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Cytobios 1983The effects have been studied of puromycin and cycloheximide on the reaggregation of ectoderm cells dissociated from Xenopus laevis blastulae. Puromycin or cycloheximide...
The effects have been studied of puromycin and cycloheximide on the reaggregation of ectoderm cells dissociated from Xenopus laevis blastulae. Puromycin or cycloheximide can inhibit reaggregation, suggesting that cell reassociation is dependent upon protein synthesis. If the cells are allowed a 3 h 'recovery' period in culture medium following dissociation, before being exposed to either puromycin or cycloheximide, higher concentrations of the inhibitors are required to prevent cell aggregation, suggesting that significant synthesis of the proteins required for reaggregation occurs in the 3 h immediately following dissociation. Lower concentrations of puromycin permit cell reaggregation but reduce the normal formation of cilia. The effects have also been observed of puromycin on the scanning electron microscopical appearance of Xenopus blastula ectoderm cells cultured singly in vitro. Puromycin reduces the normal formation of pseudopodia, suggesting that puromycin might inhibit reaggregation partly by inhibiting cell movement. Puromycin also produces some elongated cells, possibly by inhibition of cytokinesis.
Topics: Animals; Blastocyst; Cell Aggregation; Cycloheximide; Microscopy, Electron, Scanning; Puromycin; Xenopus laevis
PubMed: 6617261
DOI: No ID Found -
Pharmacology, Biochemistry, and Behavior Sep 1981A new one-session T-maze training procedure for cockroaches, in which animals were trained to turn right or left to avoid shock, is described. This paradigm was utilized...
A new one-session T-maze training procedure for cockroaches, in which animals were trained to turn right or left to avoid shock, is described. This paradigm was utilized to investigate effects of protein synthesis inhibiting drugs on learning and retention. Cycloheximide (CXM), which inhibited protein synthesis by over 90% during the training period, did not impair acquisition and did not produce retention deficits an any interval up to 1 day after training. Puromycin (PURO), which inhibited protein synthesis by about 70% during the training period, produced amnesia 5 hr after training, while acquisition was not affected. Thus invertebrates, as well as vertebrates, are susceptible to amnesic effects of puromycin. Although PURO-injected animals showed retention deficits as measured by the number of correct turns, no retention deficit occurred for the behavioral modification consisting of an increase in runway time during the training period. Therefore, PURO appears to show specificity for the different types of longer-term memories that are formed in a training situation.
Topics: Animals; Cockroaches; Cycloheximide; Learning; Male; Memory; Nerve Tissue Proteins; Periplaneta; Puromycin; Retention, Psychology
PubMed: 7291251
DOI: 10.1016/0091-3057(81)90282-3 -
Journal of Medicinal Chemistry Jul 1977
Topics: Bacterial Proteins; Depression, Chemical; Escherichia coli; Molecular Conformation; Phenylalanine; Puromycin
PubMed: 327067
DOI: 10.1021/jm00217a013 -
Pharmacology, Biochemistry, and Behavior Apr 1979Pigeons were injected intracerebrally with either puromycin (PM) or control saline solution following training for one 12-min session on a visual discrimination....
Pigeons were injected intracerebrally with either puromycin (PM) or control saline solution following training for one 12-min session on a visual discrimination. Injections were made either immediately following training, 1 hr later or 24 hr later. Retention testing 3 days after training showed that PM produced marked amnesia in the first two groups, but had no effect in the 24 hr condition. However, all PM groups were retarded subsequently in the number of days required to reach a 90% discimination criterion. This differentiation of two separate behavioral effects with different temporal gradients suggests that PM may be working through two distinct physiological mechanisms.
Topics: Animals; Behavior, Animal; Columbidae; Conditioning, Operant; Discrimination, Psychological; Injections, Intraventricular; Male; Puromycin; Time Factors
PubMed: 461482
DOI: 10.1016/0091-3057(79)90227-2 -
STAR Protocols Sep 2022Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess...
Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess translation activity in mammalian cells by incorporation of O-propargyl-puromycin (OP-Puro). OP-Puro is a puromycin analog that is incorporated into newly synthesized proteins and is detected by click chemistry reaction. We use OP-Puro labeling to assess translation activity between different cell types or cells under different growth conditions by confocal microscopy and flow cytometry. For complete details on the use and execution of this protocol, please refer to Hsu et al. (2021) and Hsu et al. (2022).
