CHEMICAL PATHOLOGY OF NEUROLOGICAL DISORDERS

用常规的体细胞杂交技术和用 小鼠半乳糖神经酰胺酶缺乏症，抽动症，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上。 将尝试对人半乳糖基神经酰胺酶进行详细的表征 采用丙烯酰胺凝胶电泳法和电聚焦技术。其目的是(1) 区分人类的酶和老鼠的酶，这样这个过程 可作为上述染色体定位的辅助手段。 研究，以及(2)获得少量相对纯度较高的酶 用作生产单抗的抗原，也可能用作 至少几个N-末端氨基酸测序的来源。更多 将尝试从胎盘和/或尿液中进行常规净化 有着相似的最终目标。新的“自杀”抑制剂的使用 β-半乳糖苷酶--半乳糖甲基-对-硝基苯基三氮烯(GAL-MNT) 将探索不同地抑制GM1-神经节苷脂 β-半乳糖苷酶。如果成功，传统的荧光衬底， 4-甲基伞形花酰基-β-半乳糖苷可用于测定 半乳糖基神经酰胺酶及其在凝胶上的定位 凝胶。这将促进半乳糖神经酰胺酶研究的方方面面， 包括对受影响患者的诊断。 将尝试诱导出一种实验性的小鼠模型 GM1-神经节苷脂代谢与Gal-MNT抑制剂的结合 GM1-神经节苷脂-β-半乳糖苷酶，而不是半乳糖基神经酰胺酶。模型 将在病理和生化方面进行详细的描述。然后, 该模型将被操纵以回答有关 大脑发育对酶的需求及与脑发育有关的酶 这组遗传性神经病的治疗干预时机 发育中的大脑的可塑性障碍。平行实验 计划使用器官型中枢神经系统培养物。 一种可能的新因素，赋予葡萄糖神经酰胺酶能力 将对葡萄糖神经酰胺的水解物进行表征。这是不一样的 长久以来被称为“高谢尔激活蛋白”的因子。就其本身而言，这 将是脂类酶学的一个重要发展。此外，它是 可想而知，类似高谢病的遗传状态可能存在于 这一因素的反常。这种可能性将在 因子得到了充分的表征。

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.