PROTEIN AND LIPID PHOSPHORYLATION AND THE CORNEA

这一建议是我们正在进行的脂质和 角膜中的蛋白磷酸化，关注细胞信号的事件 在伤口闭合和上皮细胞增殖期间的转导。 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）是磷脂酰肌醇激酶的中心组分。 角膜上皮细胞的生长控制途径。 这些细胞信号 细胞周期中释放的生长因子会激活细胞内的信号通路， 损伤，它们的持续激活最终导致 合成损伤修复所必需的蛋白质。 两种伤口模型 将使用愈合：轻度碱烧伤模型和完全清创术 上皮细胞层 角膜中的主要PKC亚型是α PKC，其促进角膜上皮细胞的增殖。 立即早期基因的转录激活并导致基因 在细胞增殖和创伤中调节细胞周期的级联反应 治愈 我们还建议鉴定PKC亚型的调节剂 作为生长因子和伤口愈合对PIK的影响。 另外我们 旨在确定生长调节基因是如何转录 激活 与伤口闭合和细胞增殖相关 将建立。 强大的分析程序，如高性能液体 色谱法（HPLC）、毛细管气液色谱法（CGLC）和 将使用快速高效液相色谱法（FPLC）。 率 将测量伤口闭合和DNA中3H胸苷掺入 作为细胞增殖和伤口愈合的指示。 在Northern印迹后完成特定mRNA的定量 存储磷光体成像。 获得的结果将定义 PKC和PIK参与角膜伤口愈合。

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.