REGULATION AND MECHANISM OF HORMONE ACTION

这项研究提案的广泛、长期目标是 对甲状腺激素作用机制和结构的认识 与激素相互作用所需的转运蛋白。这个 提出的实现这些目标的方法是研究自然发生的 人类的突变，即对甲状腺激素的普遍抵抗 (周长)和遗传性甲状腺激素结合球蛋白(TBG)缺陷。 最近开发的腰围临床诊断方法将是 适合在门诊环境中使用，并通过他们的 适用于非耐药受试者。流行病学研究将 包括通过筛查来确定腰围的流行率 新生儿和患有注意力缺陷/多动障碍的儿童，a 怀疑有高发病率的腰围的情况。这些数据将 提供腰围、纤维连接蛋白和胶原培养的组织诊断 合成及其信使核糖核酸积累。遗传连锁研究由 利用甲状腺激素受体的限制性片段多态性 探头，c-erb-aα和β将有助于定位周长和 为其异质性表型提供了解释。数据表单链接 分析将有助于确定适合于 扩增特定的甲状腺激素受体基因，以便 通过基因测序在分子水平上对缺陷进行表征。 到目前为止已确定的11个tbg突变中的其余7个将被鉴定 通过基因测序。他们的频率将在符合以下条件的受试者中确定 标准临床实验室已发现的TBG缺陷 意思是。突变的tbg分子的性质，通过直接或直接研究 物理化学法或基因转染法表达后 转化为异源细胞，将有助于推断分子需求 对其生物学功能的影响。关于监管的若干问题研究 TbG基因的表达将利用从结构中获得的信息 突变的TBG基因在正常的TBG中异常表达 分子，以及从嵌合基因表达获得的数据 在转基因的肝细胞中构建。雌激素对子宫内膜异位基因表达的影响 TBG分子的翻译后修饰将通过以下方法进行研究 TbG基因在转基因细胞中表达的寡糖分析 存在或不存在雌激素的大鼠肝细胞。删除以下内容 通过定点突变的N-糖基化的每个潜在位点 提供有关低聚糖链在 TbG多肽骨架及其在蛋白质正确折叠中的作用 分子。

The broad, long term objectives of this research proposal are to gain knowledge on the mechanisms of thyroid hormone action and the structural requirements of transport proteins for interaction with the hormone. The proposed approach to achieve these goals is to study naturally occurring mutations in man, namely, generalized resistance to thyroid hormone (GIRTH) and inherited defects of thyroxine-binding globulin (TBG). Recently developed methods for the clinical diagnosis of GIRTH will be adapted for use in an outpatient setting and validated by their application to non-resistant subjects. Epidemiological studies will include determination of the prevalence of GIRTH by the screening of newborns and children with the attention deficit/hyperactive disorder, a condition suspected of having high incidence of GIRTH. These data will provide a tissue diagnosis of GIRTH, cultures of fibronectin and collagen synthesis and their mRNA accumulation. Genetic linkage studies by restriction fragment polymorphism using the thyroid hormone receptor probes, c-erb-a alpha and beta will help localize the defect of GIRTH and provide an explanation for its heterogenous phenotype. Data form linkage analysis will help identify affected subjects suitable for the amplification of a specific thyroid hormone receptor gene in order to characterize the defect at the molecular level by gene sequencing. The remaining 7 of 11 TBG mutants so far identified will be characterized by gene sequencing. Their frequency will be determined in subjects with TBG defects which have been detected by standard clinical laboratory means. The properties of mutant TBG molecules, studies by either direct physiochemical methods or after their expression by gene transfection into heterologous cells, will serve to deduce the molecular requirements of normal TBG for its biological function. Studies on the regulation of TBG gene expression will exploit information derived from the structures of mutant TBG genes which exhibit abnormal expression of a normal TBG molecule, and data obtained from the expression of chimeric gene constructs in transfected liver cells. The effect of estrogen on the post-translational modification of the TBG molecule will be studied by analysis of the oligosaccharide moieties of TBG expressed in transfected rat liver cells in the presence or absence of estrogens. The deletion of each potential site of N-glycosylation by site-directed mutagenesis would provide information on the location of the oligosaccharide chains on the TBG peptide backbone and their roles in the proper folding of the molecule.