DETECTION OF NADH REDUCTASE KINETICS WITH [1-13C]-PYRUVATE MRSI

使用 [1-13C]-丙酮酸 MRSI 检测 NADH 还原酶动力学

• 批准号: 8169869
• 负责人: Moses Morakortoi Darpolor
• 金额: $ 1.85万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169869
• 关键词: Anabolism; B-Cell Lymphomas; Biochemical Pathway; Cell Culture Techniques; Cells; Chemicals; Complementary DNA; Computer Retrieval of Information on Scientific Projects Database; Electrons; Enzyme Kinetics; Enzymes; Experimental Designs; Funding; Genes; Goals; Grant; Healthcare; Inbred BALB C Mice; Inbred C57BL Mice; Infectious Ectromelia; Infusion procedures; Institution; Kinetics; Life; Liver; Lymphoma; Measures; Methods; Mouse Pox Virus; NADH; NADH dehydrogenase (quinone); NADP; NQO1 gene; Negative Finding; Nicotinamide adenine dinucleotide; Nuclear; Oxidoreductase; Pathway interactions; Plasmids; Proteins; Pyruvate; Pyruvates; Quinones; Rattus; Reporter; Research; Research Personnel; Resources; Signal Transduction; Source; Techniques; Technology; Testing; United States National Institutes of Health; base; cohort; data acquisition; design; electron donor; flasks; in vivo; instrument; magnetic resonance spectroscopic imaging; plasmid DNA; promoter; protein function; research study; therapeutic target

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Goal: The goal of this project is to demonstrate the utility of hyperpolarized [1-13C]-pyruvate magnetic resonance spectroscopic imaging (MRSI) as in vivo reporter of enzyme kinetics involved in the biosynthesis pathways of pyruvate. Our long-term goal is to utilize this technique for validating potential therapeutic targets that regulate protein function in vivo with the precision and control conferred by tunable gene technology, and in addition advance our understanding of basic biochemical pathways. We assume that NADH reductase is the key enzyme that regulates (rate limiting step) the NADH to NAD+ reduction. Materials and Methods: EL4 (C57BL mouse lymphoma) and A20 (BALB/c B cell lymphoma) cells are transfected with cDNA plasmid of NQO1. These cells have been tested and found negative for ectromelia virus (mousepox). The cDNA plasmid of NQO1 expresses the gene for nicotinamide adenine dinucleotide (NADH) quinone oxidoreductase 1 (NQO1). NQO1 is a ubiquitous cytosolic flavoenzyme that catalyzes two- electron reduction of various quinones, with NADH or NADPH as an electron donor. A flask of 50 million cells in 2.5 ml of RPMI 1640 medium is administered with hyperpolarized [1-13C]-pyruvate followed by data acquisition. A 3.0 Tesla Signa magnet (GE healthcare) and a dynamic nuclear polarizer (Oxford Instruments Hypersense) are utilized. Specific Aim 1: To transfect cell cultures with a NADH-reductase gene that over-expresses NADH. Experiment Design 1: We will transfect cells with cDNA plasmids that increases the levels of NADH in cell culture, and we will measure the chemical flux of [1-13C]-pyruvate to lactate as an indirect measure of the NADH levels. However, since hyperpolarized [1-13C]-pyruvate signal is short lived (~30s) we will prefer a regulated promoter gene of NADH-reductase that can be turned on appropriately prior to the infusion of hyperpolarized pyruvate. Specific Aim 2: To ectopically expressed a plasmid DNA reporter or a tunable regulated gene of NADH reductase in rat liver. Experimental Design 2: We will transfect a rat liver by hydrodynamics-based administration of plasmid DNA or by systemic delivery of conditionally regulated protein. When NADH levels are significantly higher in the liver, the hyperpolarized [1-13C]-pyruvate experiment will be performed to ascertain the conversion rates of pyruvate to lactate as an indirect measure of NADH-reductase. As a control measure, one cohort of rats will not receive the plasmid DNA or tunable form of NADH-reductase.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 目标：该项目的目标是证明超极化[1-13C] - 丙酮酸磁性的实用性 共振光谱成像（MRSI）作为参与该酶动力学的体内记者 丙酮酸的生物合成途径。我们的长期目标是利用这项技术来验证 潜在的治疗靶标，可在体内调节蛋白质功能的精度和对照 由可调基因技术授予，此外，还提高了我们对基本的理解 生化途径。我们假设NADH还原酶是调节的关键酶（速率 限制步骤）NADH至NAD+还原。 材料和方法： EL4（C57BL小鼠淋巴瘤）和A20（BALB/C B细胞淋巴瘤）细胞用cDNA转染 NQO1的质粒。这些细胞已经进行了测试，并发现对鼻孔病毒（Mousepox）阴性。 NQO1的cDNA质粒表达烟酰胺腺嘌呤二核苷酸（NADH）的基因 奎因酮氧化还原酶1（NQO1）。 NQO1是一种普遍存在的胞质黄酶，可催化两种 将各种奎因酮的电子还原，nadh或nadph作为电子供体。烧瓶50 用超极化[1-13C] - 丙酮酸施用2.5 mL RPMI 1640培养基中的百万个细胞 其次是数据获取。 3.0 Tesla Signa磁铁（GE Healthcare）和动态核 利用偏光层（牛津仪器过度）。 特定目的1：用过表达NADH的NADH-还原酶基因转染细胞培养物。 实验设计1：我们将用cDNA质粒转染细胞，以增加NADH的水平 在细胞培养中，我们将测量[1-13c] - 丙酮酸的化学通量作为间接 NADH水平的度量。但是，由于超极化[1-13c] - 丙酮酸信号短暂 （〜30s）我们更喜欢可以打开的NADH-还原酶的调节启动子基因 在输注超极化丙酮酸之前适当。 特定目的2：异位表达质粒DNA报告基因或可调节的调节基因 大鼠肝脏中的NADH还原酶。 实验设计2：我们将通过基于流体力学的给药来转染大鼠肝 质粒DNA或通过系统地递送有条件调节的蛋白质。当nadh级别 在肝脏中，将进行超极化[1-13C] - 丙酮酸实验 确定丙酮酸对乳酸的转化率是NADH-还原酶的间接度量。作为 控制度量，一组大鼠将无法接收NADH-还原酶的质粒DNA或可调形式。