PREDISPOSITIONS TO COMPLICATIONS OF ALCOHOLISM

酗酒并发症的倾向

• 批准号: 3112273
• 负责人: KWAN-FU REX SHEU
• 金额: $ 16.69万
• 依托单位: WINIFRED MASTERSON BURKE MED RES INST
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1991-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3112273
• 关键词: SDS polyacrylamide gel electrophoresis; Wernicke Korsakoff syndrome; alcoholism /alcohol abuse; chemical fingerprinting; complementary DNA; densitometry; electrofocusing; endonuclease; enzyme mechanism; enzyme structure; enzyme substrate; gel electrophoresis; gene expression; gene mutation; genetic disorder; genetic mapping; human genetic material tag; human tissue; laboratory rabbit; medical complication; messenger RNA; molecular cloning; molecular genetics; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; protein structure; spectrometry; structural genes; thiamine pyrophosphate; tissue /cell culture; transaldolase /transketolase

The long-term aim of his research is to identify genetic predispositions to complications of alcoholism. The immediate goal for this grant period is to elucidate the basis of an abnormality in the thiamin pyrophosphate (TPP)-dependent enzyme transketolase (EC 2.2.1.1). An impaired affinity to TPP (high Km) has been postulated to predispose a subpopulation of alcoholics to the development of a form of thiamin deficiency, the Wernicke-Korsakoff's syndrome. Structural and molecular genetic studies is proposed to characterize the aberrant transketolase in cultured skin fibroblasts from patients with Wernicke-Korsakoff's syndrome or from patients with documented transketolase abnormality. Structural comparisons will be performed by immunoblots of electrophoretic gels (SDS-gels, isoelectric focusing gels and native gels), by activity staining of electrophoretic gels, by peptide mapping and other immunochemical techniques. This proposition will also examine the possibility of multiple forms of human transketolase. Preliminary work has already purified human liver transketolase, raised antibodies and standardized immunochemical procedures. For molecular genetic studies, cDNA for normal human transketolase will be isolated (with the antibody probe) sequenced, and used to isolated genomic DNAs to construct a structural map for the transketolase gene. The cDNA for aberrant transketolase will also be isolated and sequenced to identify the exact site of genetic mutation. The mutation will be further tested at the genomic level using the abnormal genomic fragment amplified with the polymerase chain reaction (PCR) method. These studies will answer the following 2 questions. Is the aberration of Wernicke-Korsakoff transketolase associated with a structural abnormality? If so, is this structural abnormality determined at the genetic level?

他研究的长期目标是确定遗传易感性 酗酒的并发症。 此资助期的近期目标是 阐明焦磷酸硫胺素异常的基础 (TPP) 依赖性酶转酮醇酶 (EC 2.2.1.1)。 亲和力受损 TPP（高公里）已被假设为容易导致亚人群 酗酒者会导致某种形式的硫胺素缺乏症， 韦尼克-科萨科夫综合症。 结构和分子遗传学研究 提议表征培养皮肤中异常的转酮醇酶 来自韦尼克-科萨科夫综合征患者或来自 有转酮酶异常记录的患者。 结构比较 将通过电泳凝胶（SDS-gels， 等电聚焦凝胶和天然凝胶），通过活性染色 电泳凝胶，通过肽图谱和其他免疫化学 技术。 该提案还将探讨多种可能性 人类转酮醇酶的形式。 前期工作已经净化人体 肝脏转酮酶、抗体和标准化免疫化学 程序。 对于分子遗传学研究，正常人转酮醇酶的 cDNA 将是 分离（使用抗体探针）测序，并用于分离基因组 用于构建转酮酶基因结构图的 DNA。 cDNA 对于异常的转酮醇酶也将被分离并测序以识别 基因突变的确切位点。 该突变将在以下进一步测试 使用用扩增的异常基因组片段进行基因组水平 聚合酶链式反应（PCR）方法。 这些研究将回答以下两个问题。 是像差 Wernicke-Korsakoff 转酮醇酶与结构异常相关？ 如果是这样，这种结构异常是在基因水平上决定的吗？