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Glycolipid metabolism in single cells

单细胞中的糖脂代谢

基本信息

批准号：
7869118
负责人：
Norman J Dovichi
金额：
$ 17.52万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2010-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7869118
关键词：
Affinity Chromatography Anabolism Analytical Chemistry Animal Model Area Arts Aspirate substance Axon Behavior Binding Proteins Biochemistry Biodegradation Biological Biological Assay Biological Models Biology Blood capillaries Book Chapters Books Brain Capillary Electrophoresis Carbohydrate Chemistry Carbohydrates Cell Communication Cell Culture System Cell Culture Techniques Cell Line Cell surface Cells Chemicals Collaborations Color Confocal Microscopy Cytolysis Cytometry Cytoplasmic Granules Data Detection Development Diagnostic tests Disease Dyes Electrophoresis Enzymatic Biochemistry Enzymes Evaluation Fiber Optics Fluorescence Fluorescent Dyes Funding Funding Opportunities Gangliosides Generations Genetic Glycolipids Glycoside Hydrolases Goals Grant Human Genome Project In Vitro Incubated International Investigation Journals Label Laboratories Lasers Libraries Life Link Mass Spectrum Analysis Metabolic Metabolic Pathway Metabolism Methods Mole the mammal Molecular Monitor Motor Neurons Mus Names Nervous System Trauma Neurodegenerative Disorders Neurons Oligonucleotides Oligosaccharides Osmotic Shocks PC12 Cells Paper Pathway interactions Pharmacology Photons Population Primary Cell Cultures Principal Investigator Process Publishing Reaction Reagent Recording of previous events Regulation Research Research Personnel Resistance Rodent Role Science Series Signal Transduction Spectrum Analysis Sphingolipids Spinal Ganglia Staging Supporting Cell Surface Technology Time Universities Variant Vent Washington Work analog analytical method analytical tool base capillary carbohydrate metabolism cell determination cell fixing design embryonic stem cell experience flasks glycosyltransferase improved inhibitor/antagonist innovation instrument instrumentation interest medical schools member metabolomics neuron development novel professor programs response single cell analysis single molecule skills symposium technology development tetramethylrhodamine tool uptake

项目摘要

DESCRIPTION (provided by applicant): This proposal is in response to RFA-RM-06-010, Using Metabolomics to Investigate Biological Pathways and Networks. We quote from the RFA: "The goal of the current funding opportunity is to apply metabolomics technology to the study of pathway and network regulation in normal and disease states that are not easily approached by other technologies." This proposal employs metabolic cytometry to monitor glycolipid metabolism in single neurons. In metabolic cytometry, fluorescently labeled substrate is incubated with cells and is metabolized to a suite of catabolic and anabolic products. These products are analyzed in a single cell by capillary electrophoresis with ultrasensitive laser-induced fluorescence. As long at the fluorescent tag remains intact, all metabolic products can be detected at the zeptomole to yoctomole level. Errors in glycolipid metabolism are important in a number of serious neurodegenerative diseases, and there is ample evidence that there is dramatic cell-to-cell variation in glycolipid expression. This proposal will use highly innovative tools to characterize glycolipid metabolism in single cells. The project requires a significant synthetic effort to prepare fluorescent substrates and reagents. The project requires additional analytical technology development to simultaneously monitor glycolipid metabolism along different paths. The project requires significant biological effort to use these technologies to monitor glycolipid metabolism across populations of cultured cells and primary neurons. Ole Hindsgaul and Monica Palcic, at the Carlsberg Laboratory, serve as synthetic chemist and biochemist. They will prepare a suite of fluorescently-labeled gangliosides and members of the globoseries of glycolipids. They will also verify that these reagents are substrates for endogenous enzymes in the biosynthetic and biodegradation pathways. Norman Dovichi of the University of Washington will develop ultrasensitive capillary electrophoresis/laser-induced fluorescence technology to monitor these compounds in single cells at the low zeptomole level. Ronald Schnaar (Johns Hopkins) will incubate fluorescent substrates with primary nerve cells and embryonic stem cells at different stages of nerve cell determination, differentiation and maturation. The treated cells will be fixed and transferred to Dovichi's lab for analysis.

描述(申请人提供)：本提案响应RFA-RM-06-010，使用代谢组学研究生物途径和网络。我们引用了RFA的话：“目前资金机会的目标是将代谢组学技术应用于研究正常和疾病状态下的途径和网络调节，而其他技术不容易接近这些途径和网络调节。”这项建议使用代谢细胞术来监测单个神经元中的糖脂代谢。在代谢细胞术中，荧光标记的底物与细胞孵育，并被代谢成一系列分解代谢和合成代谢产物。这些产物在单个细胞中通过毛细管电泳超灵敏的激光诱导荧光进行分析。只要荧光标记保持完好，所有代谢产物都可以在齐托莫尔到yoctomole水平上被检测到。糖脂代谢的错误在一些严重的神经退行性疾病中是重要的，而且有充分的证据表明，糖脂的表达在细胞之间存在显著的差异。这项提议将使用高度创新的工具来表征单细胞中的糖脂代谢。该项目需要大量的合成工作来制备荧光底物和试剂。该项目需要额外的分析技术开发，以同时监测不同途径的糖脂代谢。该项目需要大量的生物学努力，以使用这些技术来监测培养细胞和原代神经元群体中的糖脂代谢。嘉士伯实验室的Ole Hindsgaul和Monica Palcic担任合成化学家和生物化学家。他们将准备一套荧光标记的神经节苷脂和糖脂球状系列成员。他们还将验证这些试剂是生物合成和生物降解途径中内源性酶的底物。华盛顿大学的诺曼·多维奇将开发超灵敏的毛细管电泳/激光诱导荧光技术，在低水平的齐托莫水平上监测单细胞中的这些化合物。罗纳德·施纳尔(Johns Hopkins)将在神经细胞确定、分化和成熟的不同阶段与原代神经细胞和胚胎干细胞孵育荧光底物。处理后的细胞将被固定并转移到Dovichi的实验室进行分析。