GLYCOLIPID METABOLISM IN SINGLE CELLS (RMI)

单细胞中的糖脂代谢 (RMI)

• 批准号: 7012599
• 负责人: Norman J Dovichi
• 金额: $ 3.2万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7012599
• 关键词: PC12 cells; biotechnology; capillary electrophoresis; chemical synthesis; fluorescent dye /probe; glycolipids; lipid metabolism; metabolomics; molecular probes; technology /technique development

DESCRIPTION (provided by applicant): This R21/R33 proposal combines the efforts of an international team to develop tools and methods to monitor a subset of the ganglioside metabolome in single mammalian cells. In the two-year duration R21 phase of the project, a set of eight fluorescent substrates and standards will be synthesized using chemical and enzymatic methods. The set consists of all known metabolites along two different glycolipid metabolic pathways. The standards and substrates will be prepared with four different fluorescent labels, so that different points in the metabolic pathway can be accessed simultaneously. NG108-15 and/or PC12 cells will be incubated with the substrates, which will undergo biosynthesis and biodegradation. Only those reaction products that preserve the fluorescent label will generate a detectable signal in our instrument, allowing careful dissection of specific metabolic pathways. A four-spectral channel laser-induced fluorescence detector will be assembled for this project. This detection system will allow the subattomole detection of four different sets of metabolic products, which will provide a particularly powerful tool for the characterization of metabolites along different pathways. During the one-year duration R33 phase of the project, this set of reagents and instrumentation will be applied to the study of metabolism in single cells. The long-term goal of this project is to monitor the complete ganglioside metabolome in single neurons.

描述（由申请人提供）：此R21/R33提案结合了国际团队开发工具和方法的努力，以监测单个哺乳动物细胞中神经节苷脂代谢组的子集。 在该项目的两年持续时间R21阶段中，将使用化学和酶学方法合成一组八组荧光底物和标准标准。该集合由沿两个不同的糖脂代谢途径的所有已知代谢产物组成。标准和底物将使用四个不同的荧光标签制备，因此可以同时访问代谢途径中的不同点。 NG108-15和/或PC12细胞将与底物一起孵育，这将进行生物合成和生物降解。只有那些保留荧光标签的反应产物才能在我们的仪器中产生可检测的信号，从而仔细解剖特定的代谢途径。该项目将组装四光通道激光诱导的荧光检测器。该检测系统将允许对四组不同的代谢产物进行下检测，这将为沿不同途径的代谢物的表征提供特别强大的工具。 在项目的一年持续时间R33阶段中，这组试剂和仪器将应用于单个细胞中的代谢研究。 该项目的长期目标是监测单个神经元中完整的神经节代谢组。