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  Data had been collected utilizing the Bio Rad CFX manager program

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jx123
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Počet príspevkov : 155
Registration date : 01.12.2014

 Data had been collected utilizing the Bio Rad CFX manager program  Empty
OdoslaťPredmet: Data had been collected utilizing the Bio Rad CFX manager program     Data had been collected utilizing the Bio Rad CFX manager program  Icon_minitimeUt máj 26, 2015 6:52 am

Both the etoposide and CH11 apoptotic agents induced apoptosis in WT Jurkat and J Lat cells, confirmed by the detection of cleaved Poly polymerase 1. a downstream target of Caspase 3, on immunoblots. However, all of the J Lat clones had reduced amounts of cleaved PARP 1 compared Amuvatinib 溶解度 to WT Jurkat cells, indicating a certain degree of resistance to apoptosis. Further more, J Lat clones were more resistant to apoptosis induced by DNA breaks than by CD95 Fas activation as we observed almost complete cleavage of PARP 1 in J Lat clones 6. 3, 8. 4 and 10. 6 treated with CH11. The resistance of J Lat cells to apoptosis was also ana lyzed using a Caspase 3 7 assay. After treating with etoposide, J Lat cell clones 6. 3 and 8. 4, but not 10. 6, had reduced caspase activity compared to treated WT Jurkat.<br><br> However, the J Lat cell lines, except for J Lat clone 8. 4, were more susceptible than WT Jurkat cells to apoptosis when it was induced by the anti fas CH11 antibody. This finding is consistent with the reduced amount of cleaved PARP 1 shown in Figure 7B for the same clones. AT-406 datasheet Our results indicate that HIV 1 infected cells are more resistant to apoptosis when it is induced by the DNA damaging agent etoposide. These data suggest that when an HIV 1 genome is integrated into the host DNA, sev eral host genes including Caspase 8, Ikaros, Aiolos and NPM B23 genes are regulated by the HIV 1 TAR miRNAs. The downregulation Caspase 8, Ikaros and NPM B23 of, and the overexpression of Aiolos, may play important roles in the cell fate after HIV 1 infection.<br><br> Discussion HIV 1 regulates the expression of many host cell genes during infection of human cells. Dysregulation of host genes occurs when viral AG-490 溶解度 molecules interact with cell components, disrupt normal cellular pathways, recruit host factors for viral replication or change the endogen ous miRNA profile of the cell. Here, we report that HIV 1 TAR miRNAs could be another mechanism by which HIV 1 regulates the expression of host genes. We demonstrated that miR TAR 5p and miR TAR 3p were incorporated into Ago complexes, which contains the effector components of miRNA targeted RNA silen cing in human cells and is required for small RNA species to mediate mRNA regulatory effects. We established stable Jurkat cell lines expressing the HIV 1 TAR miRNAs and we selected four lines that showed variations in TAR miRNA expression levels.<br><br> Based on the premises that miRNAs regulate mRNAs in a dose dependent manner, and that increased miRNA levels would yield a stronger phenotype, the Jurkat TAR 3 cell line was selected for further proteomic analyses and NPM B23 emerged as a top protein candi date downregulated by the HIV TAR miRNAs. The 2D gel analysis of a clonal population circumvented a relatively high background associated to poorly infected cells, as previously described, and allowed us to study a stable latently infected cell population, in con trast with other early infected cell studies. This approach, however, also has its intrinsic limitations, the most important of which may be the poor separation of proteins from some areas of the gel, and a fold change too small to justify mass spectrometry analyses.
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