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Science (New York, N.Y.) Dec 1966Abstract. Mice were injected in the temporal region of the brain with cycloheximide or puromycin; both agents markedly inhibit protein synthesis in the brain. Recordings...
Abstract. Mice were injected in the temporal region of the brain with cycloheximide or puromycin; both agents markedly inhibit protein synthesis in the brain. Recordings of electrical activity were made in the hippocampal region 5 hours after injection of these drugs. The amplitude and frequency observed in records from mice injected with cycloheximide were indistinguishable from those injected with saline alone. Records from puromycin-injected mice were strikingly abnormal. This finding may contribute to the differences in behavioral effects of intracerebral injections of the two inhibitors of protein synthesis studied.
Topics: Animals; Antifungal Agents; Brain Chemistry; Cycloheximide; Electroencephalography; Hippocampus; Male; Mice; Proteins; Puromycin
PubMed: 5924923
DOI: 10.1126/science.154.3756.1557 -
Proceedings of the National Academy of... Apr 2012
Topics: Alkynes; Diagnostic Imaging; Protein Biosynthesis; Puromycin
PubMed: 22447778
DOI: 10.1073/pnas.1202000109 -
Archives of Microbiology Aug 2015In Saccharomyces cerevisiae, a typical apoptotic phenotype is induced by some stress factors such as sugars, acetic acid, hydrogen peroxide, aspirin and age....
In Saccharomyces cerevisiae, a typical apoptotic phenotype is induced by some stress factors such as sugars, acetic acid, hydrogen peroxide, aspirin and age. Nevertheless, no data have been reported for apoptosis induced by puromycin, a damaging agent known to induce apoptosis in mammalian cells. We treated S. cerevisiae with puromycin to induce apoptosis and evaluated the percentage of dead cells by using Hoechst 33342 staining, transmission electron microscopy (TEM) and Annexin V flow cytometry (FC) analysis. Hoechst 33342 fluorescence images were processed to acquire parameters to use for multiparameter analysis [and perform a principal component analysis, (PCA)]. Cell viability was evaluated by Rhodamine 123 (Rh 123) and Acridine Orange microscope fluorescence staining. The results show puromycin-induced apoptosis in S. cerevisiae, and the PCA analysis indicated that the increasing percentage of apoptotic cells delineated a well-defined graph profile. The results were supported by TEM and FC. This study gives new insights into yeast apoptosis using puromycin as inducer agent, and PCA analysis may complement molecular analysis facilitating further studies to its detection.
Topics: Apoptosis; Flow Cytometry; Microscopy, Electron, Transmission; Principal Component Analysis; Protein Synthesis Inhibitors; Puromycin; Saccharomyces cerevisiae
PubMed: 25868793
DOI: 10.1007/s00203-015-1110-7 -
The Journal of Cell Biology Dec 1971The frequency of colony formation in monolayers of cultured frog cell lines treated with puromycin was compared in (a) haploid and heteroploid lines and (b)...
The frequency of colony formation in monolayers of cultured frog cell lines treated with puromycin was compared in (a) haploid and heteroploid lines and (b) mutagen-treated and nontreated haploid lines. Evidence that resistant colonies result from gene mutation was negative, since the colony frequency is independent of both ploidy and mutagen treatment. A study of five frog cell lines showed that colony formation in puromycin depends on (a) the concentration of puromycin, (b) preselection of the population with puromycin, and, particularly, (c) the capacity of the treated population to survive some exposure to puromycin. One haploid and one heteroploid strain showing stable resistance to puromycin have been isolated; comparison of those variants with sensitive populations has shown that resistance to puromycin is correlated with the cells' capacity to exclude the drug. The evidence for different levels of membrane permeability, combined with evidence for many degrees of resistance among and within cell populations, suggests a model of self-determining membrane units. The evolution of a resistant phenotype may result from changes in the proportion of specific units in the membrane population.
Topics: Animals; Anura; Cell Division; Cell Line; Cell Membrane Permeability; Cell Survival; Cells, Cultured; Clone Cells; Culture Media; Culture Techniques; Drug Resistance; Haploidy; Mutagens; Mutation; Phenotype; Polyploidy; Puromycin; Rana pipiens; Time Factors; Tritium
PubMed: 5316064
DOI: 10.1083/jcb.51.3.742 -
Angewandte Chemie (International Ed. in... Mar 2015The antibiotic puromycin, which inhibits protein translation, is used in a broad range of biochemical applications. The synthesis, characterization, and biological...
