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Chemistry & Biology Jul 2004Understanding the expression of known and unknown gene products represents one of the key challenges in the post-genomic world. Here, we have developed a new class of...
Understanding the expression of known and unknown gene products represents one of the key challenges in the post-genomic world. Here, we have developed a new class of reagents to examine protein expression in vivo that does not require transfection, radiolabeling, or the prior choice of a candidate gene. To do this, we constructed a series of puromycin conjugates bearing various fluorescent and biotin moieties. These compounds are readily incorporated into expressed protein products in cell lysates in vitro and efficiently cross cell membranes to function in protein synthesis in vivo as indicated by flow cytometry, selective enrichment studies, and Western analysis. Overall, this work demonstrates that fluorescent-puromycin conjugates offer a general means to examine protein expression in vivo.
Topics: Blotting, Western; Cell Line; Flow Cytometry; Fluorescent Dyes; Humans; Proteins; Puromycin
PubMed: 15271358
DOI: 10.1016/j.chembiol.2004.05.011 -
FEBS Letters Jul 1988The toxicity of the protein synthesis inhibitor puromycin towards a number of human and Chinese hamster cell lines has been examined. In comparison to cells of human... (Comparative Study)
Comparative Study
The toxicity of the protein synthesis inhibitor puromycin towards a number of human and Chinese hamster cell lines has been examined. In comparison to cells of human origin, Chinese hamster cells exhibited about 25-fold higher resistance towards puromycin. These differences appeared to be species related as all the cell lines from any one species showed similar sensitivity towards puromycin. The incorporation of [3H]leucine in the hamster cell lines was accordingly found to be more resistant to the inhibitory effects of puromycin as compared to human cells. Studies on the cellular uptake of [3H]puromycin showed that in comparison to human cells, the drug uptake/binding in the hamster cell lines was greatly reduced. However, protein synthesis in the extracts of hamster and human cells showed no significant differences in sensitivity towards puromycin. These results show that the observed species related differences in cellular toxicity to puromycin are due to differences in the cellular uptake/binding of the drug.
Topics: Animals; Cell Line; Cell Survival; Cricetinae; Drug Resistance; Female; HeLa Cells; Humans; Leucine; Ovary; Protein Biosynthesis; Puromycin; Species Specificity; Tumor Cells, Cultured
PubMed: 3391265
DOI: 10.1016/0014-5793(88)81320-6 -
Cell Biology International Reports Jun 1986A brief exposure of quiescent (Go) Swiss 3T3 mouse fibroblasts to inhibitors of protein synthesis can replace platelet-derived growth factor in the stimulation of... (Comparative Study)
Comparative Study
A brief exposure of quiescent (Go) Swiss 3T3 mouse fibroblasts to inhibitors of protein synthesis can replace platelet-derived growth factor in the stimulation of cellular DNA synthesis. When 3T3 cells, after a 6 hr exposure to either cycloheximide or puromycin, are incubated with platelet-poor plasma, a significant percentage of cells enters DNA synthesis. Either inhibition of protein synthesis, or platelet poor plasma by themselves are totally ineffective. A possible mechanism by which inhibitors of protein synthesis may initiate cell cycle progression is through the activation of the c-myc gene.
Topics: Animals; Cells, Cultured; Culture Media; Cycloheximide; DNA Replication; Kinetics; Mice; Platelet-Derived Growth Factor; Puromycin
PubMed: 3742620
DOI: 10.1016/0309-1651(86)90041-x -
The Journal of Biological Chemistry Jan 1969
Topics: Carbon Isotopes; Cell-Free System; Centrifugation, Density Gradient; Chemical Precipitation; Chromatography, Paper; Chromatography, Thin Layer; Electrophoresis; Hydrogen-Ion Concentration; Methionine; Methylation; Methyltyrosines; Puromycin; Spectrophotometry; Streptomyces; Time Factors; Transferases
PubMed: 5773275
DOI: No ID Found -
Journal of Molecular Biology Nov 1965
Topics: In Vitro Techniques; L Cells; Nucleosides; Puromycin; RNA; Uridine
PubMed: 5883921
DOI: 10.1016/s0022-2836(65)80229-7 -
Journal of Biochemistry Nov 1982An antibody specific for puromycin (PU) was prepared by immunization of rabbits with a PU conjugate of bovine serum albumin, which was newly synthesized by coupling PU...
