NUTRIENT REGULATION OF SUCRASE A-DEXTRINASE MATURATION

蔗糖酶 A-糊精酶成熟的营养调节

• 批准号: 2200401
• 负责人: RICARDO O CASTILLO
• 金额: $ 17.89万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2200401
• 关键词: O glycosidase; dietary carbohydrates; enzyme activity; enzyme induction /repression; gastrointestinal system; gene expression; growth /development; laboratory rat; nucleic acid probes; nutrition related tag; perfusion; tissue /cell culture

DESCRIPTION (adapted from investigator's abstract and/or aims): The gastrointestinal tract of the rat undergoes dramatic maturational changes during the third week of life resulting in major changes in cellular kinetics and enzyme activities. Sucrase-alpha-dextrinase, a glycoprotein hydrolase required for final absorption of dietary carbohydrate and located at the brush border membrane surface of the enterocyte, is absent prior to the third week of life when it abruptly appears and rises rapidly, as a major distinguishing feature of postnatal intestinal maturation. Although the mechanisms regulating S-D maturation are not known, there is clear evidence that luminal nutrients are required for full maturational expression of S-D. A unique animal model developed by the applicant which is now well established will be used in combination with advanced techniques of cellular and molecular biology in order to investigate the mechanisms of nutrient-dependent S-D maturation. Initial studies will determine the effect of luminal carbohydrate on the in-vivo synthesis, assembly and degradation of S-D in the maturing intestine using techniques of intraluminal radiolabeling of the nascent S-D peptide and investigating the enzyme at various stages of its synthesis, assembly and final insertion into the brush border. These studies will determine whether the kinetics of intracellular transport, the structure of S-D precursors or the degradation of S-D are altered by luminal carbohydrates. Subsequent studies will determine whether luminal carbohydrate regulates S-D maturation by transcription or translation by measuring specific mRNA for S-D using cDNA probes developed by the applicant using polymerase chain reaction (PCR) techniques and analyzing S-D structure, intracellular assembly and post-insertional processing using glycosidase probes, 2-dimensional gel electrophoresis and amino acid sequencing of the S-D protein. In order to determine whether luminal nutrients act directly on the enterocyte, the S-D response to luminal nutrients of the intestinal absorptive cells along the crypt-villus axis and in organ culture will be studied. These studies performed during steady-state conditions will be complemented by analogous studies of acute induction of S-D using intestinal perfusion techniques developed by the applicant for use in infant animals in order to determine whether a single mechanism or combinations of mechanisms participate in the S-D response. These studies will generate new information on the mechanisms by which intraluminal nutrients regulate S-D maturation during postnatal intestinal development.

描述（改编自研究者的摘要和/或目标）： 大鼠胃肠道发生显着的成熟变化 在生命的第三周，导致细胞发生重大变化 动力学和酶活性。 蔗糖酶-α-糊精酶，一种糖蛋白 最终吸收膳食碳水化合物所需的水解酶，位于 在肠上皮细胞的刷状缘膜表面，之前不存在 生命的第三周，它突然出现并迅速上升，作为 出生后肠道成熟的主要特征。 虽然 调节 S-D 成熟的机制尚不清楚，但有明确的 有证据表明，管腔营养物质是完全成熟所必需的 S-D的表达。 申请人开发的独特动物模型 现已成熟，将与先进的结合使用 细胞和分子生物学技术，以研究 营养依赖性 S-D 成熟机制。 初步研究将 确定腔内碳水化合物对体内合成的影响， 使用技术在成熟肠道中组装和降解 S-D 新生 S-D 肽的管腔内放射性标记的研究 处于合成、组装和最终插入各个阶段的酶 进入画笔边界。 这些研究将确定动力学是否 细胞内运输、S-D 前体的结构或 S-D 的降解被腔内碳水化合物改变。 随后的 研究将确定腔内碳水化合物是否调节 S-D 通过测量特定 mRNA 进行转录或翻译来成熟 S-D使用申请人使用聚合酶链开发的cDNA探针 反应 (PCR) 技术和分析 S-D 结构、细胞内 使用糖苷酶探针进行组装和插入后处理， S-D 的二维凝胶电泳和氨基酸测序 蛋白质。 为了确定管腔营养物质是否直接作用于 肠上皮细胞，对肠腔营养物质的 S-D 反应 沿隐窝绒毛轴和器官培养的吸收细胞将 研究过。 这些在稳态条件下进行的研究将 补充了使用 S-D 急性诱导的类似研究 申请人开发的肠道灌注技术用于 婴儿动物以确定是否是单一机制或 参与 S-D 响应的机制组合。 这些研究 将产生关于腔内的机制的新信息 营养物质在产后肠道发育过程中调节 S-D 成熟。