. These experiments suggest polymerase δ is uniquely required in yeast extracts for LLR-associated synthesis. Similar results were obtained with antisera to the clamp loader proteins Rfc3 and Rfc4, and to PCNA, i.e. LLR was inhibited just prior to gap filling for both loop removal and loop retention. Thus PCNA and RFC seem to act in LLR only during repair synthesis, in contrast to their roles at both pre- and post-excision steps of MMR. These biochemical experiments support the idea that yeast polymerase δ, RFC and PCNA are required for large loop DNA repair synthesis.     

Inhibition of protein synthesis in reovirus-infected HeLa cells with elevated levels of interferon-induced protein kinase activity

Protein synthesis was inhibited in one line of interferon-treated HeLa cells (line 2) upon infection with reovirus, but not in different HeLa cells (line 1) treated in the same way. The inhibition resulted in polysome runoff, suggesting that it was due to an impairment of peptide chain initiation. Interferon induces the synthesis of a protein kinase, which is activated in cell-free systems by double-stranded RNA and phosphorylates the alpha subunit of eukaryotic initiation factor 2, thus inhibiting the initiation of protein synthesis. Therefore, we measured the level of this protein kinase in extracts prepared from the two HeLa cell lines. Cells of line 2 showed about 3-4 times more protein kinase activity than cells of line 1. The inhibition of protein synthesis upon infection with reovirus was correlated with an increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 in interferon-treated cells labeled with 32P. The kinase was presumably activated in intact cells by viral double-stranded RNA, but this activation resulted in inhibition of protein synthesis only in cells with elevated levels of the kinase.     

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml(-1)) and by HeLa, HT29 and JM cells with IC50 in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml(-1), all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.     

Inhibition of cleavage of a plant viral polyprotein by an inhibitor activity present in wheat germ and cowpea embryos

In rabbit reticulocyte lysate, the bottom component RNA of cowpea mosaic virus directs the synthesis of a 200,000-molecular-weight precursor protein (200K protein) that is cleaved during synthesis by a reticulocyte enzyme to form a 32K protein and a 170K protein. Cleavage of the 200K protein was found to be effectively inhibited by inhibitor activity in wheat germ and cowpea embryo extracts. The inhibitor was nondialyzable, precipitatable by ammonium sulfate, and partially stable at high temperatures. The activity appeared to be specific in that it caused no inhibition of the secondary cleavage reactions (cleavage of the 170K protein) at concentrations that were sufficient to cause complete inhibition of the primary cleavage reaction (cleavage of the 200K protein).     

RNA biosynthesis in mitochondria under conditions of prolonged inhibition of protein biosynthesis in rat liver cytoplasm]

When cytoplasmic protein synthesis is inhibited by cycloheximide (CHI) in vivo synthesis of water-soluble mitochondrial proteins and of mitochondrial RNA is decreased. These changes measured in isolated rat liver mitochondria are similar to those observed in vivo and correlate with the changes the synthesis of water-soluble proteins in mitochondria. When the cytoplasmic fraction (30,000 g-supernatant) had been added to the mitochondria showing decreased RNA synthesis, the RNA synthesis increased to the control level (the incubation conditions were favourable for the protein transport from microsomes to mitochondria). RNA synthesis in mitochondria was not stimulated by cytoplasmic fractions from the CHI-pretreated rats. After prolonged dialysis these fraction stimulated RNA synthesis even to a greater extent than cytoplasmic fractions from the untreated animals. Mitochondrial RNA polymerase activity (measured in mitochondrial extracts supplemented with exogenous DNA) was higher in extracts of mitochondria from livers of normal rats than in extracts of mitochondria from livers of animals injected with CHI.     

Effect of chloride and glutamate ions on in vitro protein synthesis by the moderate halophile Vibrio costicola.

