CORE--SCIENTIFIC FACILITIES

对细胞内信号转导有更深入的了解 基底节区多巴胺能神经传递的机制 神经节可以为治疗高血压带来新的治疗靶点 精神分裂症和退行性神经疾病，如 帕金森氏症和亨廷顿氏症。一个多学科的 将在目前的提案中采取方法，调查 依赖于磷酸化的关键分子的功能调节 信号通路。研究将在几个层面上进行 组织复杂性，从体外生化研究到 纯化分子对表型鉴定的影响 靶向删除整个动物体内的关键信号转导分子。 该项目的总体目标是提供一个科学核心设施 供计划项目的所有成员使用。责任 将包括主要试剂的生产和供应以及 执行例行但关键的任务，这些任务将需要 完成本计划其他部分所述的学习 项目。将日常任务集中起来将有助于 以高效率、高成本效益的方式确保供应充足的必需品 所有项目的材料和试剂。 核心将维持一个组织培养设施，以提供持续的 培养细胞的供应，包括原代神经元培养， 重组的培养物和永生的细胞系。它还将保持 纯化的蛋白激酶、蛋白磷酸酶、抗体和 底物蛋白质。标准的净化方案将用于 从天然来源获得这些酶，并采用重组技术 也将被用于大量生产特定的蛋白质 足以进行详细的酶学和结构研究。 核心将负责扩展和维护 缺乏DARPP-32，抑制物-1， 蛋白磷酸酶-1α1和γ亚型，以及蛋白磷酸酶-1 2B，以及携带磷酸化位点突变的转基因动物 DARPP-32..畜牧业将包括建立适当的 动物的交配和断奶。核心还将负责 用聚合酶链式反应和Southern方法检测后代的基因分型 涂抹战略。 核心将负责产生磷酸化状态- 针对DARPP-32、抑制物-1、蛋白上特定位点的特异性抗体 磷酸酶-1，Na+，K+-ATPaseα亚基亚型与Na+、Ca~(2+)的关系 频道。检测和量化状态变化的能力 这些关键底物蛋白的磷酸化将是非常有用的。 在研究中提出了阐明它们的功能意义。

A greater understanding of the intracellular signal transduction mechanisms which underlie dopaminergic neurotransmission in the basal ganglia can lead to novel therapeutic targets for the treatment of schizophrenia, and degenerative neurological disorders such as Parkinson's disease and Huntington's disease. A multi-disciplinary approach will be undertaken in the current proposal to investigate the phosphorylation-dependent functional regulation of key molecules in these signaling pathways. Studies will be performed at several levels of organizational complexity, ranging from in vitro biochemical studies with purified molecules to the characterization of phenotypes resulting from targeted deletion of key signal transduction molecules in whole animals. The overall goal of this project is to provide a Scientific Core facility for use by all the members of the Program Project. The responsibilities of the Core will include the production and supply of key reagents and the performance of routine, yet critical tasks that will be required to accomplish the studies described in the other sections of this Program Project. The centralization of routine tasks will facilitate an efficient, cost-effective means to ensure an adequate supply of essential materials and reagents to all the Projects. The Core will maintain a tissue culture facility to provide a continuous supply of cultured cells, including primary neuronal cultures, reaggregated cultures and immortalized cell lines. It will also maintain stocks of purified protein kinases, protein phosphatases, antibodies, and substrate proteins. Standard purification protocols will be used to obtain these enzymes from native sources, and recombinant technologies will also be employed to produce specific proteins in quantities sufficient to carry out detailed enzymological and structural studies.. The Core will be responsible for the expansion and maintenance of the colonies of "knockout" mice that are deficient in DARPP-32, inhibitor-1, protein phosphatase-1 alpha1 and gamma isoforms, and protein phosphatase- 2B, and transgenic animals harboring phosphorylation site mutations in DARPP-32.. Animal husbandry will include the setting up of appropriate matings and weaning of animals. The Core will also be responsible for the determination of the genotype of the offspring, using PCR and Southern blot strategies. The Core will be responsible for the production of phosphorylation state- specific antibodies to specific sites on DARPP-32, inhibitor-1, protein phosphatase-1, Na+,K+-ATPase alpha subunit isoforms and Na+ and Ca2+ channels. The ability to detect and quantitate changes in the state of phosphorylation of these key substrate proteins will be of great utility in the studies proposed to elucidate their functional significance.