CHEMICAL PATHOLOGY OF NEUROLOGICAL DISORDERS

采用传统的体细胞杂交技术和 鼠类半乳糖神经酰胺酶缺乏症、抽搐者、人类 编码半乳糖神经酰胺酶的基因将被映射到特定的染色体。 将尝试人类半乳糖神经酰胺酶的详细表征 通过丙烯酰胺凝胶电泳和电聚焦。 目标是 (1) 区分人类酶和小鼠酶，以便该程序 可作为上述染色体定位的补充手段 研究，（2）获得少量相对纯的酶 用作单克隆抗体生产的抗原，也可能用作 至少几个 N 端氨基酸的测序来源。 更多 将尝试从胎盘和/或尿液中进行常规纯化 具有相似的最终目标。 新型“自杀”抑制剂的用途 β-半乳糖苷酶 -- 半乳糖基甲基-对硝基苯基三氮烯 (gal-MNT) -- 将探索差异性抑制 GM1-神经节苷脂 β-半乳糖苷酶。 如果成功的话，传统的荧光底物， 4-甲基伞形基β-半乳糖苷可用于测定 半乳糖神经酰胺酶及其在电泳上的定位 凝胶。 这将促进半乳糖神经酰胺酶研究的各个方面， 包括对受影响患者的诊断。 将尝试诱导实验小鼠模型 GM1-神经节苷脂沉积症与 gal-MNT 抑制剂可特异性抑制 GM1-神经节苷脂β-半乳糖苷酶，但不是半乳糖神经酰胺酶。 型号 将在病理学和生化方面进行详细表征。 然后， 该模型将被操纵来回答有关的问题 大脑发育及相关疾病对酶的需求 这组遗传性神经病患者的治疗干预时机 发育中大脑的可塑性障碍。 平行实验 计划使用器官型中枢神经系统培养物。 赋予葡萄糖神经酰胺酶能力的一个可能的新因素 将表征水解葡萄糖神经酰胺。 这不一样 长期被称为“戈谢激活蛋白”的因子。 就其本身而言，这 这将是脂质酶学的一个重要发展。 此外，它是 可以想象，类似戈谢病的遗传状态可能存在，因为 导致该因素异常。 当 因素得到充分表征。

With the conventional somatic cell hybridization technique and with the murine galactosylceramidase-deficiency disease, the twitcher, the human gene coded for galactosylceramidase will be mapped to a specific chromosome. Detailed characterization of human galactosylceramidase will be attempted by acrylamide gel electrophoresis and electrofocusing. The aim is (1) to distinguish the human enzyme from the mouse enzyme so that the procedure can be used as a supplementary means for the above chromosomal localization study, and (2) to obtain a small amount of relatively pure enzyme to be used as the antigen for monoclonal antibody production and also possibly as the source for sequencing of at least several N-terminal amino acids. More conventional purification will be attempted from the placenta and/or urine with similar eventual aims. The use of the new "suicide" inhibitor of beta-galactosidase -- galactosylmethyl-p-nitrophenyltriazene (gal-MNT) -- will be explored to differentially inhibit GM1-ganglioside beta-galactosidase. If successful, the conventional fluorogenic substrate, 4-methylumbelliferyl beta-galactoside could be used for assays of galactosylceramidase and also for its localization on the electrophoretic gel. This will facilitate all aspects of the galactosylceramidase studies, including diagnosis of affected patients. Attempts will be made to induce an experimental mouse model of GM1-gangliosidosis with the gal-MNT inhibitor which specifically inhibits GM1-ganglioside beta-galactosidase but not galactosylceramidase. The model will be characterized in detail pathologically and biochemically. Then, the model will be manipulated to answer questions concerning the requirement for the enzyme by developing brain and those concerning the timing of therapeutic intervention in this group of genetic neurological disorders vis-a-vis plasticity of developing brain. Parallel experiments are planned with the use of the organotypic CNS cultures. A possible new factor that confers to glucosylceramidase the capacity to hydrolyze glucosylceramide will be characterized. This is NOT the same factor long known as the "Gaucher activator protein". By itself, this would be an important development in lipid enzymology. Furthermore, it is conceivable that genetic status resembling Gaucher disease might exist due to abnormality of this factor. Such a possibility will be tested when the factor is adequately characterized.