IN VIVO REGULATION OF CORNEAL ENDOTHELIAL CELL FUNCTION

了解生长过程中调节RNA合成的分子机制 角膜上皮细胞的分化在发育中是重要的 更有效地治疗角膜损伤引起的缺陷，或 疾病 正在进行的研究是第一次深入的努力， 表征哺乳动物角膜中转录的核酶 上皮细胞，并确定其活性的机制， 监管. 我们以前的研究提供了证据， 核苷酸介导的毒蕈碱胆碱能调节RNA聚合酶 活性在兔角膜上皮细胞中。 该委员会的具体目标 提出的研究如下：（1）为了表征RNA聚合酶I， II和III的兔角膜上皮（包括电泳和 进一步的生化/动力学表征 纯化的酶）。 (2)确定监管机制， 胆碱能激动剂氨甲酰胆碱增强RNA聚合酶活性 在兔角膜上皮中。 有待检验的假设 观察到的RNA聚合酶活性增强的介导机制， 氨甲酰胆碱包括药物的以下潜在作用， 其中一个暂时与我们的动力学数据一致， RNA聚合酶在分离纯化的粗酶研究中的应用 细胞核：（a）对RNA翻译后磷酸化的影响 环核苷酸依赖性蛋白聚合酶 激酶（“靶向假设”）;（B）对其他激酶磷酸化的影响 染色质相关蛋白（组蛋白，HMG非组蛋白） 影响DNA模板对RNA聚合酶的可及性;（c）影响 （d）对RNA聚合酶相对比例的影响 动力学和代谢上不同的RNA聚合酶同工酶， 不同的底物亲和力。 实验方法包括 磷酸化和核蛋白合成的电泳分析 药物处理和对照细胞中的蛋白质， RNA的层析纯化和电泳表征 聚合酶I、II和III、组蛋白和HMG蛋白。 远程 本研究的目的包括（1）阐明分子调控 胆碱能药物可能影响基因表达的机制 角膜上皮细胞生长过程中的转录水平， 区分和（2）确定胆碱能剂的适用性， 单独使用或与其他药物联合使用，用于管理各种类型的 眼睛表面的损伤和疾病。

Understanding molecular mechanisms regulating RNA synthesis during growth and differentiation of corneal epithelial cells is important in developing more effective treatment of defects arising from corneal injury or disease. The research in progress represents the first in-depth effort to characterize nuclear enzymes of transcription in mammalian corneal epithelium and to determine mechanisms by which their activity is regulated. Our previous studies have provided evidence for cyclic nucleotide-mediated muscarinic cholinergic regulation of RNA polymerase activity in rabbit corneal epithelial cells. The specific aims of the proposed research are as follows: (1) To characterize RNA polymerases I, II and III of rabbit corneal epithelium (including electrophoretic and further biochemical/kinetic characterization of chromatographically purified enzymes). (2) To determine regulatory mechanisms by which the cholinergic agonist carbamylcholine enhances activity of RNA polymerases in rabbit corneal epithelium. Hypotheses to be tested concerning mechanisms mediating observed enhancement of RNA polymerase activity by carbamylcholine include the following potential effects of the drug, each of which is tentatively consistent with our kinetic data for one or more RNA polymerases in studies of crude enzymes from isolated, purified nuclei: (a) Effects on posttranslational phosphorylation of RNA polymerases by cyclic nucleotide-dependent (cGMP-dependent) protein kinases ("targeted hypothesis"); (b) Effects on phosphorylation of other chromatin-associated proteins (histones, HMG nonhistone proteins) influencing accessibility of DNA template to RNA polymerases; (c) Effects on RNA polymerase synthesis; (d) Effects on relative proportions of kinetically and electrophoretically distinct RNA polymerase isozymes with different substrate affinities. Experimental approaches include electrophoretic analysis of phosphorylation and synthesis of nuclear proteins in drug-treated and control cells, in conjunction with chromatographic purification and electrophoretic characterization of RNA polymerases I, II and III, histones and HMG proteins. Long-range objectives of this research include (1) elucidating molecular regulatory mechanisms by which cholinergic agents may influence gene expression at the transcriptional level during corneal epithelial cell growth and differentiation and (2) determining the suitability of cholinergic agents, alone or in combination with other drugs, for management of various types of injury and disease of the ocular surface.