PREDISPOSITIONS TO COMPLICATIONS OF ALCOHOLISM

酗酒并发症的倾向

• 批准号: 3112272
• 负责人: KWAN-FU REX SHEU
• 金额: $ 15.43万
• 依托单位: WINIFRED MASTERSON BURKE MED RES INST
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3112272
• 关键词: Wernicke Korsakoff syndrome; enzyme activity; enzyme structure; gene expression; gene mutation; genetic disorder; human genetic material tag; human tissue; molecular cloning; molecular pathology; northern blottings; nucleic acid sequence; nutrient drug interaction; nutrition related tag; polymerase chain reaction; protein sequence; protein structure function; thiamine pyrophosphate; tissue /cell culture; transaldolase /transketolase; vitamin B deficiency; western blottings

The long-term goal of this investigation is to identify genetic predispositions to complications of alcoholism. An abnormality of the thiamin pyrophosphate (TPP)-dependent enzyme transketolase (EC 2.2.1.1) has been identified in patients with Wernicke-Korsakoff syndrome, a disease found primarily in a subgroup of alcohol abusers who respond favorably on thiamin treatment. In the past one and a half years we have identified a functionally impaired transketolase protein in cultured skin fibroblasts from three patients with Wernicke-Korsakoff syndrome, by enzymatic and immunochemical techniques. Molecular cloning for the transketolase cDNA is well underway. It has been proposed that the abnormality of transketolase is caused by an alteration of the transketolase gene. We plan to elucidate the molecular basis of the transketolase abnormality in these three fibroblast lines that the protein is already shown to be abnormal, an to other patients and non-symptomatic family members. The results may lead to a better understanding of the genetic predisposition, as well as the interaction of gene and nutrition (thiamin) in the development of Wernicke-Korsakoff syndrome. We plan to further describe the abnormality of transketolase enzymatically and immunochemically at the protein level in these three Wernicke-Korsakoff cells, and extend these biochemical work to other fibroblasts from Wernicke-Korsakoff patients and non-symptomatic alcoholic controls. The protein sequence will be obtained by conventional molecular biological techniques. Complementary DNA will be isolated and cloned from the human fibroblast cDNA expression library using the antibody probe, and verified by matching for partial amino acid sequence we already obtained. The transketolase cDNA in the Wernicke-Korsakoff and non-symptomatic alcoholic control fibroblasts will be amplified by polymerase chain reaction (PCR) method, using the mRNA/cDNA hybrid prepared form these cells and well-spaced oligonucleotide primers. The transketolase cDNA from patient and control cells will be compared by direct sequencing of the PCR-amplified, total heterozygous cDNA, and each of the allelic, clonal cDNA. The abnormality of transketolase in these Wernicke-Korsakoff fibroblasts will be interpreted based on the genetic alteration of protein sequence.

这项研究的长期目标是确定遗传因素 酗酒并发症的倾向。 的异常 焦磷酸硫胺素 (TPP) 依赖性酶转酮醇酶 (EC 2.2.1.1) 已在韦尼克-科尔萨科夫综合征患者中发现， 该疾病主要发现于有反应的酗酒者亚群中 对硫胺素治疗有利。 在过去的一年半里，我们 在培养皮肤中发现功能受损的转酮醇酶蛋白 来自三名 Wernicke-Korsakoff 综合征患者的成纤维细胞 酶促和免疫化学技术。 分子克隆 转酮醇酶 cDNA 的研究进展顺利。 有人建议 转酮酶异常是由于转酮酶的改变引起的 转酮醇酶基因。 我们计划阐明其分子基础 这三种成纤维细胞系中的转酮醇酶异常 对于其他患者来说，蛋白质已经被证明是异常的 无症状的家庭成员。 结果可能会带来更好的结果 了解遗传倾向以及相互作用 基因和营养（硫胺素）在 Wernicke-Korsakoff 发育中的作用 综合症。 我们计划进一步描述转酮醇酶的异常情况 在这三种蛋白质水平上进行酶促和免疫化学分析 Wernicke-Korsakoff 细胞，并将这些生化工作扩展到其他 来自 Wernicke-Korsakoff 患者和无症状患者的成纤维细胞 酒精控制。 蛋白质序列将通过以下方式获得 常规分子生物学技术。 互补DNA将是 从人成纤维细胞 cDNA 表达文库中分离克隆 使用抗体探针，并通过部分氨基酸匹配进行验证 我们已经获得了序列。 转酮酶 cDNA 位于 Wernicke-Korsakoff 和无症状酒精控制成纤维细胞将 通过聚合酶链反应（PCR）方法进行扩增，使用 由这些细胞制备的 mRNA/cDNA 杂合体且间隔良好 寡核苷酸引物。 来自患者和对照的转酮醇酶 cDNA 细胞将通过 PCR 扩增的直接测序进行比较，总 杂合 cDNA，以及每个等位基因、克隆 cDNA。 异常情况 这些 Wernicke-Korsakoff 成纤维细胞中的转酮醇酶将是 根据蛋白质序列的遗传改变进行解释。