Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability
|
Author:
Date:
2020-08-20
[Abstract] Stopped-Flow Light Scattering (SFLS) is a method devised to analyze the kinetics of fast chemical reactions that result in a significant change of the average molecular weight and/or in the shape of the reaction substrates. Several modifications of the original stopped-flow system have been made leading to a significant extension of its technical applications. One of these modifications allows the biophysical characterization of the water and solute permeability of biological and artificial membranes. Here, we describe a protocol of SFLS to measure the glycerol permeability of isolated human red blood cells (RBCs) and evaluate the pharmacokinetics properties (selectivity and potency) of isoform-specific inhibitors of AQP3, AQP7 and AQP9, three mammalian aquaglyceroporins ...
[摘要] [摘要] 停流光散射(SFLS)是一种用于分析快速化学反应动力学的方法,该化学反应导致平均分子量和/或反应底物形状发生显着变化。已对原始停流系统进行了几处修改,从而大大扩展了其技术应用范围。这些修饰之一允许水的生物物理表征以及生物膜和人造膜的溶质渗透性。
在这里,我们描述了一种SFLS协议,用于测量分离的人红细胞(RBC)的甘油渗透性,并评估AQP3,AQP7和AQP9的同种型特异性抑制剂的药代动力学特性(选择性和效价),这三种哺乳动物的水甘油糖蛋白允许转运甘油跨膜。在20°C的SFLS设备中,将RBC的悬浮液(1%的血细胞比容)暴露于100 mM甘油的向内定向,并在530 nm的单色波长下记录120 s的散射光强度变化。该设备的死区时间为1.6毫秒,混合效率在不到1毫秒的时间内达到99%。数据拟合到单指数函数和有关的时间constan 吨( ,对应于伴随甘油的进入红细胞的水渗透运动的光散射的细胞膨胀相的秒)进行测定。RBC 的甘油渗透系数(P gly ,cm / s)通过以下公式计算:
P gly = 1 / [[((S / V) ]
其中 (s)是拟合的指数时间常数,S / V是分析的RBC 样本的表面积与体积之比(cm -1 ...
|
|
Stable Isotope Resolved Metabolomics Studies in ex vivo TIssue Slices
|
Author:
Date:
2016-02-05
[Abstract] An important component of this methodology is to assess the role of the tumor microenvironment on tumor growth and survival. To tackle this problem, we have adapted the original approach of Warburg (Warburg, 1923), by combining thin tissue slices with Stable Isotope Resolved Metabolomics (SIRM) to determine detailed metabolic activity of human tissues. SIRM enables the tracing of metabolic transformations of source molecules such as glucose or glutamine over defined time periods, and is a requirement for detailed pathway tracing and flux analysis. In our approach, we maintain freshly resected tissue slices (both cancerous and non- cancerous from the same organ of the same subject) in cell culture media, and treat with appropriate stable isotope-enriched nutrients, e.g., 13 ...
[摘要] 这种方法的一个重要组成部分是评估肿瘤微环境对肿瘤生长和存活的作用。为了解决这个问题,我们采用Warburg(Warburg,1923)的原始方法,通过将薄组织切片与稳定同位素解析代谢组学(SIRM)组合来确定人体组织的详细代谢活性。 SIRM使得能够在定义的时间段内跟踪诸如葡萄糖或谷氨酰胺的来源分子的代谢转化,并且是详细的途径追踪和通量分析的要求。在我们的方法中,我们保持在细胞培养基中新鲜切除的组织切片(来自同一受试者的相同器官的癌性和非癌性),并用适当的稳定的富含同位素的营养物,例如葡萄糖或13 C 15葡萄糖或13 C 15葡萄糖或13 C 15葡萄糖, - 谷氨酰胺。这些切片可存活至少24小时,并使得有可能消除对靶组织代谢的系统影响,同时保持原始的3D细胞结构。因此,它是用于评估治疗剂对靶组织代谢的影响及其对个体患者的治疗功效的极好的临床前平台(Xie等人,2014; Sellers等人,/em>,2015)。
|
|