PROTEIN AND LIPID PHOSPHORYLATION AND THE CORNEA

这项建议是我们正在进行的关于血脂和 角膜中的蛋白质磷酸化，关注细胞信号事件 伤口闭合过程中的信号转导和上皮细胞的增殖。我们 将检验以下假设：蛋白激酶C(PKC)亚种和 磷脂酰肌醇激酶(PIK)是 角膜上皮细胞的生长调控途径。这些细胞信号 通路被激活，以响应在过程中释放的生长因子 损伤，并且它们的持续激活最终导致 合成损伤修复所必需的蛋白质。两种创伤模型 治疗将使用：轻度碱烧伤模型和完全清创 上皮层。 角膜中一个主要的PKC亚种是αPKC，它促进 即刻早期基因的转录激活和导致基因 在细胞增殖和创伤中调节细胞周期的级联反应 治愈。我们还建议确定PKC亚种的调节子 以及生长因子和创面愈合对PIK的影响。另外，我们 意在定义生长调节基因是如何转录的 激活了。伤口闭合与细胞增殖的相关性 将会建立起来。 强大的分析程序，如高性能液体 毛细管气相色谱(CGLC)、高效液相色谱(HPLC)和 将使用快速高效液相色谱(FPLC)。利率 将测量伤口闭合和DNA中的~3H胸腺嘧啶核苷掺入 作为细胞增殖和伤口愈合的指标。 特定mRNAs的定量将在北向杂交后完成 通过存储磷光体成像。所获得的结果将定义 PKCs和PIKs在角膜创伤愈合中的作用

This proposal is a continuation of our ongoing studies of lipid and protein phosphorylation in cornea, focusing on the events of cell signal transduction during wound closure and epithelial cell proliferation. We will test the following hypothesis: Protein kinase C (PKC) subspecies and phosphatidylinositol kinases (PIK) are central components of the growth-control pathway in corneal epithelial cells. These cell-signaling pathways are activated in response to growth factors released during injury, and their sustained activation ultimately results in the synthesis of proteins necessary for injury repair. Two models of wound healing will be used: a mild alkali burn model and a complete debridement of the epithelial layer. A major PKC subspecies in the cornea is alphaPKC, which promotes the transcriptional activation of immediate early genes and leads to gene cascades that modulate the cell cycle in cellular proliferation and wound healing. We propose to identify the modulators of PKC subspecies as well as the effect of growth factors and wound healing on PIKs. Also, we intend to define how growth-regulated genes are transcriptionally activated. Correlation with wound closure and cellular proliferation will be established. Powerful analytical procedures such as high performance liquid chromatography (HPLC), capillary gas liquid chromatography (CGLC), and fast performance liquid chromatography, (FPLC), will be used. The rate of wound closure and 3H thymidine incorporation in DNA will be measured as an indication of cellular proliferation and wound healing. Quantification of specific mRNAs will be accomplished after norther blots with storage phosphor imaging. The results obtained will define the involvement of PKCs and PIKs in corneal wound healing.