Core--Analytical and Biomolecular

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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.