Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Most approaches to studying messenger RNA (mRNA) decay in vivo lack sufficient sensitivity to identify decay intermediates. The identification of such intermediates using in vitro decay systems can provide suggestive evidence for endonuclease-mediated degradation in vivo; to validate conclusions drawn from in vitro experiments one must demonstrate cleavage of the mRNA in vivo. Primer extension or S1 nuclease protection assays work best on relatively abundant mRNAs and even then require long exposure times. We describe a facile approach using ligation-mediated polymerase chain reaction to identify in vivo mRNA decay intermediates. In this procedure, total cellular RNA is ligated to a primer bearing a 5' phosphate and 3' amino group. Reverse transcription is primed using a complementary primer, and mRNA-specific decay intermediates are identified by polymerase chain reaction amplification using a 5'- [32P]-labeled gene-specific primer. Products generated in this manner are gel purified, reamplified, and the 3' end of each decay intermediate is identified by the sequence junction of the specific mRNA and the initial ligation primer. We show an example of the time-course of appearance of several specific decay intermediates of c-myc mRNA in differentiating murine erythroleukemia cells.
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PMID:Application of ligation-mediated reverse transcription polymerase chain reaction to the identification of in vivo endonuclease-generated messenger RNA decay intermediates. 1477 8

Sanguinarine, a benzophenanthridine alkaloid, has anticancer potential through induction of cell death. We previously demonstrated that sanguinarine treatment at a low concentration (1.5 microg/ml) induced apoptosis in K562 human erythroleukemia cells, and a high concentration (12.5 microg/ml) induced the morphology of blister formation or oncosis-blister cell death (BCD). Treatment of cells at an intermediate sanguinarine concentration (6.25 microg/ml) induced diffuse swelling or oncosis-diffuse cell swelling (DCS). To assess the underlying mechanism of sanguinarine-induced apoptosis and oncosis-BCD in K562 cells, we studied their response to pre-treatment with two chemical compounds: aurintricarboxylic acid (ATA) and cycloheximide (CHX). The pretreatment effects of both chemical compounds on apoptosis and oncosis-BCD were evaluated by measuring multiple parameters using quantitative morphology, electron microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling and annexin-V-binding. ATA, a DNA endonuclease inhibitor, efficiently prevented DNA nicking and inhibited apoptosis almost completely and oncosis-BCD by about 40%, while CHX, a protein synthesis inhibitor, failed to inhibit both apoptosis and oncosis-BCD. These results demonstrate, first, the importance of endonuclease in sanguinarine-induced apoptosis and to some extent in oncosis-BCD and, second, that this inhibition does not require de novo protein synthesis.
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PMID:Aurintricarboxylic acid inhibits protein synthesis independent, sanguinarine-induced apoptosis and oncosis. 1736 25


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