Analytic Core

分析核心

• 批准号: 10005394
• 负责人: Danyelle M. Townsend
• 金额: $ 15.32万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2022-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10005394
• 关键词: Analytical Biochemistry; Antibodies; Antioxidants; Biochemical; Biochemistry; Biological Assay; Biological Models; Biology; Biomedical Research; Cell membrane; Cell surface; Cells; Centers of Research Excellence; Collaborations; Complex; Computer software; Coupled; Data; Detection; Development; Doctor of Philosophy; Energy Metabolism; Equilibrium; Event; Faculty; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Fluorescent Probes; Gamma-glutamyl transferase; Glutathione Disulfide; Glutathione S-Transferase; Goals; Grant; High Pressure Liquid Chromatography; Homeostasis; Hydrogen Peroxide; Immunoblot Analysis; Immunohistochemistry; In Situ; In Vitro; Individual; Intracellular Membranes; Kinetics; Label; Measurement; Measures; Mediating; Membrane Potentials; Membrane Proteins; Mentors; Methods; Modification; Oxidants; Oxidation-Reduction; Post-Translational Protein Processing; Proteins; Proteomics; Publications; Reaction; Research; Research Personnel; Resolution; Resource Sharing; SKIL gene; Serum; Signal Transduction; South Carolina; Stress; Structure; Sulfhydryl Compounds; Superoxide Dismutase; Surface; Techniques; Technology; Time; Tissues; antioxidant enzyme; base; catalase; cohort; design; enzyme activity; glutathione peroxidase; in silico; instrumentation; interdisciplinary approach; millisecond; molecular modeling; nitrosative stress; oxidant stress; peroxiredoxin; protein protein interaction; release of sequestered calcium ion into cytoplasm; success

CORE E: ANALYTICAL REDOX BIOCHEMISTRY Director: Danyelle Townsend, PhD ABSTRACT Understanding the complexities of redox mediated signaling events requires a multidisciplinary approach. The SC COBRE in Oxidants, Redox Balance and Stress Signalling has assembled a cohort of promising junior faculty with expertise in relevant biomedical model systems. Analytical biochemistry specific to the detection and quantification of redox sensitive molecules and coordinate protein changes that drive homeostasis is a unique niche. As such, development of the Analytical Redox Biology Core (ARBC) is important. The primary objective of the Core is to provide comprehensive analytical redox biochemistry methods and mentoring support for the COBRE junior faculty with the goal to advance their research endeavors, publications and fundability. The specific aims of the ARBC are: 1) Provide ROS/RNS identification and quantification using state-of-the-art techniques; 2) Perform quantitative analysis of ROS/RNS (redox molecules and metabolites), including those associated with calcium mobilization and changes in energy metabolism; 3) Provide expertise and technology for in depth biochemical analysis of thiol-centered enzyme activities and define protein:protein interactions. Since oxidative (nitrosative) stress often is associated with a conditional increase in antioxidant protection, the Core has established methods to detect and measure various antioxidant enzyme activities as a function of oxidant stress/antioxidant protection equilibrium. Comprehensive analysis of redox status also includes measurement of intracellular GSH, GSSG, protein surface and “buried” thiols utilizing both endpoint and/ or real-time kinetic measurements with millisecond resolution. In complex studies of redox signaling, certain protein:protein interactions appear to be redox dependent and attributed to post-translational modifications, including S-nitrosylation and S-glutathionylation. The ARBC has developed fluorescent labeling and FRET analysis to evaluate redox dependent protein:protein interactions with subsequent in silico molecular modeling using ZDOCK, GOLD Suite (v 5.2) software. Collectively, these technologies will provide a multidisciplinary approach to advance the understanding of redox mediated signaling events specific to the model systems presented by the junior faculty in their research.

核心E：分析性氧化还原生物化学 主任：Danyelle汤森，博士 摘要 理解氧化还原介导的信号事件的复杂性需要多学科的 approach. SC COBRE在氧化剂，氧化还原平衡和应力信号已经组装了一个 一群在相关生物医学模型系统方面拥有专业知识的有前途的初级教师。分析 生物化学特异于检测和定量氧化还原敏感分子和协调 驱动体内平衡的蛋白质变化是一个独特的生态位。因此，开发分析 氧化还原生物学核心（ARBC）是重要的。核心的主要目标是提供 全面的分析氧化还原生物化学方法和COBRE初级指导支持 教师的目标是推进他们的研究工作，出版物和资金。具体 ARBC的目标是：1）使用最先进的技术提供ROS/RNS鉴定和定量 2）进行ROS/RNS（氧化还原分子和代谢物）的定量分析， 包括与钙动员和能量代谢变化相关的那些; 3）提供 专业知识和技术，用于对巯基为中心的酶活性进行深入的生化分析， 定义蛋白质相互作用。由于氧化（亚硝化）应激通常与 有条件地增加抗氧化保护，核心已经建立了检测和 测量作为氧化应激/抗氧化保护的函数的各种抗氧化酶活性 均衡氧化还原状态的综合分析还包括细胞内GSH的测量， 利用终点和/或实时动力学的GSSG、蛋白质表面和“埋藏”硫醇 毫秒级分辨率的测量。在氧化还原信号的复杂研究中，某些 蛋白质：蛋白质相互作用似乎是氧化还原依赖性的，并归因于翻译后 修饰，包括S-亚硝基化和S-谷胱甘肽化。ARBC开发了荧光 标记和FRET分析，以评估氧化还原依赖性蛋白质：蛋白质与随后的 使用ZDOCK，GOLD Suite（v 5.2）软件进行计算机分子建模。总的来说，这些 技术将提供一个多学科的方法，以促进对氧化还原的理解， 介导的信号事件特定于模型系统提出的初级教师在他们的 research.