An antibody specific for puromycin (PU) was prepared by immunization of rabbits with a PU conjugate of bovine serum albumin, which was newly synthesized by coupling PU to mercaptosuccinylated bovine serum albumin via a cross-linker, N-(gamma-maleimidobutyryloxy)succinimide. Enzyme labeling of PU was performed using beta-D-galactosidase [EC 3.2.1.23] via N-(m-maleimidobenzoyloxy) succinimide. An ultrasensitive and specific enzyme immunoassay for PU was developed utilizing these reagents by a double antibody technique. The standard curve of the assay was linear in the range of 2 pg to 100 pg, and the lower limit of detection was 28.2 pm (2 pg/tube); so the enzyme immunoassay was found to be approximately 326,000 times more sensitive than a microbiological assay. Further, the enzyme immunoassay is free from interference by any purine or pyrimidine analogs, or by other drugs commonly used for the inhibition of protein synthesis. Using this assay, drug levels were easily determined in rat tissue following PU administration. Since PU is extensively available as an inhibitor of protein synthesis, the enzyme immunoassay should provide useful tool for developing biochemical and toxicity studies of PU.
Topics: Animals; Antibodies; Immunoenzyme Techniques; Kinetics; Puromycin; Rabbits; Rats; Rats, Inbred Strains; Tissue Distribution; beta-Galactosidase
PubMed: 6818227
DOI: 10.1093/oxfordjournals.jbchem.a134085 -
Journal of Medicinal Chemistry Jul 1970
Topics: Methods; Puromycin
PubMed: 5452454
DOI: 10.1021/jm00298a058 -
Pulsating electromagnetic field stimulation prevents cell death of puromycin treated U937 cell line.Journal of Physiology and Pharmacology... Apr 2010Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities...
Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities starting from 1 x 10(6) cells/ml to 0.0625 x 10(6) cells/ml, were exposed to a pulsating magnetic field 50 Hz, 45+/-5 mT three times for 3 h per each stimulation with 24 h intervals. Proliferation has been studied by counting number of cells stimulated and non-stimulated by PEMF during four days of cultivation. Viability of cells was analyzed by APC labeled Annexin V and 7-AAD (7-amino-actinomycin D) dye binding and flow cytometry. Growing densities of cells increase cell death in cultures of U937 cells. PEMF exposition decreased amount of cells only in higher densities. Measurement of Annexin V binding and 7-AAD dye incorporation has shown that density-induced cell death corresponds with decrease of proliferation activity. PEMF potentiated density-induced death both apoptosis and necrosis. The strongest influence of PEMF has been found for 1 x 10(6)cells/ml and 0.5 x 10(6) cells/ml density. To eliminate density effect on cell death, for further studies density 0.25 x 10(6) cells/ml was chosen. Puromycin, a telomerase inhibitor, was used as a cell death inducer at concentration 100 microg/ml. Combined interaction of three doses of puromycin and three fold PEMF interaction resulted in a reduced of apoptosis by 24,7% and necrosis by 13%. PEMF protects U937 cells against puromycin- induced cell death. PEMF effects on the human lymphoid cell line depends upon cell density. Increased density induced cells death and on the other hand prevented cells death induced by puromycin.
Topics: Antimetabolites, Antineoplastic; Apoptosis; Cell Proliferation; Cell Survival; Electromagnetic Fields; Humans; Lymphoma, Large B-Cell, Diffuse; Necrosis; Puromycin; U937 Cells
PubMed: 20436221
DOI: No ID Found -
The Journal of Organic Chemistry Apr 2011We are reporting on the utility of commercial vinyl isocyanate for a practical synthetic route from adenosine to N(6)-bis-demethylpuromycin in seven steps and 65%...
We are reporting on the utility of commercial vinyl isocyanate for a practical synthetic route from adenosine to N(6)-bis-demethylpuromycin in seven steps and 65% overall yield. A clean one-pot conversion of 3'-bromo-2'-carbamoyl derivative 8 to 3'-amino-3'-deoxyadenosine derivative 10 is the main feature of this synthetic pathway. This synthesis is the shortest synthetic route toward 3'-(aminoacylamido)deoxyadenosines to date.
Topics: Deoxyadenosines; Indicators and Reagents; Magnetic Resonance Spectroscopy; Molecular Structure; Puromycin; Stereoisomerism; Structure-Activity Relationship
PubMed: 21361316
DOI: 10.1021/jo102178h -
Biochemical Pharmacology Apr 1970
Topics: Adenine Nucleotides; Amines; Animals; Chromatography, Ion Exchange; Chromatography, Paper; Deoxyadenosines; Deoxyribonucleosides; Deoxyribonucleotides; Glucosyltransferases; Glycogen; Hydrolysis; Kidney; Liver; Liver Glycogen; Male; Nucleosides; Nucleotidases; Nucleotides; Purines; Puromycin; Rats; Spectrophotometry; Time Factors; Ultraviolet Rays; Venoms
PubMed: 5315021
DOI: 10.1016/0006-2952(70)90065-1