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Clarification of mechanism of cell degeneration using Drosophila and mouse mutant

利用果蝇和小鼠突变体阐明细胞变性机制

基本信息

批准号：
02680205
负责人：
INOUE Hiroko
金额：
$ 1.41万
依托单位：
Waseda University (1991)The University of Tokyo (1990)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

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.

1.为了验证果蝇rdgA（retinal degeneration A）基因的产物是否为甘油二酯激酶（diacylglycerol kinase，DG激酶），本研究采用生物化学方法，试图纯化果蝇DG激酶。从正常蝇头中提取DG激酶，并通过连续柱层析纯化。由于果蝇头部中的酶量少且通过纯化程序的产率低，因此所实现的纯化不完全，但与酶活性相关的115 kd蛋白质。虽然双向电泳分离并从凝胶中提取了一个115 kd的蛋白质，但该蛋白量不足以确定其氨基酸序列.用免疫荧光染色法观察了小脑变性小鼠和大鼠的肌醇磷脂代谢。在浦肯野细胞变性（Purkinje cell degeneration，pcd）小鼠小脑颗粒细胞中检测到磷脂酰肌醇二磷酸（Phosphatidylinositol bisphosphate，PIP_2），但未检测到肌醇三磷酸结合蛋白（inositol trisphosphate binding protein，I-PKC）。PIP_2在药物性小脑变性大鼠小脑中的表达与正常大鼠相似.当磷脂酰肌醇（PI）脂质体被引入到培养基中时，c-myc未表达的人肾癌细胞的存活率降低，而c-myc表达的细胞的存活率不降低。PI脂质体导致c-myc未表达细胞死亡的原因是细胞内钙的异常积累^[2]。PI脂质体处理导致PKC活性在所有检查的细胞中的降低，并且在c-myc未表达的细胞中颗粒部分的活性的降低是显著的。