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At least 163 records · Page 9Linked to original sources
PubMed1975

Stimulation of derepressed enzyme synthesis in bacteria by growth on sublethal concentrations of chloramphenicol.

Culturing of Salmonella typhimurium or Escherichia coli cells in the presence of low concentrations (</=1 mug/ml) of chloramphenicol (CAP) permitted exponential growth, but at doubling times up to twice those of controls. When such cultures were subsequently starved for uracil or arginine, derepression of aspartate transcarbamylase (ATCase) or ornithine transcarbamylase, respectively, was enhanced three- to 10-fold as compared to cultures not exposed to CAP. Enhancement of beta-galactosidase synthesis by prior exposure to CAP was also observed in uracil-starved E. coli cultures. Stimulation of enzyme synthesis appeared to be a specific effect of CAP; low levels of erythromycin, puromycin, sparsomycin, tetracycline, and rifampin did not show such effects. Derepression of ATCase synthesis in exponentially growing cells in the presence of CAP did not result in stimulation of enzyme synthesis by CAP. A prior history of growth of a culture in the presence of CAP was shown to be necessary for enhancement of enzyme synthesis by CAP; furthermore, continued presence of CAP in the medium during starvation was not necessary for enhanced enzyme synthesis and inhibited it in some instances. Enhanced enzyme synthesis in starving, CAP-treated cultures could be blocked by rifampin, which suggested that CAP treatment allows prolonged or more extensive messenger ribonucleic acid synthesis.

Bacteria↗
PubMed1982

Sequences of ribosome binding sites from the large size class of reovirus mRNA.

Ribosome-protected fragments from two of the large-sized reovirus mRNAs were recovered from sparsomycin-blocked 80S initiation complexes. The sequence of each protected oligonucleotide was determined. The ribosome binding site of each message includes the m7G cap and a centrally positioned AUG codon. An adenine residue occurs three nucleotides upstream from the initiator codon, thus conforming to the pattern [AG]NNAUG observed for nearly all eucaryotic initiation sites.

Base Sequence↗
PubMed1978

Inhibition, by selected antibiotics, of protein synthesis in cells growing in tissue cultures.

A large number of compounds including actinobolin, adrenochrome, amicetin, anisomycin, aurintricarboxylic acid, blasticidin S, chartreusin, chlortetracycline, cycloheximide, doxycycline, edeine A1, edeine complex, emetine, fusidic acid, gougerotin, GppCH2p, oxytetracycline, pactamycin, polydextran sulphate, puromycin, pyrocatechol violet, sparsomycin and tubulosine have been tested for inhibitory effects on protein synthesis in cultured cells from both mouse fibroblasts (3T6 cells) and chick embryo fibroblasts (CEF). Essentially, similar results were obtained with both cell types with the most effective inhibitors being pactamycin, emetine, tubulosine, anisomycin and cycloheximide and with no significant inhibitory activity being detected with edeine complex, edeine A1, GppCH2p, polydextran sulphate, aurintricarboxylic acid, pyrocatechol violet and adrenochrome. The concentration of pactamycin required to produce 50% inhibition of protein synthesis approximated 5 X 10(-9) M, but for most of the inhibitors it ranged from 5 X 10(-6) M to 5 X 10(4) M. The molecular basis underlying these differences may be related, in addition to their intrinsic inhibitory power, to differences in permeability of the cells towards the various drugs tested. Alternatively, active accumulation of the drugs by the cells may be the variable parameter.

Animals↗
PubMed1988

Higher order structures of the 5'-proximal region decrease the efficiency of translation of the porcine pro-opiomelanocortin mRNA.

The SP6 polymerase/promoter system was used to synthesize porcine pro-opiomelanocortin mRNAs with nucleotide sequence deletions in the 5'- as well as 3'-untranslated and coding regions. The translational efficiency of the mutant mRNAs was evaluated by cell-free translation or by monitoring the rate and extent of ribosome binding in the presence of sparsomycin. The results of these experiments indicate that specific nucleotide sequences in the 5'-untranslated and coding regions of the pro-opiomelanocortin mRNA decrease its rate of translation. Structure mapping of the mRNA with double-strand and single-strand specific nucleases suggests that these sequences can form stable secondary structures.

