GASTROINTESTINAL SIGNIFICANCE OF GASTRIN REGULATION

胃泌素调节的胃肠意义

• 批准号: 3153130
• 负责人: Tadataka Yamada
• 金额: $ 19.57万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3153130
• 关键词: Zollinger Ellison syndrome; amidation /deamidation; carboxyl group; cell transformation; clone cells; complementary DNA; fibroblasts; gastrins; genetic translation; hormone biosynthesis; hormone regulation /control mechanism; messenger RNA; methylprednisolone; orphan disease /drug; peptic ulcer; secretion; thymidine kinase; tissue /cell culture

Although the physiological importance of gastrin in regulating gastric acid secretion has been established, and thus the relationship between gastrin and the pathogenesis of peptic ulcer disease implied, little is known about the mechanisms by which the synthesis of this gastrointestinal hormone is regulated. Recent development of molecular biological and cell culture techniques has made it possible to examine the control of hormone synthesis directly. I will use these techniques to study the biosynthesis, post-translational processing and secretion of gastrin. To determine the structure of the human gastrin precursor, gastrin cDNA will be synthesized from human antral and Zollinger-Ellison tumor mRNA with reverse transcriptase using the gastrin-specific synthetic oligodeoxynucleotide primer, d(G-A-A-G-T-C-C-A-T-C-C-A). The cDNA will be sequenced and used as a probe for selection of full-length gastrin cDNA clones from a cDNA library constructed using antral and Zollinger-Ellison tumor mRNA. The biosynthesis of gastrin will be examined with a newly developed antibody (GL-9), which recognized gastrin precursor, and with antibody 5135, which is specific for the carboxyl terminus of gastrin. Three systems will be used for biosynthetic studies: 1) cell-free translation of mRNA with wheat germ extract, 2) primary cultures of isolated canine antral gastrin cells, and 3) gastrin secreting single cell clones formed by hybridization of Zollinger-Ellison tumor cells with thymidine kinase deficient mouse fibroblasts. The critical step in post-translational processing of gastrin precursor to its active form is the amidation of the carboxyl terminal Phe. The enzyme responsible for this amidation will be identified and purified using a synthetic probe (Tyr-Gly-Trp-Met-Asp-Phe-Gly-Arg-Arg), an antibody specific for this probe (GL-9), and an antibody (5135) specific for amidated gastrin. Intermediates formed during amidation will be identified by high pressure liquid chromatography. The pathophysiological importance of alterations in gastrin synthesis and processing will be examined in vivo in rats treated chronically with methyl prednisolone, or in vitro in chronically stimulated somatic cell hybrids. In the vivo studies, induced alterations in gastrin mRNA content, biosynthesis, processing, and release will be examined and related to the development of gastric ulcers.

虽然胃泌素在调节胃酸方面的生理重要性 分泌已经建立，因此胃泌素之间的关系 以及消化性溃疡病的发病机制， 这种胃肠激素的合成机制是 监管. 分子生物学与细胞培养研究进展 新技术使研究激素合成的控制成为可能 直接. 我将用这些技术来研究生物合成， 翻译后加工和分泌胃泌素。 确定 人胃泌素前体的结构，胃泌素cDNA将被合成 从人胃窦和Zollinger-Ellison肿瘤mRNA中， 使用胃泌素特异性合成寡脱氧核苷酸的转录酶 引物，d（G-A-A-G-T-C-C-A-T-C-C-A）。 将对cDNA进行测序并用作 用于从cDNA中选择全长胃泌素cDNA克隆的探针 使用胃窦和Zollinger-Ellison肿瘤mRNA构建文库。 的 胃泌素的生物合成将用一种新开发的抗体进行检测 （GL-9），其识别胃泌素前体，和抗体5135，其 对胃泌素的羧基端具有特异性。 三个系统将 用于生物合成研究：1）小麦mRNA的无细胞翻译 细菌提取物，2）分离的犬胃窦胃泌素细胞的原代培养物， 和3）通过杂交形成的分泌胃泌素的单细胞克隆， 胸苷激酶缺陷小鼠Zollinger-Ellison肿瘤细胞 成纤维细胞 胃泌素翻译后加工的关键步骤 其活性形式的前体是羧基末端的酰胺化 菲 负责这种酰胺化的酶将被鉴定， 使用合成探针（Tyr-Gly-Trp-Met-Asp-Phe-Gly-Arg-Arg）纯化， 对该探针特异的抗体（GL-9）和对该探针特异的抗体（5135 用于酰胺化胃泌素。 酰胺化过程中形成的中间体将被 通过高压液相色谱法鉴定。 的病理生理 胃泌素合成和加工的改变的重要性将是 在用甲基强的松龙长期治疗的大鼠中进行体内检查，或 在体外慢性刺激的体细胞杂种中。 在体内 研究，诱导胃泌素mRNA含量，生物合成， 处理和释放将被审查和相关的发展， 胃溃疡