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PubMed1981

Regulation of protein synthesis: translational control by procollagen-derived fragments.

Previous studies have shown that a type I procollagen-derived peptide, called pN alpha 1(I)-Col 1, selectively inhibits collagen synthesis by fibroblasts in culture and the translation of procollagen mRNA in a rabbit reticulocyte lysate system. We prepared the 10,700-dalton peptide from dermatosparactic calf skin, which contained high levels of incompletely processed type I procollagen, by collagenase digestion. Time-course and dose-response studies showed that the peptide specifically inhibited the translation of procollagen mRNA in preparations of human fibroblast RNA while not affecting the translation of globin mRNA or of other messenger RNAs in fibroblast RNA. After reduction and alkylation, the peptide lost its specificity but became a nonspecific inhibitor of translation. Enzymatic cleavage enabled us to localize the nonspecific activity to a short sequence -Pro-Thr-Asp-Glu, an assignment confirmed by peptide synthesis. Using pactamycin, a specific inhibitor of translational initiation, we showed that the intact peptide acts on procollagen mRNA translation by inhibition of polypeptide chain elongation or termination, or both, whereas the nonspecific inhibitory activity of the unfolded peptide and its derivatives can be attributed to an inhibition of chain initiation. Although the native peptide may function in feedback regulation of collagen synthesis, the physiological role of the lower molecular weight fragments, if any, is uncertain.

Amino Acid Sequence↗
PubMed1998

The phosphorylation of protein S6 modulates the interaction of the 40 S ribosomal subunit with the 5'-untranslated region of a dictyostelium pre-spore-specific mRNA and controls its stability.

AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively as shown by nuclear run-off experiments and by fusing its promoter to the luciferase reporter gene. The same mRNA disappears quickly from disaggregated cells. If the 5'-untranslated region (5'UTR) of the constitutively expressed Actin 15 mRNA is substituted for the 5'UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If the 5'UTR of AC914 mRNA is substituted for the 5'UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin, but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting a role of 40 S subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines whether 40 S subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.

5' Untranslated Regions↗
PubMed1977

Prolonged inhibition of protein and glycoprotein synthesis in tumor cells treated with muconomycin A.

Several agents were compared for their ability to inhibit protein synthesis for long periods in tumor cells growing in culture. Mouse B16 melanoma cells, treated with high concentrations of cycloheximide or pactamycin for 1 hour and then washed repeatedly, recovered their ability to incorporate [3H]leucine into protein in about 4 hours, while cells treated with emetine recovered in 12 hours. After similar treatment with muconomycin A, however, incorporation of [3H]leucine remained inhibited for at least 30 hours. During this time the cells remained attached to the culture dishes, were able to exclude trypan blue dye, and retained nearly normal levels of rubidium-86 content. When another, untreated, population of cells was added to the muconomycin-treated cells, protein synthesis was not inhibited in the untreated population; action of the drug was thus shown to be confined to the treated cells. In melanoma cells treated with neuraminidase and muconomycin, measurement of glycoprotein synthesis (as determined by sialic acid analysis) showed that muconomycin also inhibited restoration of sialic acid content. Brief treatment with muconomycin, therefore, appeared to be sufficient for prolonged inhibition of protein and glycoprotein synthesis.

Anti-Bacterial Agents↗
PubMed1976

Characterization of the minor polypeptides in the foot-and-mouth disease particle.

In addition to the four major polypeptides VP1 and VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 X 10(3) (P40) and 52 X 10(3) (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.

Animals↗
PubMed1984

Theiler's virus-specified polypeptides made in BHK-21 cells.

This study was initiated to determine whether the Theiler's murine encephalomyelitis viruses (TMEV) use the same strategy of gene expression as other picornaviruses. The precursor-product relationships apparent from pulse-chase experiments indicated that the virus-specified polypeptides of the GDVII strain are generated by post-translational cleavages. Pactamycin mapping experiments also showed a gene order for GDVII virus similar to that derived for other picornaviruses by this technique. Finally, six other TMEV induced species of polypeptides similar to those of GDVII virus, but there were minor differences in some of their electrophoretic mobilities.

Animals↗
PubMed1988

The P2 and P3 regions of the poliovirus genome are preferentially translated at alkaline pH in infected HeLa cells.

HeLa cells infected with poliovirus exclusively synthesized proteins coded by the P2 and P3 regions of the viral genome when they were placed in an alkaline medium lacking sodium ions. The amount of viral protein synthesized was augmented by increasing the concentration of KCl. Also in the presence of KCl, the cells continued synthesizing this altered pattern of proteins for longer times. This effect occurred in the presence of guanidine, it was reversible, and the normal pattern of poliovirus proteins reappeared when control medium was added, even when guanidine was present. These findings suggest that truncated viral RNA is not made under these conditions. Pactamycin and sodium fluoride, two known inhibitors of the initiation of translation, blocked protein synthesis in cells placed in alkaline medium, indicating the possibility that initiation at internal sites of the viral mRNA may take place under these conditions. Finally, the proteins synthesized were analysed by two-dimensional gel electrophoresis. The proteins migrated as authentic viral proteins indicating that if internal initiation takes place, at least some of these initiation events occur in phase with the initiation codon present in the poliovirus genome.