Topics: Animals; Cell Line; Click Chemistry; Mammals; Proteomics; Puromycin
PubMed: 36072758
DOI: 10.1016/j.xpro.2022.101654 -
The Journal of Biological Chemistry Sep 1983Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in...
Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.
Topics: Electrophoresis, Polyacrylamide Gel; Escherichia coli; Macromolecular Substances; Microscopy, Electron; Puromycin; Ribosomes
PubMed: 6350302
DOI: No ID Found -
Nature Communications Jun 2019A major challenge in biology is that genetically identical cells in the same environment can display gene expression stochasticity (noise), which contributes to...
A major challenge in biology is that genetically identical cells in the same environment can display gene expression stochasticity (noise), which contributes to bet-hedging, drug tolerance, and cell-fate switching. The magnitude and timescales of stochastic fluctuations can depend on the gene regulatory network. Currently, it is unclear how gene expression noise of specific networks impacts the evolution of drug resistance in mammalian cells. Answering this question requires adjusting network noise independently from mean expression. Here, we develop positive and negative feedback-based synthetic gene circuits to decouple noise from the mean for Puromycin resistance gene expression in Chinese Hamster Ovary cells. In low Puromycin concentrations, the high-noise, positive-feedback network delays long-term adaptation, whereas it facilitates adaptation under high Puromycin concentration. Accordingly, the low-noise, negative-feedback circuit can maintain resistance by acquiring mutations while the positive-feedback circuit remains mutation-free and regains drug sensitivity. These findings may have profound implications for chemotherapeutic inefficiency and cancer relapse.
Topics: Animals; Antimetabolites, Antineoplastic; CHO Cells; Computer Simulation; Cricetulus; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Feedback, Physiological; Gene Expression Regulation; Gene Regulatory Networks; Models, Genetic; Neoplasms; Puromycin; Stochastic Processes
PubMed: 31235692
DOI: 10.1038/s41467-019-10330-w -
Science (New York, N.Y.) Sep 1974Puromycin fails to alter minimal oxygen consumption of rats treated with thyroxine, provided the rectal temperatures of these rats are maintained at 37.8 degrees to 38.1...
Puromycin fails to alter minimal oxygen consumption of rats treated with thyroxine, provided the rectal temperatures of these rats are maintained at 37.8 degrees to 38.1 degrees C. The previously reported puromycin-induced decline in basal metabolic rate of thyroxine-treated rats may have been due to the hypothermia produced by this drug.
Topics: Animals; Basal Metabolism; Body Temperature; Oxygen Consumption; Puromycin; Rats; Thyroxine
PubMed: 4853979
DOI: 10.1126/science.185.4156.1060 -
ELife Aug 2020Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide...
Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.
Topics: Animals; Cell Line, Tumor; Mice; Peptide Chain Elongation, Translational; Protein Synthesis Inhibitors; Proteins; Puromycin; RNA, Messenger; RNA, Transfer, Amino Acyl; Ribosomes
PubMed: 32844746
DOI: 10.7554/eLife.60048 -
The Journal of Organic Chemistry Mar 2009The mechanism by which the ribosome catalyzes peptide bond formation remains controversial. Here we describe the synthesis of a nucleoside that can be used in Brønsted...
The mechanism by which the ribosome catalyzes peptide bond formation remains controversial. Here we describe the synthesis of a nucleoside that can be used in Brønsted experiments to assess the transition state of ribosome catalyzed peptide bond formation. This substrate is the nucleoside 3'-amino-3'-deoxy-3'-[(3''R)-3-fluoro-l-phenyl-alanyl]-N(6),N(6)-dimethyladenosine, which was prepared from (1R,2R)-2-amino-1-phenylpropane-1,3-diol. This substrate is active in peptide bond formation on the ribosome and is a useful probe for Brønsted analysis experiments on the ribosome.
Topics: Catalysis; Fluorine; Peptides; Puromycin; Ribosomes
PubMed: 19284740
DOI: 10.1021/jo802611t