Animals↗
PubMed1989

In vitro synthesis, phosphorylation, and localization on 48 S initiation complexes of human protein synthesis initiation factor 4E.

Complementary DNA for human eukaryotic initiation factor 4E (eIF-4E) was transcribed in vitro and the transcripts used to direct protein synthesis in a cell-free reticulocyte translation system. The predominant translation product was 25 kDa, was bound to a m7GTP-Sepharose affinity column, and was specifically eluted with m7GTP. Both phosphorylated (P) and unphosphorylated (U) forms of eIF-4E were synthesized, and the P/U ratio increased as a function of incubation time in the reticulocyte lysate system. Both forms were quantitatively retained on m7GTP-Sepharose. When translation reactions were resolved on sucrose density gradients, the 35S-labeled eIF-4E sedimented predominantly at 3-4 S. However, in the presence of edeine or guanylyl imidodiphosphate, both of which cause accumulation of 48 S initiation complexes, eIF-4E was detected in the 48 S region. In the presence of sparsomycin, used to accumulate 80 S initiation complexes, no eIF-4E was observed in the 80 S region. No change in the eIF-4E distribution was caused by m7GTP. These results are consistent with a model whereby eIF-4E is transferred to the 43 S initiation complex together with mRNA and is released from the initiation complex when the 60 S ribosomal subunit joins.

Animals↗
PubMed1982

Poly(dT) inhibition of globin synthesis in the rabbit reticulocyte lysate system. Reversal of the inhibition by poly(dT)-binding protein.

The mechanism of inhibition of globin synthesis by poly(dT) was studied in the rabbit reticulocyte lysate system. When the lysate was incubated with [14C]poly(dT), poly(dT) was found to bind with the native 40S ribosomal subunit and the "supernatant factor." But the binding to the native 40S ribosomal subunits was not directly related to the poly(dT) inhibition. Ribosomal subunits were prepared from rabbit reticulocytes and tested for their binding with poly(dT) and their effect on the poly(dT) inhibition. Poly(dT) was found to bind with the derived 40S ribosomal subunit, but not with the derived 60S subunit, and the poly(dT) inhibition was slightly reversed by the derived 40S ribosomal subunit. Under conditions such that the elongation of nascent chains was inhibited by sparsomycin, the formation of the 80S/Met-tRNAf complex was inhibited by poly(dT) and the inhibition was greater at high concentration of KOAc. However, the formation of the 40S/Met-tRNAf complex was inhibited to the same extent at 70 mM and 200 mM KOAc in the presence of GMPPCP. A factor (TF) that reverses the poly(dT) inhibition was partially purified from the KCl-wash of rabbit reticulocyte ribosomes by ammonium sulfate fractionation, and Sephadex G-150 and DEAE-cellulose column chromatographies. From the Sephadex G-150 and DEAE-cellulose column chromatography of TF, the molecular weight of TF was estimated to be 81,000-102,000. TF reversed the poly(dT) inhibition of 80S/[3H]mRNA/Met-tRNAf complex or that of 40S/[3H]mRNA/Met-tRNAf complex. TF bound to [14C]poly(dT) or 3H-labeled globin mRNA. SDS/polyacrylamide slab gel electrophoresis of the complexes between the factor and [14C]poly(dT) or [3H]mRNA showed common polypeptide bands of 22,500, 25,000, and 49,000 daltons. These data can be explained by assuming that poly(dT) binds to a factor which is required for the binding of 40S/Met-tRNAf complex with mRNA to form inactive complexes, and thus inhibits globin synthesis. The relationship between the poly(dT)-binding protein and known initiation factors is discussed.

Animals↗
PubMed1994

Activation and release of enzymatically inactive, full-length rhodanese that is bound to ribosomes as peptidyl-tRNA.