Vibrio costicola grown in the presence of different NaCl concentrations contains cell-associated Na+ and K+ ions whose sum is equal to or greater than the external Na+ concentration. In the presence of 0.5 M NaCl, virtually no in vitro protein is synthesized in extracts of cells grown in 1.0 M NaCl. However, we report here that active in vitro protein synthesis occurred in 0.6 M or higher concentrations of Na2SO4, sodium formate, sodium acetate, sodium aspartate, or sodium glutamate, whereas 0.6 M NaF, NaCl, or NaBr completely inhibited protein synthesis as measured by polyuridylic acid-directed incorporation of [14C]phenylalanine. Sodium glutamate, sodium aspartate, and betaine (0.3 M) counteracted the inhibitory action of 0.6 M NaCl. The cell-associated Cl- concentration was 0.22 mol/kg in cells grown in 1.0 M NaCl. Of this, the free intracellular Cl- concentration was only 0.02 mol/kg. Cells contained 0.11 mol of glutamate per kg and small concentrations of other amino acids. All of the negative counterions for cell-associated Na+ and K+ have not yet been determined. In vitro protein synthesis by Escherichia coli was inhibited by sodium glutamate. Hybridization experiments with ribosomes and the soluble (S-100) fractions from extracts of E. coli and V. costicola showed that the glutamate-sensitive fraction was found in the soluble, not the ribosomal, part of the system. The phenylalanyl-tRNA synthetase of V. costicola was not inhibited by 0.5 M or higher concentrations of NaCl; it was slightly more sensitive to high concentrations of sodium glutamate. Therefore, this enzyme was not responsible for the salt response of the V. costicola in vitro protein-synthesizing system.     

Highly efficient translation of messenger RNA in cell-free extracts prepared from L-cells.

Micrococcal nuclease was used to eliminate endogenous protein synthesis in extracts prepared from L cells. The nuclease can be inhibited subsequently with 2'-deoxythymidine-3', 5'-diphosphate. Nuclease-treated extracts primed with exogenous reovirus mRNA, synthesized full length polypeptides with linear kinetics for almost two hours leading to stimulation of the order of 10(4) times over endogenous background. On the average, between 40 and 50 molecules of polypeptide were synthesized per molecule of mRNA.     

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC₅₀ (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml⁻¹) and by HeLa, HT29 and JM cells with IC₅₀ in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml⁻¹, all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.     

Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola.

In vitro protein synthesis was studied in extracts of the moderate halophile Vibrio costicola by using as mRNAs the endogenous mRNA of V. costicola and the RNA of the R17 bacteriophage of Escherichia coli. Protein synthesis (amino acid incorporation) was dependent on the messenger, ribosomes, soluble cytoplasmic factors, energy source, and tRNA(FMet) (in the R17 RNA system) and was inhibited by certain antibiotics. These properties indicated de novo protein synthesis. In the V. costicola system directed by R17 RNA, a protein of the same electrophoretic mobility as the major coat protein of the R17 phage was synthesized. Antibiotic action and the response to added tRNA(FMet) showed that protein synthesis in the R17 RNA system, but not in the endogenous messenger system, absolutely depended on initiation. Optimal activity of both systems was observed in 250 to 300 mM NH4+ (as glutamate). Higher salt concentrations, especially those with Cl- as anion, were generally inhibitory. The R17 RNA-directed system was more sensitive to Cl- ions than the endogenous system was. Glycine betaine stimulated both systems and partly overcame the toxic effects of Cl- ions. Both systems required Mg2+, but in lower concentrations than the polyuridylic acid-directed system previously studied. Initiation factors were removed from ribosomes by washing with 3.0 to 3.5 M NH4Cl, concentrations about three times as high as that needed to remove initiation factors from E. coli ribosomes. Washing with 4.0 M NH4Cl damaged V. costicola ribosomes, although the initiation factors still functioned. Cl- ions inhibited the attachment of initiation factors to tRNA(FMet) but had little effect on binding of initiation factors to R17 RNA.     

Action of antibiotic combinations with furacilin on the synthesis of proteins and nucleic acids in intact NAG-vibrio cells]

