Core--Analytical and Biomolecular

过去15年的研究已经记录了AA级联中P450花生四烯酸单加氧酶分支的产物和酶的生化和功能相关性。这一代谢途径的大部分产物已经被合成并进行了功能表征，在大鼠、小鼠和人类肾脏中，2C和4A P450亚型被鉴定为主要的AA环氧化酶和omega/omega-1羟化酶。合成化学、蛋白质化学和重组DNA技术现在提供了高效和常规的途径，以获得大多数P450类20烷类化合物、特异性抑制剂、EET和HETE类似物、拮抗剂和激动剂、纯化的P450异构体、P450抗体、cdna，以及编码AA环氧化酶和omega/omega-1羟化酶的质粒和病毒载体。为了支持项目1-6和优化生产相互作用和资源利用，Core B将继续采用已建立的类二十烷提取、纯化、HPLC分析、GC/MS、LC/MS、蛋白质纯化和重组DNA操作方法，用于：a)生物样品中类二十烷酸的检测和定量，b)细胞、亚细胞或纯化蛋白孵育产生的代谢物的生化表征，c)合成标准物、特异性抑制剂、激动剂和拮抗剂的储存、纯化和记录，d)免疫特异性探针的储存和记录，e)重组酶的部分纯化，f)克隆cdna的扩增、纯化和记录。这个

Studies of the last 15 years have documented the biochemical and functional relevance of the products and enzymes of the P450 arachidonic acid (AA) monooxygenase branch of the AA cascade. Most of the products of this metabolic pathway have been synthesized and functionally characterized, and 2C and 4A P450 isoforms identified as the predominant AA epoxygenase and omega/omega-1 hydroxylases in the rat, mouse, and human kidney. Synthetic chemistry, protein chemistry, and recombinant DNA techniques provide now efficient and routine access to most P450 eicosanoids, specific inhibitors, EET and HETE analogs, antagonists and agonist, purified P450 isoforms, P450 antibodies, cDNAs, as well as plasmid and viral vectors coding for AA epoxygenase and omega/omega-1 hydroxylases. In support of projects 1-6 and, to optimize productive interactions and resources utilization, Core B will continue to apply established methods of eicosanoid extraction, purification, HPLC analysis, GC/MS, LC/MS, protein purification, and recombinant DNA manipulation, for: a) the detection and quantification of eicosanoids in biological samples, b) the biochemical characterization of metabolites generated by cellular, subcellular, or purified protein incubates, c) the storage, purification, and documentation of synthetic standards, specific inhibitors, agonist, and antagonists, d) the storage, and documentation of immunospecific probes, e) the partial purification of recombinant enzymes, and f) the amplification, purification and documentation of cloned cDNAs. The centralization of these routine tasks in Core B eliminates unnecessary and costly duplications, improves reproducibility, and provides projects 1-6 with efficient and timely access to synthetic standards, biospecific probes and modern bioanalytical techniques.