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Biomedical subjects

S Pestka

Publications and source records attributed to S Pestka.

At least 253 records · Page 14Linked to original sources
PubMed1976

Binding of N-substituted erythromycyclamines to ribosomes.

Several N-substituted erythromycylamines were evaluated for their ability to inhibit the binding of [(14)C]erythromycin to ribosomes. The association and dissociation constants for the binding of each compound to Escherichia coli ribosomes were determined. These studies have resulted in the development of three types of probes for topological studies of the erythromycin-binding site and the ribosome: the chemically reactive bromoacetamido, the photoreactive N-(2)-nitro-4-azidophenyl)glycinamido, and the fluorescent fluorescein isothiocyanate derivatives of 9(S)-erythromycylamine.

Anti-Bacterial Agents↗
PubMed1976

Binding of thiostrepton to ribosomes from thiostrepton-sensitive and -resistant Bacillus subtilis strains.

Binding of [(35)S]thiostrepton to ribosomes from thiostrepton-sensitive and -resistant strains of Bacillus subtilis was studied. Ribosomes from thiostrepton-resistant strains bound relatively little thiostrepton compared with ribosomes from thiostrepton-sensitive B. subtilis. In addition, ribosomes from revertant strains that were obtained as thiostrepton-sensitive revertants from thiostrepton-resistant strains bound [(35)S]thiostrepton similarly to ribosomes from the sensitive parental strain.

Anti-Bacterial Agents↗
PubMed1975

Aromatic esters of 5-O-desosaminylerythronolide A oxime.

Several substituted aromatic esters of the C-3 hydroxyl of 5-O-desosaminylerythronolide A oxime were prepared. Ribosomal binding studies showed that meta substituents on the aromatic ring gave the most active analogs. The esters described were all inactive in vivo at the maximum level tested.

Bacillus subtilis↗
PubMed1975

Cell-free translation of immunoglobulin messenger RNA from MOPC-315 plasmacytoma and MOPC-315 NR, a variant synthesizing only light chain.

Total poly(A)-containing mRNA was isolated from the MOPC-315 and MOPC-315 NR plasmacytomas. The RNA was further fractionated on sodium dodecyl sulfate-sucrose gradients. The MOPC-315 mRNA fractions directed the synthesis of both the heavy chain and light chain precursor of the MOPC-315 IgA protein in a cell-free extract of Ehrlich ascites tumor cells. None of the MOPC-315 NR mRNA fractions tested programmed the synthesis of the heavy chain in this system. Analysis of cell-free products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoprecipitation demonstrated that no translatable heavy chain mRNA could be extracted from the MOPC-315 NR variant plasmacytoma.

Animals↗
PubMed1975

Cell-free synthesis of human interferon.

With mRNA prepared from induced human fibroblasts biologically active human interferon was synthesized de novo in a cell-free extract from mouse cells. The identity of the antiviral activity as human interferon was demonstrated by its species and antigenic specificity.

Animals↗
PubMed1975

Inhibition of protein synthesis in intact HeLa cells.

Polysome analysis has proved to be a sensitive probe for the mode of action of inhibitors of protein synthesis in intact HeLa cells. To classify the active compounds as inhibitors of initiation, elongation, or termination, their effects on the cellular polyribosome pattern were compared under three conditions. These conditions tested (i) their direct effect on the polyribosome profile; (ii) their effect on ribosome run-off produced by hypertonicity; and (iii) their effects on recovery from hypertonicity. Using this technique, diacetoxyscirpenol, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, and three alkaloids, harringtonine, isoharringtonine, and homoharringtonine, were found to be inhibitors of initiation. Polysome analysis indicated that in HeLa cells 7.8 x 10(-7) M pactamycin, which inhibited protein synthesis 94%, interfered with elongation as well as initiation under these conditions. Emetine, anisomycin, cycloheximide, and trichodermin each gave polysome patterns consistent with inhibition of elongation. Fusidic acid and aurintricarboxylic acid inhibited incorporation of [(14)C]leucine into intact HeLa cells, but polysome analysis did not localize any specific inhibitory effects to the initiation, elongation, or termination steps of protein synthesis. The use of specific inhibitors of initiation of protein synthesis has indicated that most, if not all, mammalian messenger ribonucleic acids contain a single initiation site.

Amino Acids↗
PubMed1974

Antibiotics as probes of ribosome structure: binding of chloramphenicol and erythromycin to polyribosomes; effect of other antibiotics.

