MRS ANALYSIS OF POLYAMINE METABOLISM IN INTACT CELLS

完整细胞中多胺代谢的 MRS 分析

基本信息

批准号：
2097396
负责人：
KATHY ANN MCGOVERN
金额：
$ 10.13万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-05-01 至 1998-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2097396
关键词：
biophysics bioreactors carbon cell cycle chemical kinetics heat high performance liquid chromatography intracellular metabolism neoplastic cell nuclear magnetic resonance spectroscopy physiologic stressor polyamines radionuclides stable isotope stress tissue /cell culture

项目摘要

Polyamines are intimately involved in cell growth, cellular response to stress and transformation processes. Polyamine metabolism changes significantly in cells at different stages of cell growth, in cells before and after a specific stress, and in cells before and after transformation from a normal to a neoplastic phenotype. These changes in intracellular polyamine metabolism result in measurable differences in the total amounts of specific polyamines and their metabolites in a given cell as well as specific activities of many enzymes of the polyamine pathway. The function of polyamines in these processes remains unclear. The unifying hypothesis of this proposal is simply stated: changes in intracellular compartmentation of polyamine metabolites play a key role in both the control of polyamine metabolism and polyamine function. It is the relative amounts of polyamine metabolites in a specific compartment which is relevant to these processes. The proposed research will test this hypothesis using nuclear magnetic resonance spectroscopy (NMR/MRS) to non-invasively examine polyamine metabolism in intact cells under well-defined perfusion conditions. By growing cells in defined media, or using specific inhibitors of the polyamine pathway, normal polyamines can be replaced with polyamines labelled with NMR-visible isotopes. This will permit careful characterization of intracellular polyamine pools as defined by specific NMR parameters. Relevant measurements include chemical shifts, signal intensities, and relaxation times for each detectable metabolite, and given appropriate kinetics, reaction rates through specific pathways. Using a variety of labelled compounds and experimental strategies, NMR parameters of intracellular polyamine pools will be determined in a single cell line during exponential growth and at confluence. The same cell line will be further examined while in exponential growth before, during, and after heat shock. Used in conjunction with standard biochemical assays, NMR spectroscopic data obtained from intact cells will provide unique insight into intracellular polyamine metabolism and function. The labelling strategies and methods developed herein will be widely applicable to the study of polyamine metabolism in other cells and tissues; the potential to extend these techniques to intact animals exists. Interference with polyamine metabolism and function has proven a valuable approach to the treatment of neoplasms and other diseases. Further characterization of polyamine metabolism in the intact cell will likely suggest novel therapies as well as further refinement to current approaches.

多胺与细胞生长、细胞对细胞的反应密切相关压力和转变过程。多胺代谢变化在细胞生长的不同阶段的细胞中显着在特定压力之前和之后，以及在细胞中之前和之后从正常表型向肿瘤表型的转变。这些变化细胞内多胺代谢导致可测量的差异给定的特定多胺及其代谢物的总量细胞以及多胺的许多酶的特定活性途径。多胺在这些过程中的功能仍不清楚。该提案的统一假设很简单：多胺代谢物的细胞内区隔发挥关键作用控制多胺代谢和多胺功能。它是特定物质中多胺代谢物的相对量与这些过程相关的隔间。拟议的研究将使用核磁共振波谱法检验这一假设 (NMR/MRS) 非侵入性检查完整细胞中的多胺代谢在明确的灌注条件下。通过在规定的范围内培养细胞介质，或使用多胺途径的特定抑制剂，正常多胺可以用核磁共振可见标记的多胺代替同位素。这将允许仔细表征细胞内由特定 NMR 参数定义的多胺池。相关的测量包括化学位移、信号强度和弛豫每种可检测代谢物的时间，并给出适当的动力学，通过特定途径的反应速率。使用各种标记化合物和实验策略，细胞内NMR参数多胺池将在单个细胞系中测定指数增长和汇合。相同的细胞系将进一步在加热前、加热期间和加热后指数生长期间进行检查震惊。与标准生化测定、NMR 结合使用从完整细胞获得的光谱数据将提供独特的见解参与细胞内多胺代谢和功能。标签本文开发的策略和方法将广泛适用于其他细胞和组织中多胺代谢的研究；潜力将这些技术扩展到完整的动物是存在的。干扰多胺代谢和功能已被证明是一种有价值的方法治疗肿瘤和其他疾病。进一步表征完整细胞中的多胺代谢可能会提示新的疗法以及对当前方法的进一步完善。