QUANTITATIVE IN VIVO IN VITRO STUDIES OF CELL PROCESSES

描述（来自申请人的摘要）：在这个项目中， 研究人员和同事建议量化：1） 细胞质和周质溶质的类型和数量以及细胞数量 和隔室水，其允许E.大肠杆菌在非常广泛的范围内生长， 外部渗透压;和2）伴随的膨压的巨大变化 穿过细胞壁。通过与溶质的体外作用比较，这些 细胞质溶质和生物聚合物浓度的较大变化应 单独地干扰大多数细胞过程，但集体地不干扰。同样 显著的是细胞质水量的巨大变化， 膨压的测量显示， 水活动。长期目标是：1）了解E。大肠杆菌作为一种化学物质 和渗透系统; 2）涉及溶质-生物聚合物相互作用的研究， 对体外和体内生物聚合物过程的体外至溶质效应;以及3） 了解细胞过程的整体补偿机制， 缓冲溶质浓度的变化，以及 代谢惰性“植物保护剂”对生长速率刺激作用 溶质。 其具体目的是：1）测定其生理生化指标， E的反应。大肠杆菌对高、低渗透压环境胁迫的耐受性; 2） 以定量E.大肠杆菌渗透物、其他溶质和拥挤 试剂与生物聚合物在体外，以获得结构 溶质对生物聚合物过程的影响的预测/解释;以及3） 通过定量体内和体外研究来测试全球 补偿机制，涉及在下列因素的不稳定影响之间取得平衡： 累积的溶质，排除的溶质的稳定作用，以及 大分子拥挤维持生物聚合物的结构和功能， 细胞生长在很宽的渗透压范围内， 游离细胞质水的量。体内研究使用标准 溶质的分析方法和放射性同位素差示分析 隔室水;主要研究人员提出了一个定量的 应用基因芯片技术，分析 单个mRNA与生长渗透压。他们使用了一种新的 用于溶质-生物聚合物相互作用的体外研究的渗透压测定法及其应用 快速淬灭混合和放射性同位素和酶测定来表征 溶质对生物聚合物过程的影响。 对E.大肠杆菌，最好的特点 生命系统，既具有现实意义，又具有根本意义，既作为一个 肾脏和许多其他真核生物的体积和渗透调节模型 细胞，并在打击两个害虫诱导的毒力和耐受性， 致病性E.大肠杆菌和鼠伤寒沙门氏菌干燥，加热， 过氧化物和尿素。

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.