Analytical and Biomolecular Core

分析和生物分子核心

• 批准号: 8521253
• 负责人: Jorge Capdevila
• 金额: $ 19.86万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2016-04-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8521253
• 关键词: Agonist; Alkane 1-monooxygenase; Anabolism; Antibodies; Arachidonic Acids; Binding; Biochemical; Biological; Code; Complementary DNA; Cytochrome P450; Detection; Documentation; Eicosanoids; Enzymes; Funding; Genes; Genetic Models; High Pressure Liquid Chromatography; Human; Hydroxyeicosatetraenoic Acids; Hypertension; Incubated; Isotopes; Kidney; Literature; Maintenance; Metabolic; Metabolism; Methodology; Methods; Mixed Function Oxygenases; Molecular Biology; Mus; Organ; Pathway interactions; Phase; Plasmid Cloning Vector; Plasmids; Play; Productivity; Protein Chemistry; Protein Isoforms; Proteins; Publishing; Quality Control; Radiolabeled; Rattus; Recombinant DNA; Recombinants; Renal function; Reproducibility; Research Personnel; Resources; Role; Sampling; Scientist; Subcellular Fractions; Synthesis Chemistry; Techniques; Time; Viral; Viral Vector; analog; cDNA Library; cDNA Probes; cell preparation; cytochrome P-450 CYP2C subfamily; experience; expression vector; improved; inhibitor/antagonist; member; programs; protein purification; radiotracer; tandem mass spectrometry; tool

The role of the P450 arachidonic acid (AA) monooxygenase as a major pathway for the metabolism of endogenous AA is now well established, as is the functional relevance of its products and of the enzymes responsible for their biosynthesis. Studies with purified proteins, genetic models of hypertension, or mice carrying disrupted P450 genes have identified members of the CYP2C and CYP4A gene subfamily of P450s as the predominant, and functionally relevant AA epoxygenase and omega hydroxylases in the rat, mouse, and human kidney, respectively. 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 CYP2C AA epoxygenase and CYP 4A omega-hydroxylases. In support of projects 1- 5 and, to optimize productive interactions and resources utilization. Core B will continue to apply established methods of eicosanoid extraction, purification, HPLC analysis, UPLC/MS/MS characterization, 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 cDNAs probes. The centralization of these routine tasks in Core B eliminates unnecessary and costly duplications, improves reproducibility, and provide projects 1-5 with efficient and timely access to synthetic standards, biospecific probes and state of the art bioanalytical techniques.

P450花生四烯酸（AA）单加氧酶作为代谢的主要途径 现在已经确定了内源性AA，其产品及其酶的功能相关性也是如此 负责其生物合成。纯化蛋白质，高血压的遗传模型或小鼠的研究 携带破坏的P450基因已经鉴定了P450S的CYP2C和CYP4A基因的成员 作为大鼠小鼠，小鼠中的主要和功能相关的AA环氧酶和欧米茄羟化酶 和人类肾脏。合成化学，蛋白质化学和重组DNA技术 现在提供有效且常规的访问权限，可访问大多数P450 eicosanoids，特定抑制剂，EET和Hete 类似物，拮抗剂和激动剂，纯化的P450同工型，P450抗体，cDNA以及质粒和 编码CYP2C AA环氧酶和CYP 4A欧米茄羟基酶的病毒载体。支持项目1- 5并且，以优化生产互动和资源利用。核心B将继续应用建立 类eicosanoid提取，纯化，HPLC分析，UPLC/MS/MS表征，蛋白质的方法 纯化和重组DNA操作，因为：a）在 生物样品，b）由细胞，亚细胞或 纯化的蛋白质孵化，c）合成标准的存储，纯化和文献，特定 抑制剂，激动剂和拮抗剂，d）免疫特异性探针的存储和记录，e） 重组酶的部分纯化，以及f）放大，纯化和文档 cDNA探针。这些常规任务在核心B中的集中化消除了不必要和昂贵的 重复，提高可重复性并为1-5提供有效及时的合成访问权限 标准，生物探针和艺术生物分析技术的状态。