Cycloheximide↗
PubMed1980

The induction of suppressor T cells by concanavalin A is independent of cellular proliferation and protein synthesis.

Factors that govern the induction of suppressor T cells after stimulation with concanavalin A (Con A) were investigated in a two-stage culture system. Normal mouse spleen cells were incubated with Con A in the presence of a variety of drugs and then assayed for suppressive activity by means of a secondary anti-sheep erythrocyte response in vitro. The inclusion of inhibitors of mitosis (vinblastine sulphate or mytomycin C) or protein synthesis (cycloheximide or pactamycin) into normal spleen cell cultures containing Con A failed to inhibit the subsequent development of suppressor cells. Furthermore, spleen cells from mice previously irradiated with 900 rad or injected with cyclophosphamide expressed a level of suppressor activity after Con A stimulation which was equivalent to that of normal spleen cells. However, the inclusion of drugs that inhibit microtubule or microfilament function (colchicine or cytocholasin B) did prevent suppressor cell induction. Kinetic studies also revealed that significant suppressor activity was detectable in normal spleen cells after only 3 h exposure to Con A. These results indicate that the induction of suppressor T cells in this system is a maturation event involving changes in the cell membrane and is entirely independent of protein synthesis and cellular proliferation.

Animals↗
PubMed1993

Resistance of lambda cI translation to antibiotics that inhibit translation initiation.

The lambda cI lysogenic transcript is unusual in having no leader. Expression of a cI-lacZ protein fusion was relatively resistant to kasugamycin and pactamycin, which inhibit translation initiation on transcripts with leaders. Our data imply that there are distinct differences in translation initiation between the two classes of transcripts.

Aminoglycosides↗
PubMed1977

Analysis of intracellular feline leukemia virus proteins II. Generation of feline leukemia virus structural proteins from precursor polypeptides.

The synthesis and processing of feline leukemia virus (FeLV) polypeptides were studied in a chronically infected feline thymus tumor cell line, F-422, which produces the Rickard strain of FeLV. Immune precipitation with antiserum to FeLV p30 and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to isolate intracellular FeLV p30 and possible precursor polypeptides. SDS-PAGE of immune precipitates from cells pulse-labeled for 2.5 min with [35S]methionin revealed the presence of a 60,000-dalton precursor polypeptide (Pp60) as well as a 30,000-dalton polypeptide. When cells were grown in the presence of the proline analogue L-azetidine-2-carboxylic acid, a 70,000-dalton precursor polypeptide (Pp70) was found in addition to Pp60 after a 2.5-min pulse. The cleavage of Pp60 could be partially inhibited by the general protease inhibitor phenyl methyl sulfonyl fluoride (PMSF). This partial inhibition was found to occur only if PMSF was present during pulse-labeling. Intracellular Pp70 and Pp60 and FeLV virion p70, p30, p15, p11, and p10 were subjected to tryptic peptide analysis. The results of this tryptic peptide analysis demonstrated that intracellular Pp70 and virion p70 were identical and that both contained the tryptic peptides of FeLV p30, p15, p11, and p10. Pp60 contained the tryptic peptides of FeLV P30, P15, and P10, but lacked the tryptic peptides of P11. The results of pactamycin gene ordering experiments indicated that the small structural proteins of FeLV are ordered p11-p15-p10-p30. The data indicate that the small structural proteins of FeLV are synthesized as part of a 70,000-dalton precursor. A cleavage scheme for the generation of FeLV p70, p30, p15, p11, and p10 from precursor polypeptides is proposed.

Azetidinecarboxylic Acid↗
PubMed1977

Common precursor for Rauscher leukemia virus gp69/71, p15(E), and p12(E).