Furacillin in combination with such antibiotics as tetracycline, levomycetin or neomycin inhibited the synthesis of proteins in the cells of NAG-vibrios. Combination of 8 gamma/ml of furacillin and 0.125 gamma/ml of tetracycline inhibited the protein synthesis by 58.8 per cent, 8 gamma/ml of furacillin and 0.5 gamma/ml of levomycetin inhibited the synthesis by 61 per cent, 8 gamma/ml of furacillin and 4 gamma/ml of neomycin inhibited it by 59.5 per cent. At the same time furacillin alone in concentrations of 16 and 8 gamma/ml inhibited the protein synthesis by 69.1 and 37 per cent respectively, tetracycline alone in doses of 0.25 and 0.125 gamma/ml inhibited it by 51.3 and 34.7 per cent respectively, levomycetin alone in doses of 1 and 0.5 gamma/ml inhibited it by 54.4 and 33.2 per cent, enomycin in doses of 8 and 4 gamma/ml inhibited it by 54.4 and 22.6 per cent respectively. Therefore, when the above antibiotics were used in combination with furacillin the inhibitory effect of the drugs on the protein synthesis was summarized. When furacillin was combined with tetracycline or levomycetin in the above concentrations, the inhibitory effect on RNA synthesis (42 or 32 per cent respectively) was lower than that of furacillin alone (50.5 per cent). When furacillin was used in combination with neomycin, the inhibition of RNA synthesis increased up to 69 per cent. The increase in the inhibitory effect was also noted with respect to the synthesis of DNA. Combination of furacillin with tetracycline, levomycetin or neomhcin decreased the synthesis of DNA by 79.7, 85 or 85.8 per cent respectively as compared to the inhibitory effect of 8 gamma/ml of furacillin equal to 61 per cent.     

Isolation and partial purification of catfish pancreatic islet messenger RNA.

Poly(a)-rich mRNA has been isolated from catfish pancreatic islet total nucleic acid. Cell-free translation of the mRNA by wheat germ extracts yielded a protein of 11 000-12 000 molecular weight, estimated by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. This peptide is larger than catfish proinsulin, but contains tryptic peptides of proinsulin. Its synthesis comprises up to 23% of the cell-free product, depending on the conditions of cell-free synthesis. Synthesis is inhibited by 7-methylguanosine 5'-monophosphate suggesting the presence of a 7-methylguanosine cap on the 5' end of catfish proinsulin mRNA. Sucrose gradient centrifugation of the islet poly(A)-rich mRNA yielded 8S and 12S peaks. These fractions were translated with wheat germ extracts and it was determined that over 60% of the islet mRNA-dependent protein from the 8S fraction was preproinsulin. The 8S mRNA fraction was electrophoresed on 3% agarose-6 M urea gels and demonstrated to be several bands, ranging from 100 000-200 000 molecular weight.     

The effect of hemin and of allyl isopropyl acetamide on protein synthesis in rat hepatocytes

Protein synthesis was measured in incubated hepatocytes. While hemin brings about a slight stimulation, allyl isopropyl acetamide (a compound that destroys the heme bound to cytochrome P450) inhibits protein synthesis by a mechanism that appears to result exclusively from depletion of cytoplasmic heme. Indications that in hepatocytes, as in reticulocytes, protein synthesis may be in part regulated by heme at the level of initiation are: i) that inhibition is accompanied by polysome breakdown; ii) that the protein synthesis inhibitor already isolated from rat liver, is hemin reversible iii) that hepatocyte extracts contain a Mr 38,000 phosphoprotein which comigrates with the Mr 38,000 subunit of rabbit initiation factor 2 and iv) that the phosphorylation of both of these subunits is inhibited by hemin.     

无机钼酸在棕色固氮菌(Azotobacter vinelandii)体内的累积和转化

醋酸铵培养的棕色固氮菌(Azotobacter vinelandii),经超声击碎高速离心制备粗提取液、DEAE-纤维素柱层析表明,体内~(99)MoFe蛋白合成受到阻遏,在0.15 M NaGl洗脱分部中,除~(99)Mo储存蛋白峰外,还存在一个无机~(99)MoO_4~=组分。醋酸铵培养的棕色固氮菌经去阻遏后,在体内固氮活性出现的同时,可观察到原先菌体内累积的~(99)Mo储存蛋白峰降低,无机~(99)MoO_4~=的组分几乎消失以及~(99)MoFe蛋白合成。若去阻遏过程存在氯霉素,则菌体不显示固氮活性,(99)MoFe蛋白不再合成,储存蛋白和无机铝酸组分中~(99)Mo的转移停止。     

Use of a cell-free protein synthesizing system from cells of ascites carcinoma Krebs-2 for RNA translation of plant viruses

