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Oxidation of PUFA initiates a chain reaction producing an abundance of ROS, including lipid aldehyde radicals. In addition, the process of RPE phagocytosis is itself an oxidative stress and results in the generation of endogenous ROS. Furthermore, RPE cells contain an abundance of photosensitizers, and exposure to intense visible lights induces generation of ROS. To cope with these toxic oxygen intermediates, the RPE has evolved effective defenses against oxidative damage.However, with increasing age, the RPE antioxidative capability appears to be reduced.For example, catalase activity and hemeoxygenase level decrease in aging RPE cells. Thus, it is likely that aging RPE cells may be more susceptible to oxidative damage.One disadvantage regarding these techniques is the requirement for large quantities of DNA, which can be limiting when specic tissue or primary cell cultures are used.The techniques also require isolation of the mitochondria before DNA pur ication, wh ich prov ides additional opportunity for oxidation to occur during sample preparation.To this end, a novel genespecic quantitative PCR assay has been developed to measure oxidative DNA damage and repair. Both inactively and actively transcribed genes were chosen for detection of nuclear DNA damage.A kb fragment from the mitochondrial genome was amplied for detection of mtDNA damage.Oxidative DNA damage includes single and doublestrand breaks, abasic sites, and base damages. The damage measured by the QPCR assay inc ludes bo thstrandbreaks and base modication.This genespecic assay technique works on the premise that a lesion on the DNA template will block a thermostable polymerase and result in a decreased amplication of the target sequence. Therefore, only those DNA templates that do not contain polymerase blocking lesions will be amplied.DNA lesions, such as strand break, base modication and apurinic site, are all capable of blocking the progression of the polymerase.Thus, quantication of DNA lesions per genomic fragment is inversely proportional to the resultant PCR product.The current detection limits are lesions nucleotides with ng mammalian DNA. Because differences in QPCR amplication may at times be related to template copy number differences or simply due to the DNA quality unrelated to in vivo or in vitro mediated damage, a quantitative amplication of a small region is performed as a quality control.Small target regions in the DNA are unlikely to suffer any insult, and, thus, can serve as indicators of relative copy number and PCR quality of the genomic extract.Transformed RPE cells overcome these problems while maintaining morphological and functional characteristics of primary cells.Additional studies indicate that these cells express and respond to oxidative stress in a fashion similar to that primary cultured human RPE cells. Several approaches have been used to induce oxidative stress of RPE cells, including exposure to chemical Targetmol’s Xylitol oxidants, hyperoxia, feeding with rod outer segments or lipofuscin, and photoillumination.We chose to use the SV transformed fetal human RPE cells to study oxidative stress.RPE cells were exposed to various concentrations, and mt and nDNA damages were assessed by QPCR.We found mtDNA was signicantly damaged by all concentrations of HO. The time course analysis showed that signicant damage to mtDNA was detectable within min of exposure.

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