Rauscher murine leukemia virus glycoprotein gp69/71 and non-glycosylated p15(E) are synthesized by way of a 90,000-dalton precursor glycoprotein, termed Pr2a+b. Peptide mapping experiments showed that Pr2a+b contains all the tyrosine-containing tryptic peptides of gp69/71. Two additional tyrosine-containing tryptic peptides in Pr2a+b that are not detected in gp69/71 are found in p15(E). Thus, gp69/71 and p15(E) peptide sequences account for all the tyrosine tryptic peptides of Pr2a+b. The gene order of the two proteins was determined by pulse-labeling infected cells in the presence and absence of pactamycin at concentrations of the inhibitor that prevent initiation of translation, but not elongation. The gene order was found to be: (2)HN-gp69/71-p15(E)-COOH. A newly identified major viral protein, termed p12(E), migrates in sodium dodecyl sulfate-polyacrylamide gels in the "p12" region. It is related to p15(E) as determined by tryptic mapping experiments. p15(E) and p12(E) are not phosphorylated, and both can be separated from phosphoprotein p12 by guanidine hydrochloride-agarose chromatography. p12(E) and p15(E) elute in the void volume fraction, whereas phosphoprotein p12 elutes between p15 and p10. The two p12 proteins can also be separated from each other by two-dimensional gel electrophoresis involving isoelectric focusing in the first dimension and sodium dodecyl sulfate-gel electrophoresis in the second dimension.

Genes↗
PubMed1978

Requirement of protein synthesis for the degradation of host mRNA in Friend erythroleukemia cells infected wtih herpes simplex virus type 1.

We describe experiments which demonstrate that shortly after infection of Friend erythroleukemia cells with herpes simplex virus (HSV), polyribosomes dissociate and cellular mRNA degrades. Analysis of infected cell extracts on sucrose density gradients demonstrates that the majority of the polyribosomes have dissociated to monoribosomes at 2 h postinfection. Physical measurements of infected-cell RNAs support this conclusion and demonstrate that the polyadenylated RNAs decrease in size. The degradation of mRNA is apparently a stochastic process as judged by the failure to detect a shift in the Crt1/2 when polyadenylated RNA extracted from infected cells at different times is hybridized to globin complementary DNA. In experiments designed to determine whether dissociation of polyribosomes is sufficient to cause degradation of globin mRNA, the amount of globin mRNA in uninfected cells did not change when cells were treated with NaF or pactamycin at concentrations sufficient to dissociate all polyribosomes. In cells infected with UV-irradiated virus polyribosomes dissociate but globin mRNA does not degrade, suggesting that it is possible to separate dissociation from degradation.

Animals↗
PubMed1980

Specificity of protein synthesis inhibitors in the inhibition of encephalomyocarditis virus replication.

Effect of protein synthesis inhibitors on encephalomyocarditis virus production in L-cells was studied. Inhibition of initiation by hypertonicity, harringtonine, or pactamycin decreased viral protein synthesis to a lesser extent than that of host. Virus yield was unaffected or actually enhanced by low concentrations of these inhibitors. On the contrary, the elongation inhibitors cycloheximide, anisomycin, and emetine, shown previously to inhibit viral protein synthesis preferentially, had a greater effect on virus yield than on overall protein synthesis. These results support our earlier proposal that the antiviral activity of cycloheximide derives from its specific effect on the rate of elongation of protein synthesis, and that elongation inhibitors in general may show varying degrees of specific antiviral activity.

Animals↗
PubMed1987

Translational requirement of La Crosse virus S-mRNA synthesis: in vivo studies.

By using methods to isolate cytoplasmic RNAs which limit degradation, the effect of drugs which inhibit protein synthesis on the accumulation of La Crosse virus plus-strand S RNAs in vivo has been studied. Cycloheximide and puromycin treatment of infected cultures caused an abortive transcript of ca. 205 nucleotides (nt) to accumulate, whereas pactamycin led to the appearance of an RNA which was slightly shorter (ca. 200 nt). Both the 205- and 200-nt RNAs contained the same range of host primers at their 5' end, but their 3' ends mapped at ca. positions 175 and 165, respectively. Examination of the sequence in this region and at the mature mRNA termination site (position 886) suggests that the sequence YAAAAAT(A)GCAG is involved in transcription termination.

Animals↗
PubMed1979

Characterisation of protein synthesis in cell-free extracts from different mammalian cells by their sensitivity to inhibitors of polypeptide-chain initiation.

Plasma cells and reticulocytes are differentiated mammalian cell systems specialized in the synthesis of distinct proteins. To study whether a cell specificity of polypeptide-chain initiation in such cell systems can be detected through inhibitors, the sensitivity to several drugs interfering with initiation was compared in cell-free systems with S-30 extracts from these cells. The following was indicated by the experiments: (1) under the selected conditions, the different cell-free systems are comparable with respect to their activity of initiation, and that aurintricarboxylic acid inhibits mRNA binding to the ribosome in plasma cell tumours the same as in reticulocytes. (2) The sensitivity of translation of endogenous mRNAs in reticulocytes and plasma cell tumours to inhibitors of mRNA binding to the ribosome, aurintricarboxylic acid and the homopolynucleotides poly(U) and poly(A), are different. (3) Inhibition of the succeeding reactions of peptide-chain initiation by sodium fluoride and pactamycin was not selective. (4) In both cell systems translation of poly(U) is equally sensitive to aurintricarboxylic acid. (5) In extracts from TEPC 15 myeloma cells synthesis of immunoglobulin L-chains compared with that of the other myeloma proteins is partially resistant to aurintricarboxylic acid, whereas in reticulocytes, no differential sensitivities of individual proteins could be observed. The different susceptibility of the mRNA binding reaction in plasma cell tumours and reticulocytes suggests that the predominant mRNAs of these cells have different affinities to ribosome binding sites.

