ENVIRONMENTAL LIGHT AND RETINAL MEMBRANE DEVELOPMENT

环境光与视网膜膜发育

• 批准号: 6178544
• 负责人: DANIEL T ORGANISCIAK
• 金额: $ 29.26万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6178544
• 关键词: DNA damage; SDS polyacrylamide gel electrophoresis; aging; albino rat; apoptosis; circadian rhythms; cysteine endopeptidases; cytochrome c; electron microscopy; enzyme activity; gene induction /repression; gene mutation; genetic susceptibility; immunocytochemistry; laboratory rat; light adverse effect; light microscopy; membrane biogenesis; northern blottings; oxidative stress; retina degeneration; retinal pigment epithelium; rhodopsin; thiourea; visual photoreceptor; western blottings

DESCRIPTION (Adapted from applicant's abstract): This study seeks to determine the mechanisms of intense light-mediated photoreceptor cell degeneration. The hypotheses to be tested are: (1) that there is a sensitive period within the circadian cycle which results in enhanced susceptibility to retinal light damage and which is mediated by the expression of intrinsic factors in the retina (aim 1) and (2) that susceptibility to light damage is affected by genetic inheritance, which can enhance the rate of photoreceptor cell loss from environmental insult in the form of dim cyclic light exposure or acute intense light treatment (aim 2). This proposal will focus on identifying the circadian expression of intrinsic factors in the retina that enhance or prevent light-induced cell damage leading to apoptotic cell death. The investigator will study the interactions of light-and dark rearing conditions and the timing of intense light treatments in vivo that produce synchronous photoreceptor cell damage in normal albino rats and in transgenic rats with mutations in the rhodopsin primary amino acid sequence. Photoreceptor cell loss will be assayed using rhodopsin and photoreceptor DNA measurements, electrophoretic analysis of DNA fragmentation, and light and electron microscopy of light-damaged retinas. The circadian expression of melatonin and dopamine will be measured and Western and Northern analysis will be used to identify potential modulators of retinal damage. These measurements will be complimented by 2D gel electrophoresis of retinal proteins and ordered differential display of mRNA from retinas at different times of the day and night. The synthetic antioxidant dimethylthiourea will be used as a probe to uncover early events in the mechanism of cell death, including mitochondrial involvement and the potential for oxidative stress to induce apoptosis. In addition, the time course of the appearance of cytoplasmic cytochrome C and the activation of cellular caspases will be determined by antibody-based techniques, immunocytochemistry and enzymatic analysis.

描述（改编自申请人摘要）：本研究旨在 确定强光介导的感光细胞的机制 退化要检验的假设是：（1）有一个 昼夜节律周期内的敏感期，导致增强 对视网膜光损伤的敏感性，并由 视网膜中内在因子的表达（aim 1）和（2）， 对光损伤的敏感性受遗传影响， 可以提高感光细胞从环境中的损失率， 以昏暗的周期性光照或急性强光照射形式的损伤 治疗（目标2）。这项提案将集中在确定昼夜节律 增强或阻止视网膜中的内在因子的表达 光诱导的细胞损伤导致细胞凋亡。的 研究人员将研究光饲养和暗饲养的相互作用 条件和时间的强光治疗在体内， 在正常白化病大鼠中产生同步感光细胞损伤， 在视紫红质一级氨基酸突变的转基因大鼠中， 顺序感光细胞损失将使用视紫红质测定， 光感受器DNA测量，DNA电泳分析 碎片，光损伤的光学和电子显微镜 视网膜褪黑激素和多巴胺的昼夜节律表达将是 将使用西方和北方分析来确定 视网膜损伤的潜在调节剂。这些测量将是 补充视网膜蛋白质的2D凝胶电泳和有序 一天中不同时间视网膜mRNA差异显示 和夜晚。合成的抗氧化剂二甲基硫脲将被用作 一种揭示细胞死亡机制早期事件的探针， 包括线粒体的参与和潜在的氧化 应激诱导细胞凋亡。此外， 细胞质细胞色素C的出现和细胞内 半胱天冬酶将通过基于抗体的技术来测定， 免疫细胞化学和酶分析。