On the Molecular Architecture of the Epitherial Desmosom and Its an Etiological Role

上皮桥粒的分子结构及其病因学作用

基本信息

批准号：
02670474
负责人：
SHIDA Hisato
金额：
$ 1.34万
依托单位：
Yamanashi Medical College (Yamanashi Ikadaigaku)
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

For the purpose of investigating the molecular organization of the desmosome, quantitative analysis of the immunoelecton microscopy (IEM) using immuno-gold method with anti-D mono/polyclonal antibodies (Abs) was carried out. According to the analysis, such useful tool as position of peaks, shape of curves obtained from length/frequency curves which were specific to the DG and DP molecules are recognized in the first year. However, theoretical meanings were remained to be uncertain. From the second year, computer aided distributions were investigated as the simulated models of immunoelectron microscopy. By applying fitting analysis to the histogram, a combined curve of two normal distribution curves with the different value of half width and the same value of peak position were appeared as a least square curve and from this combined curve, the resolution was estimated as 60 A. Basing on the theoretical analysis and the quantitative immunoelectron microscopy of desmosome, a three dimensional model of cytoskeletal-D proteins complex at the desmosome was proposed.A two dimensional architecture of D proteins and E-cadherin (ECAD) was also analyzed by IEM using isolated D pre-treated with 6 M guanidine HCl (G-HCl). At the proper region of D, density of DG1, DP1/2, DG2/3 was decreased in this order forming a random distribution. The considerable amount of DP3 was extracted by the G-HCl treatment. Only a few amount of E-CAD was detected at the peripheral region of D. The difference of the localization pattern between the D molecules and E-CAD was also confirmed in cultured keratinocytes. Though the break-down of cell-to-cell adhesion of the epitherial cells was observed under the presence of anti-DG1 and/or DG2/3 Abs for 27 hs incubation, non of the effect of pepstatin was observed.

为了研究桥粒的分子组织，使用免疫金法和抗D单/多克隆抗体（Abs）进行免疫电子显微镜（IEM）定量分析。根据分析，第一年就认识到了诸如峰位置、从长度/频率曲线获得的曲线形状等有用的工具，这些工具是 DG 和 DP 分子特有的。然而，理论意义仍不确定。从第二年开始，计算机辅助分布作为免疫电子显微镜的模拟模型进行了研究。通过对直方图进行拟合分析，出现半宽度值不同且峰位置值相同的两条正态分布曲线的组合曲线，作为最小二乘曲线，并且根据该组合曲线，估计分辨率为 60 A基于桥粒的理论分析和定量免疫电镜，提出了桥粒处细胞骨架-D蛋白复合物的三维模型。还分析了D蛋白和E-钙粘蛋白（ECAD）的二维结构。通过 IEM 使用经 6 M HCl 胍 (G-HCl) 预处理的分离 D 进行检测。在D适当区域，DG1、DP1/2、DG2/3的密度依次减小，形成随机分布。通过 G-HCl 处理提取了大量的 DP3。在D的周边区域仅检测到少量的E-CAD。在培养的角质形成细胞中也证实了D分子和E-CAD之间的定位模式的差异。尽管在抗DG1和/或DG2/3抗体存在下孵育27小时观察到上皮细胞的细胞间粘附的破坏，但未观察到胃酶抑素的作用。