Clarification of mechanism of cell degeneration using Drosophila and mouse mutant

1. 为了检验果蝇rdgA （retinal degeneration A）基因的产物是否为二酰基甘油（diacylglycerol， DG）激酶，我采用了生化方法，尝试纯化果蝇的DG激酶。从正常蝇头中提取DG激酶，用序列柱层析法纯化。由于该酶在果蝇头部中含量少，纯化过程中产量低，因此纯化并不完全，但一个115 kd的蛋白与该酶的活性相关。虽然用双向电泳分离得到了一个115 kd的蛋白，并从凝胶中提取，但该蛋白的数量不足以确定其氨基酸序列。用免疫荧光染色法检测小鼠和大鼠小脑变性后肌醇磷脂代谢。浦肯野细胞变性（Purkinje cell degeneration, pcd）突变小鼠小脑颗粒细胞中检测到磷脂酰肌醇二磷酸（PIP_2），但未检测到肌醇三磷酸结合蛋白和I型蛋白激酶C （PKC）。药物作用下大鼠小脑PIP_2的染色与正常大鼠相似。将磷脂酰肌醇（PI）脂质体引入培养基后，未表达c-myc的人肾癌细胞的活力降低，而表达c-myc的细胞的活力则没有降低。PI脂质体导致c-myc未表达细胞死亡的原因是细胞内Ca^2的异常积累。PI脂质体处理导致所有细胞PKC活性降低，并且在c-myc未表达的细胞中颗粒部分活性显著降低。

1. To check whether the product of rdgA (retinal degeneration A) gene of Drosophila is diacylglycerol (DG) kinase, I have adopted a biochemical approach by attempting to purify the DG kinase of Drosophila. DG kinase was extracted from normal fly heads, and purified by sequential column chromatography. The purification achieved was not complete because of the small amount of the enzyme in Drosophila heads and its low yield through the purification procedures, but a 115 kd protein correlated with the enzyme activity. Although a 115 kd protein was separated with two dimensional electrophoresis and extracted from the gel, amount of the protein was not sufficient to determine its amino acid sequences.2. Inositol phospholipid metabolism in cerebellum degenerated mouse and rat was examined by imunofluorescent staining. Phosphatidylinositol bisphosphate (PIP_2) was detecteted in granule cells of pcd (Purkinje cell degeneration) mutant mouse cerebellum but inositol trisphosphate binding protein and type I protein kinase C (PKC) were not. The staining of PIP_2 in cerebellum degenerated rat by drugs was similar to that in normal.3. When phosphatidylinositol (PI) liposomes were introduced into culture media, viability of c-myc unexpressed human renal cancer cells were reduced, while that of c-myc expressed cells was not. Death of c-myc unexpressed ells by PI liposomes was found to be caused by abnormally accumulated intracellular Ca^2. PI liposome treatment caused decrease of PKC activity in all cells examined, and reduction of activity of particulate fraction was significant in c-myc unexpressed cells.