Synthesis of rhodanese in a cell-free coupled transcription/translation system derived from Escherichia coli leads to an accumulation of full-length rhodanese protein on the ribosomes as well as to enzymatically active protein that is released from the ribosomes into the supernatant fraction. The ribosome-bound protein is enzymatically inactive but can be activated and released from the ribosomes without additional protein synthesis by subsequent incubation in the presence of the added chaperones DnaJ, DnaK, GrpE, GroEL, and GroES plus ATP. Efficient activation requires that all of the chaperones are present together during incubation which yields fully active rhodanese. Incubation in the presence of DnaJ only inhibits release whereas incubation with only GroES or DnaK promotes the release of enzymatically inactive protein. Incubation of the ribosome with puromycin leads to the release of enzymatically inactive protein whereas release and activation in the presence of all of the chaperones is blocked by sparsomycin. The effect of these antibiotics provides very strong evidence that enzymatically inactive, full-length rhodanese is bound to the ribosomes as peptidyl-tRNA and that the peptidyl transferase reaction is required for its release. Considered together, the data indicate that chaperone-mediated late stages of rhodanese folding into the enzymatically active, native conformation are intimately associated with the process of termination and release that occurs as part of the reaction cycle of protein synthesis.

Animals↗
PubMed1995

Preclinical antitumor activity of ethyldeshydroxysparsomycin in combination with cisplatin.

The efficacy of cisplatin (CDDP) in combination with the protein synthesis inhibitor ethyldeshydroxysparsomycin (EDSM) has been tested in two tumor models at various schedules. Mice with L1210 leukemia or B16 melanoma were treated with CDDP alone or in combination with EDSM. Against L1210 leukemia, which is sensitive to CDDP, combinations elicited increases in life-span for all treatment schedules compared to those achieved with the corresponding dose of CDDP. Moreover, the combination of EDSM with this platinum compound yielded a cure rate > 80%, compared to < 35% for single CDDP treatment. Although the B16 melanoma is rather resistant to both CDDP and EDSM, combinations of these agents against B16 melanoma showed schedule dependent efficacy and in certain schedules significant therapeutic advantage over individual drug treatment, but cures were not observed. Our results suggest that EDSM has significant synergistic capabilities in both animal tumor models, but strong therapeutic enhancement of cisplatin efficacy is only seen when the tumor is sensitive to CDDP.

Animals↗
PubMed1975

Studies on the in vitro synthesis of ppGpp and pppGpp.

The effect of a number of inhibitors of protein synthesis on ppGpp and pppGpp synthesis in vitro has been examined. As expected from in vivo results, chloramphenicol is without effect on this reaction. Aurintricarboxylic acid and chlortetracycline, on the other hand rapidly and specifically inhibit ppGpp synthesis. Fusidic acid in the presence of saturating amounts of EF G also inhibits the reaction completely, suggesting that an empty ribosomal acceptor site is necessary for this reaction. On the other hand, the 50-S subunit proteins L7 and L12 are not required for stringent factor activity. Ribosomes from Pseudomonas fluorescens can replace those from Escherichia coli in the complete system, while ribosomes from Ehrlich ascites cannot. A small but reproducible synthesis of ppGpp is observed when the ribosomal wash from E. coli is complemented with ribosomes from wheat germ cytoplasm.

Animals↗
PubMed2003

A yeast assay for high throughput screening of natural anti-viral agents.

Over the last decade the yeast Saccharomyces cerevisiae has become a popular organism for studying heterologous gene expression and in vivo protein-protein interactions. Many variations of these basic systems have originated over the years. Besides these vast and varied applications of the yeast expression system, S. cerevisiae has also been used extensively in fundamental research as a model simple eukaryote. We have used the S. cerevisiae system to design a high throughput screen for anti-viral agents from natural sources. The design of the assay rests on the ability of the L-A helper virus and the M(1) satellite virus to detect small variations in -1 ribosomal frameshifting. A minor change in frameshifting efficiencies can be detected and clearly shown phenotypically in terms of zones of clearing on an agar plate. Using such a process, we have initiated a high throughput screening process for natural anti-viral agents.

Anisomycin↗