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Biochemical Basis for the Histo-& Cytochemical Applications of Endogenous Lectins.

组织的生化基础

基本信息

批准号：
63870001
负责人：
HIRANO Hiroshi
金额：
$ 9.28万
依托单位：
Kyorin University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B).
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63870001/
关键词：
Endogenouse lecithin Histo- & cytochemistry Chick embryo Development and differentiation Gene cloning 遺伝子クローニング

项目摘要

1) Production of recombinant proteins of both human 14K and chick 16K lecithins in E. coli has been attained by recombinant DNA techniques. This made it possible to provide sufficient amount of proteins required for a variety of biochemical studies.2) Substitution experiments of amino acid residues in human 14K lectin by site-directed mutagenesis of its expression plasmid which enables to assess significance of residues was done. Some residues which had been considered essential for sugar-binding activity, such as tryptophan and several cysteins, proved to be unessential. On the other hand, essential arginine and histidine residues were identified.(3) Expression of two chick isolectins (14K and 16K) in various organs during development of embryo was studied at the levels of both transcription of their mRNAs and translation to proteins by using cDNA proves and specific antibodies. Though results were too complicated to give a simple explanation, a tendency that 16K lecithin plays roles … More at earlier stages while 14K lecithin at later stages.4) We succeeded in cloning of cDNA for human 29K beta-galactoside-binding lecithin which is a human high-molecular weight lecithin. Revealed complete primary structure indicated that the C-terminal half of 29K lecithin is homologous to 14K lectin while the N-terminal half has an unrelated structure. This structural studies made it possible to discuss molecular evolution of vertebrate beta-galactoside-binding lecithin family.5) Though venoms of some species of snake were known to contain beta-galactoside-binding lecithins, it had not been clarified whether these lecithins have any structural relationship with Ca-independent vertebrate lectins which we have been studying extensively. Primary structure of a lecithin purified from rattlesnake venom clearly showed that it belongs to another big family of animal lecithin, that is, Ca-dependent lecithin family.6) The 14K lecithin gene expression was visualized by HRP-labeling method using in situ hydridization techniques, in which sulfonated cDNA was employed as a probe. The 14K lecithin gene expression was detected mainly in the intermediate layer of the epidermis : faintly in 13-day-old embryo, graduallly increased in intensity during epidermal differentiation, and intensely positive in 17-day-old embryo. The expression of the 14K lecithin gene was supressed by vitamin A which induced the mucous metaplasia of the epidermis. Less

1)在E.大肠杆菌中获得了重组DNA。这使得有可能提供各种生化研究所需的足够量的蛋白质。2）通过定点诱变其表达质粒进行人14 K凝集素中氨基酸残基的取代实验，这使得能够评估残基的意义。一些被认为是糖结合活性所必需的残基，如色氨酸和几个半胱氨酸，被证明是非必需的。另一方面，鉴定了必需的精氨酸和组氨酸残基。(3)利用cDNA探针和特异性抗体，从mRNA转录水平和蛋白质翻译水平研究了14 K和16 K两种鸡异凝集素在胚胎发育过程中不同器官的表达。虽然结果过于复杂，无法给出简单的解释，但16 K卵磷脂起作用的趋势 ...更多信息 4）成功克隆了人高分子量卵磷脂29 K β-半乳糖苷结合卵磷脂的cDNA。揭示完整的一级结构表明，29 K卵磷脂的C-末端的一半是同源的14 K凝集素，而N-末端的一半有一个不相关的结构。5）虽然已知某些种类的蛇的毒液中含有β-半乳糖苷结合卵磷脂，但这些卵磷脂与我们一直在广泛研究的非钙依赖性脊椎动物凝集素之间是否存在结构上的联系尚不清楚。从响尾蛇毒液中纯化的卵磷脂的一级结构清楚地表明它属于动物卵磷脂的另一个大家族，即Ca依赖性卵磷脂家族。6）使用原位杂交技术通过HRP标记方法观察14 K卵磷脂基因表达，其中使用磺酸化cDNA作为探针。14 K卵磷脂基因主要在表皮中间层表达，13日龄胚表达微弱，表皮分化期表达逐渐增强，17日龄胚表达强阳性。维生素A可抑制14 K卵磷脂基因的表达，诱导表皮粘液化。少