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 而角膜上皮细胞的分化在发育过程中非常重要 更有效地治疗因角膜损伤或其他原因引起的缺陷 疾病。正在进行的研究代表着第一次深入努力 哺乳动物角膜中转录核酶的特征 并确定它们的活动机制 受监管的。我们之前的研究已经提供了周期性的证据 核苷酸介导的M胆碱能调节RNA聚合酶 兔角膜上皮细胞的活性。《公约》的具体目标 拟开展的研究工作如下：(1)为鉴定RNA聚合酶I， 兔角膜上皮细胞的II和III(包括电泳法和 进一步的色谱学生化/动力学表征 纯化的酶)。(2)确定监管机制， 胆碱能激动剂氨甲胆碱增强RNA聚合酶活性 在兔角膜上皮细胞中。需要检验的假设是关于 介导观察到的RNA聚合酶活性增强的机制 氨基甲酰胆碱包括该药物的以下潜在作用，每种 其中一个或多个与我们的动力学数据暂时一致 RNA聚合酶在分离、纯化粗酶研究中的应用 核：(A)对RNA翻译后磷酸化的影响 环核苷酸依赖(cGMP依赖)蛋白的聚合酶 (B)对其他蛋白的磷酸化的影响 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 对RNA聚合酶合成的影响；(D)对 动力学和电泳学上不同的RNA聚合酶同工酶 不同的底物亲和力。实验方法包括 磷酸化与核合成的电泳法分析 药物治疗和对照细胞中的蛋白质，与 核糖核酸的层析纯化及电泳学性质研究 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 胆碱能药物影响基因表达的机制 角膜上皮细胞生长和发育过程中转录水平的变化 区分和(2)确定胆碱能药物的适宜性， 单独或与其他药物合用，用于各种类型的管理 眼表的损伤和疾病。

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.