QUANTITATIVE IN VIVO IN VITRO STUDIES OF CELL PROCESSES

描述(摘自申请人的摘要)：在本项目中，委托人 研究人员和同事们建议量化：1)主要变化 细胞质和周质溶质的类型和数量以及细胞数量 和隔间水，允许大肠杆菌在非常广泛的范围内生长 体外渗透压；以及2)伴随而来的膨胀压力的巨大变化 穿过牢房墙。通过与溶质的体外效应比较，这些 细胞质溶质和生物聚合物浓度的较大变化应该 个体扰乱了大多数细胞过程，但总体上不会。同样 引人注目的是细胞质水分的巨大变化，这是 膨胀压力的测量显示没有伴随着较大的变化 水的活动性。长期目标是：1)将大肠杆菌理解为一种化学物质 和渗透系统；2)有关溶质-生物聚合物相互作用的研究 体外解决对生物聚合物过程的体外和体内影响；3) 要了解单元进程所依据的全局补偿机制 对溶质浓度的变化有缓冲作用，分子基础 代谢惰性“渗透保护剂”对生长速度的促进作用 溶质。 具体目标是：1)生理生化指标的测定 大肠杆菌对高、低渗透压环境胁迫的响应2) 为了量化大肠杆菌渗透分子、其他溶质和拥挤之间的相互作用 在体外用生物聚合物试剂来获得结构 预测/解释溶质对生物聚合过程的影响；以及3) 以定量的体内和体外研究来检验全球 涉及不稳定影响之间的平衡的补偿机制 累积的溶质，排除的溶质的稳定作用，以及 大分子拥挤维持了生物聚合物的结构和功能，使得 细胞在大范围渗透压下的生长速度随着 细胞质水的游离量。体内研究使用的是标准 溶质分析方法和示差放射性同位素分析 隔间水；首席研究员提出了一种定量的 基因芯片技术在分析细胞数量变化中的应用 个体mRNA与生长渗透压的关系。他们使用了一种新的应用程序 渗透压法在体外研究溶质-生物聚合物相互作用及其应用 快速淬火混合及放射性同位素和酶分析表征 生物聚合过程中的溶质效应。 了解大肠杆菌的渗透行为，最好的特征是 生命系统，既具有现实意义，也具有根本意义，既是一种 肾脏和许多其他真核生物的体积和渗透调节模型 细胞以及在对抗渗透诱导的毒力和耐受性方面 大肠杆菌和鼠伤寒沙门氏菌的致病菌株对干燥，热， 过氧化氢和尿素。

DESCRIPTION(from applicant's abstract): In this project the principal investigator and colleagues propose to quantify: 1) the major changes in the types and amounts of cytoplasmic and periplasmic solutes and in amounts of cell and compartment water which allow E. coli to grow over a very wide range of external osmolalities; and 2) the accompanying large changes in turgor pressure across the cell wall. By comparison with in vitro effects of solutes, these large changes in concentrations of cytoplasmic solutes and biopolymers should individually perturb most cell processes, but collectively do not. Equally striking are the large changes in amount of cytoplasmic water, which measurements of turgor pressure show are not accompanied by large changes in water activity. The long term goals are: 1) to understand E. coli as a chemical and osmotic system; 2) to relate studies of solute-biopolymer interactions in vitro to solute effects on biopolymer processes in vitro and in vivo; and 3) to understand the global compensation mechanisms by which cell processes are buffered against changes in solute concentrations, and the molecular basis for the stimulatory effect on growth rate of metabolically-inert "osmoprotectant" solutes. The specific aims are: 1) to determine the physiological and biochemical responses of E. coli to the stress of high and low osmolality environments; 2) to quantify the interactions of E. coli osmolytes, other solutes and crowding agents with biopolymers in vitro in order to obtain structural predictions/interpretations of solute effects on biopolymer processes; and 3) to test by quantitative in vivo and in vitro studies the proposal that global compensation mechanisms involving balances between destabilizing effects of accumulated solutes, stabilizing effects of excluded solutes, and macromolecular crowding maintain biopolymer structure and function, allowing cell growth over a wide range of osmolalities at a rate which increases with the amount of free cytoplasmic water. The in vivo studies use standard analytical methods for solutes and a differential radioisotope assay for compartment water; the principal investigator proposes a quantitative application of gene array technology to analyze changes in amounts of individual mRNAs vs. growth osmolality. They use a novel application of osmometry for in vitro studies of solute-biopolymer interactions and use rapid-quench mixing and radioisotope and enzymatic assays to characterize solute effects on biopolymer processes. An understanding of the osmotic behavior of E. coli, the best characterized living system, is of practical as well as fundamental significance, both as a model for volume and osmotic regulation in kidney and many other eucaryotic cells and in combating both osmotically-induced virulence and tolerance of pathogenic strains of E. coli and Salmonella typhimurium to desiccation, heat, peroxide and urea.