Cell-free translation in Krebs-2 extracts was optimized for RNAs of two plant viruses; potato virus X (PVX, potexvirus), and tobacco mosaic virus (TMV, tobamovirus). PVX and TMV RNAs programmed synthesis of similar sets of polypeptides in both the Krebs-2 extracts and the rabbit reticulocyte lysates, major virus-specific products being the same in molecular weight in both in vitro systems. PVX structural protein (p29) was absent among polypeptides synthesized in the Krebs-2 system but was readily identified by immuno-precipitation among the ones synthesized in the reticulocyte lysate system. The "cap" analog, m7Gpp, inhibited the synthesis of all the polypeptides programmed by PVX RNA in the Krebs-2 system. The synthesis of only a few of the most high molecular weight products in the reticulocyte lysate system was inhibited, the synthesis of a number of low molecular weight products (and among them p29) was even stimulated. Thus, the PVX capped messengers derived from PVX genomic RNA due to its fragmentation with endogenous nuclease activities. The use of the Krebs-2 system allows to avoid activation of internal PVX genes.     

Further studies on the biosynthesis of gramicidin S and proteins in a cell-free system from Bacillus brevis

1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.     

In vitro replication of mouse hepatitis virus strain A59.

An in vitro replication system for mouse hepatitis virus (MHV) strain A59 was developed using lysolecithin to produce cell extracts. In extracts of MHV-infected cells, radiolabeled UMP was incorporated at a linear rate for up to 1 h into RNA, which hybridized to MHV-specific cDNA probes and migrated in denaturing formaldehyde-agarose gels to the same position as MHV genomic RNA. The incorporation of [32P]UMP into genome-sized RNA in vitro correlated with the observed increase of [3H]uridine incorporation in MHV-infected cells labeled in vivo. Incorporation of [32P]UMP into genome-sized RNA was inhibited when extracts were incubated with puromycin. The addition to the assay of antiserum to the MHV-A59 nucleocapsid protein N inhibited synthesis of genome-sized RNA by 90% compared with the addition of preimmune serum. In contrast, antiserum to the E1 or E2 glycoproteins did not significantly inhibit RNA replication. In vitro-synthesized RNA banded in cesium chloride gradients as a ribonucleoprotein complex with the characteristic density of MHV nucleocapsids isolated from virions. These experiments suggest that ongoing protein synthesis is necessary for replication of MHV genomic RNA and indicate that the N protein plays an important role in MHV replication.     

An increase in the intracellular pH of fertilized eggs of Xenopus laevis is associated with inhibition of protein and DNA syntheses and followed by an arrest of embryonic development

In many systems, events participating in cell division are controlled by intracellular pH (pHi). In Xenopus eggs, fertilization is accompanied by an increase in pHi which occurs concomitantly with an increase in protein synthesis and a reinitiation of DNA synthesis, leading the embryo to cell division. In this paper, we have shown that increasing pHi of fertilized eggs from 7.8 to 8.2 by using weak bases produced an arrest in embryonic development. Such a change in pHi was accompanied by a severe inhibition of both protein and DNA syntheses. In order to discriminate between a direct effect of pHi and a pH-independent effect of weak bases on these biosyntheses, the situation was studied in vitro. For this purpose, cytoplasmic extracts were used in which weak base addition did not produce any change in pH. Under these conditions, protein synthesis was not inhibited, suggesting that pH is probably one of the events implicated in the regulation of protein synthesis. On the other hand, DNA synthesis was inhibited by weak bases in vitro, without any change in pH intervening.     

Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint

BACKGROUND: The DNA replication checkpoint ensures that mitosis is not initiated before DNA synthesis is completed. Recent studies using Xenopus extracts have demonstrated that activation of the replication checkpoint and phosphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DNA polymerase alpha, and it has been suggested that the ATR kinase-so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase-may be an upstream component of this response. It has been difficult to test this hypothesis as an ATR-deficient system suitable for biochemical studies has not been available. RESULTS: We have cloned the Xenopus laevis homolog of ATR (XATR) and studied the function of the protein in Xenopus egg extracts. Using a chromatin-binding assay, we found that ATR associates with chromatin after initiation of replication, dissociates from chromatin upon completion of replication, and accumulates in the presence of aphidicolin, an inhibitor of DNA replication. Its association with chromatin was inhibited by treatment with actinomycin D, an inhibitor of RNA primase. There was an early rise in the activity of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon depletion of ATR was accompanied by the loss of Chk1 phosphorylation. CONCLUSIONS: ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.