Antibiotics were used as probes of ribosome topology and function. Studies of [(14)C]chloramphenicol and [(14)C]erythromycin binding to ribosomes and polyribosomes revealed the following features. The requirement of high K(+) concentration (150 mM) for [(14)C]chloramphenicol binding to NH(4)Cl-washed ribosomes resulted from the washing procedure. Neither native 70S ribosomes nor polyribosomes require K(+) greater than 30 mM for [(14)C]chloramphenicol binding. Whereas [(14)C]chloramphenicol binds to both ribosomes and polyribosomes, [(14)C]erythromycin binds essentially only to ribosomes. After removal of peptidyl-transfer ribonucleic acid (tRNA) from polyribosomes, [(14)C]erythromycin could then be bound. The effects of a number of antibiotics on [(14)C]chloramphenicol binding to ribosomes and polyribosomes was assessed. It was found that most of the macrolides (erythromycin, carbomycin, spiramycin III, niddamycin, oleandomycin, and tylosin) and streptogramins A and B (vernamycin A, PA114A, vernamycin Balpha, and PA114B) inhibited chloramphenicol binding to NH(4)Cl-washed and native 70S ribosomes, but not to polyribosomes. After removal of peptidyl-tRNA from polyribosomes, [(14)C]chloramphenicol binding was then inhibited. In contrast, sparsomycin and althiomycin inhibited chloram-phenicol binding to polyribosomes, but not to ribosomes. After removal of peptidyl-tRNA from polyribosomes, sparsomycin and althiomycin were then ineffective. The presence of peptidyl-tRNA on polyribosomes apparently is required for binding of sparsomycin and althiomycin, but prevents binding of most macrolides and streptogramins. The lincosaminides (lincomycin and celesticetin) and methymycin (a small macrolide) inhibited [(14)C]chloramphenicol binding to NH(4)Cl-washed and native 70S ribosomes best, but also inhibited the binding to polyribosomes. The amino nucleosides and other antibiotics tested do not seem to interact strongly with the major chloramphenicol-binding site. These results provide knowledge of the interrelationships between antibiotic and substrate ribosome binding sites which should eventually contribute to a map of ribosomal topology.

Anti-Bacterial Agents↗
PubMed1974

Inhibition of [14C]chloramphenicol binding to Escherichia coli ribosomes by erythromycin derivatives.

The effect of erythromycin A and 35 analogues of erythromycin A on [(14)C]chloramphenicol binding to Escherichia coli ribosomes was evaluated. Substitutions on various portions of the erythromycin molecule were made with retention of ability to bind to ribosomes. Specifically, substantial activity in interference with [(14)C]chloramphenicol binding was retained upon removal of the cladinose and various substitutions on the 3-hydroxyl, the oxime, and 2-hydroxyl groups. Erythromycin analogues with relatively poor binding activity to ribosomes could be detected. This assay can be used alone or in conjunction with microbiological assays for screening of active analogues. It permits an estimate of the general binding activity of compounds rapidly and directly. The assay reflects the ability of the compounds to interact with their target organelle, the ribosome, and may serve as a useful adjunct in developing new compounds.

Anti-Bacterial Agents↗
PubMed1974

Binding of [14C]erythromycin to Escherichia coli ribosomes.

Erythromycin binding to Escherichia coli ribosomes required K(+) and Mg(2+). Under optimal conditions, the dissociation constant for erythromycin binding to E. coli ribosomes was found to be 1.0 x 10(-8) M and 1.4 x 10(-8) M at 24 C and 5 C, respectively. One molecule of [(14)C]erythromycin was bound to each 70S ribosome at equilibrium. Binding of erythromycin to ribosomes was rapid and reversible. The specific rate constants for the forward and reverse reactions were 1.7 x 10(7) liters per mol per min and 0.15 per min, respectively.

Carbon Radioisotopes↗
PubMed1974

Effect of erythromycin analogues on binding of [14C]erythromycin to Escherichia coli ribosomes.

The relative ability of 44 erythromycin analogues to bind to ribosomes was determined by their effect on [(14)C]erythromycin binding to Escherichia coli ribosomes. The association and dissociation constants of each of these erythromycin derivatives were determined as well as their interaction coefficient for their binding to ribosomes. Substitutions were made on various portions of the erythromycin molecule with retention of substantial activity as measured by inhibition of [(14)C]erythromycin binding to ribosomes. Since the effect of erythromycin analogues on [(14)C]erythromycin binding to ribosomes provides a relatively sensitive assay for these compounds, erythromycin analogues with relatively little affinity for ribosomes could be detected. Compounds with association constants of 10(4) M(-1) were detectable; the association constant for erythromycin binding to ribosomes was approximately 10(8) M(-1). Thus, compounds with 0.0001 the association constant of erythromycin were detectable. This assay could be used alone or in conjunction with microbiological assays for primary screening of active analogues or other compounds which interfere with [(14)C]erythromycin binding to ribosomes. It permits an estimate of the general activity of compounds rapidly and directly. Variables such as metabolic modifications of the compounds and permeability are excluded. The present assay reflects the ability of the compounds to interact directly with their target organelle and may serve as a useful adjunct in developing new compounds.

Carbon Radioisotopes↗