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Connexin abnormality in perpharal hervons system dicease

外周 Hervons 系统疾病中的连接蛋白异常

基本信息

批准号：
08670713
负责人：
YOSHIMURA Takeo
金额：
$ 1.34万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670713/
关键词：
charcot-Marie-Tooth connexin gap Junction Charcot-Marie-Tooth病 connexin 遺伝性ニューロパチー connexin32 connexin43

项目摘要

Connexin32 (Cx32) is a gap junction protein and its gene mutations are responsible for X-linked Charcot-Marie-Tooth disease. We examined the functional abnormality of C6 glioma cells transfected with mutant (C53S and Pl72R) Cx32 genes. Nontransfected C6 glioma cells did not express Cx32 mRNA or protein. Northern and Western blot analyzes showed Cx32 mRNA and protein in cells transfected with the wild-type gene as well as with the mutant Cx32 genes. An immunocytochemical study of cells with the wild-type gene showed that Cx32 is present in the cell membrane and cytoplasm. In cells transfected with C53S or P172R mutant gene, however, no immunoreactivity was found in the cell membrane. The scrape-loading and dye transfer method produced effective dye transfer in cells with the wild-type gene but not in those with mutant genes. A cell proliferation assay showed no differences in nontransfected cells, cells transfected with the wild-type gene and those with the mutant genes. Messenger RNA expression for myelin proteolipid protein did not change in any of these cells. These findings suggest that Cx32 gene mutation results in loss of cell-to-cell communication because of failure to incorporate Cx32 protein in the cell membrane where gap junctions are formed. The mutations do not, however, interfere with cell proliferation or myelin-specific gene expression, at least in C6 glioma cells.Cx32 mRNA and protein expression was examined using Schwann cells cultured with dorsal root ganglion neurons. The apparance of Cx32 mRNA and protein was I week after the start of culture whereas those of myelin basic protein and Po protein were 2 days after the start. The function of Cx32 appears to maintain the myelin membrane rather than to form myelin.

缝隙连接蛋白32（Cx32）是一种缝隙连接蛋白，其基因突变与X连锁腓骨肌萎缩症有关。我们研究了C6胶质瘤细胞转染突变体（C53S和P172R）Cx32基因的功能异常。未转染的C6胶质瘤细胞不表达Cx32 mRNA或蛋白。北方和Western印迹分析显示，在转染野生型基因以及与突变型Cx 32基因的细胞中，Cx 32 mRNA和蛋白。野生型基因的细胞的免疫细胞化学研究表明，Cx32存在于细胞膜和细胞质中。而转染C53S或P172R突变基因的细胞膜上未见免疫反应性。刮片加载和染料转移方法在具有野生型基因的细胞中产生有效的染料转移，但在具有突变基因的细胞中不产生。细胞增殖试验显示，在未转染的细胞，细胞转染野生型基因和那些与突变基因没有差异。在这些细胞中，髓鞘蛋白脂质蛋白的信使RNA表达没有变化。这些研究结果表明，Cx32基因突变导致细胞间通讯的丧失，因为Cx32蛋白不能掺入细胞膜中，而细胞膜上形成了间隙连接。然而，突变并不干扰细胞增殖或髓鞘特异性基因表达，至少在C6胶质瘤细胞中是如此。使用与背根神经节神经元一起培养的雪旺细胞检测Cx32 mRNA和蛋白表达。Cx32 mRNA和蛋白在培养开始后1周出现，而髓鞘碱性蛋白和Po蛋白在培养开始后2天出现。Cx32的功能似乎是维持髓鞘膜而不是形成髓鞘。