Animals↗
PubMed1989

Early effects of refeeding a complete diet on the translational initiation activity in rat liver slices.

A complete diet containing 23% protein was fed to two-day-fasted rats. The effects of this diet on protein biosynthesis, through the examination of liver slices, was investigated. (1) Refeeding enhanced the protein-synthesizing activity with better preservation of heavy polysomes in the liver slices. Protein in the diet was necessary to induce, through refeeding, the activation of protein synthesis in the liver. (2) The incorporation of [3H]methionine into 40S subunits and 80S subunits, where "run-off" 40S and 60S subunits had been accumulated by preincubation with 2 x 10(-6) M pactamycin, was much higher in the liver slices of refed rats than in the liver slices of fasted rats. By using chain initiation inhibitors, it was shown that labeled 40S and 80S subunits were 40S and 80S initiation complexes, respectively.

Animals↗
PubMed1976

Studies on the mechanism of lymphocyte-mediated cytolysis. VI. A reappraisal of the requirement for protein synthesis during T cell-mediated lysis.

The role of protein synthesis in the mechanism of T cell-mediated cytolysis has been re-investigated. Cytolytically active (C57BL/L anti-DBA/2) spleen cells treated with pactamycin (10-7 M to 10-6 M) exhibited suppressed protein synthesis (100 +/- 5%), but unimpeded lytic activity. Drug-treated effector cells, incubated for prolonged (up to 24 hr) periods of time in the presence and absence of antigen, showed no siginificant diminution of lytic activity although the incorporation of 3H-leucine into protein was totally ablated. Studies with emetine, another irreversible inhibitor of protein synthesis, gave identical results. These findings are difficult to reconcile with the hypothesis that effector T cells lysis via a soluble protein mediator.

Animals↗
PubMed1976

Role of changes in protein degradation in the growth of regenerating livers.

The significance of changes in rates of synthesis, export, and degradation of proteins during liver regeneration was assessed. (a) Proteins were pulse labeled by the intravenous injection of radioactive leucine and, 5 min later, pactamycin (an inhibitor of the initiation of protein synthesis). One-half of the protein radioactivity was lost from the normal liver within 3 hours. From the radioactivity of the plasma proteins at that time and a study of the disappearance of these proteins from the circulation, it was calculated that 28% of the newly synthesized proteins were exported. Serum albumin accounted for a third of the exported proteins. Thirty-six hours after partial hepatectomy the proportion of albumin to total protein synthesis remained constant, while that of the other plasma proteins increased by 50%. The fraction of the newly synthesized proteins retained by the liver after 3 hours decreased by 20%. (b) During the first 36 hours of liver regeneration the average rates of protein degradation slowed down to one-half the normal values. This was determined either by the loss of radioactivity from total protein (or the guanidino-C of protein-bound arginine) in livers labeled with [14C]bicarbonate, or calculated as the balance between protein synthesis and net protein gain. (c) From these results, and those of our previous study of the protein synthetic machinery of normal and regenerating livers (Scornik, O.A. (1974)J. Biol. Chem. 249, 3876-3883), we conclude that changes in the rate of protein degradation are the single most important factor determining the increase in protein content during liver compensatory growth.

Animals↗
PubMed1977

Suppression of lymphoproliferation by high concentrations of normal human mononuclear leukocytes.

Mononuclear leukocytes (MNL) include cells that suppress lymphoproliferation in unstimulated and antigen-stimulated cultures. Suppression is demonstrated by increasing the concentration of cells added to cultures and does not require preactivation of suppressor cells. Suprression of 3H-thymidine incorporation occurred if the high concentrations of MNL were added to cultures before the proliferative responses commenced. This suppressive effect of high cell concentration upon 3H-thymidine incorporation is removed by depleting MNL present in high numbers of cells that adhere to foreign surfaces or by preincubating these cells with cycloheximide, puromycin, or pactamycin. The suppressor cell, which only functions when present in a viable state, is radioresistant, adheres to foreign surfaces, remains active through 5 days in culture, and equates with the presence of a cell that is rich in cytoplasmic esterase. The suppressor cell may be of the monocyte series and did not appear to belong to either the T or the B lymphocyte series. This study provides additional evidence that normal immune reactivity in man is under regulatory control. The suppressor mechanism identified herein with normal human MNL is probably related to a similar type of suppression (but at a much lower cell concentration) that has been described with Hodgkin's disease and solid tumors.

Aging↗