The antibiotic puromycin, which inhibits protein translation, is used in a broad range of biochemical applications. The synthesis, characterization, and biological applications of NVOC-puromycin, a photocaged derivative that is activated by UV illumination, are presented. The caged compound had no effect either on prokaryotic or eukaryotic translation or on the viability of HEK 293 cells. Furthermore, no significant release of ribosome-bound polypeptide chains was detected in vitro. Upon illumination, cytotoxic activity, in vitro translation inhibition, and polypeptide release triggered by the uncaging of NVOC-puromycin were equivalent to those of the commercial compound. The quantum yield of photolysis was determined to be 1.1±0.2% and the NVOC-puromycin was applied to the detection of newly translated proteins with remarkable spatiotemporal resolution by using two-photon laser excitation, puromycin immunohistochemistry, and imaging in rat hippocampal neurons.
Topics: Animals; Benzaldehydes; Cell Survival; HEK293 Cells; Hippocampus; Humans; Microscopy, Fluorescence; Peptides; Photolysis; Protein Biosynthesis; Puromycin; Rats; Ultraviolet Rays
PubMed: 25656536
DOI: 10.1002/anie.201410940 -
Nucleic Acids Research Mar 2000Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we...
Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 microM) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0. 04-1.0 microM bonded to 7-21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling.
Topics: Amino Acid Sequence; Binding Sites; Cell-Free System; Escherichia coli; Humans; Molecular Sequence Data; Protein Binding; Proteins; Puromycin
PubMed: 10666460
DOI: 10.1093/nar/28.5.1176 -
Journal of General Microbiology Nov 1985Ribosomes from Streptomyces alboniger are sensitive in vitro to puromycin and, to a lesser extent, to the puromycin-precursor O-demethyl-puromycin. The...
Ribosomes from Streptomyces alboniger are sensitive in vitro to puromycin and, to a lesser extent, to the puromycin-precursor O-demethyl-puromycin. The puromycin-inactivating enzyme (puromycin N-acetyltransferase) from S. alboniger also N-acetylates O-demethyl-puromycin. This finding indicates that in certain antibiotic-producing organisms the antibiotic-inactivating enzymes may play a role in self-defence against toxic precursor molecules.
Topics: Acetylation; Acetyltransferases; Chromatography, Thin Layer; Drug Resistance, Microbial; Peptide Biosynthesis; Peptides; Puromycin; Ribosomes; Streptomyces
PubMed: 4093761
DOI: 10.1099/00221287-131-11-2877 -
FEBS Letters Jan 1992AcPhe2-tRNA(Phe) synthesized in 70S ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is...
AcPhe2-tRNA(Phe) synthesized in 70S ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. A detailed study of the kinetics of the puromycin reaction, its comparison with that of spontaneous translocation, the use of antibiotic viomycin as an effective inhibitor of spontaneous translocation revealed that, besides spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction per se triggers conformational changes in the ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl-site of the peptidyltransferase center. This is detected functionally as the ability of such an A site bound peptidyl-tRNA to react with puromycin. This reaction is highly pronounced at elevated (25 degrees C) temperature but can be hardly detected at 0 degrees C.
Topics: Binding Sites; Models, Biological; Protein Biosynthesis; Puromycin; RNA, Transfer, Amino Acyl; RNA, Transfer, Phe; Ribosomes
PubMed: 1733779
DOI: 10.1016/0014-5793(92)80380-y -
BMC Microbiology Apr 2021Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in...
BACKGROUND
Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin.
RESULTS
We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways.
CONCLUSIONS
We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed.
Topics: Imipramine; Protein Biosynthesis; Puromycin; Saccharomycetales; Surface-Active Agents
PubMed: 33879049
DOI: 10.1186/s12866-021-02185-3 -
Journal of Medicinal Chemistry Nov 1986The carbocyclic analogue of puromycin was prepared by the coupling of N-(benzyloxycarbonyl)-p-methoxy-L-phenylalanine to the racemic aminonucleoside (+/-)-9-[3...
The carbocyclic analogue of puromycin was prepared by the coupling of N-(benzyloxycarbonyl)-p-methoxy-L-phenylalanine to the racemic aminonucleoside (+/-)-9-[3 beta-amino-2 beta-hydroxy-4 alpha-(hydroxymethyl)cyclopent-1 alpha-yl]-6-(dimethylamino)purine, followed by separation of the diastereomers and subsequent removal of the Cbz blocking group. Kinetic studies indicate that carbocyclic puromycin is an excellent substrate for the peptidyltransferase reaction with both prokaryotic and eukaryotic ribosomes. A comparison of carbocyclic puromycin with previously synthesized analogues indicate that the furanosyl ring oxygen and the hydroxymethyl group of puromycin do not contribute to ribosomal binding, but both moieties contribute to the rate of product formation from the enzyme-substrate complex. Carbocyclic puromycin was equal to puromycin when evaluated for cytotoxicity using P-388 mouse lymphoid leukemia cells in culture.
Topics: Animals; Leukemia P388; Mice; Protein Biosynthesis; Puromycin; Ribosomes; Structure-Activity Relationship
PubMed: 3783599
DOI: 10.1021/jm00161a044