| Determination of Intracellular Ca2+ Concentration in the Human Pathogens Trypanosomatids Leishmania mexicana and Trypanosoma cruzi by the Use of the Fluorescent Ca2+ Indicator Fura-2
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Author:
Date:
2020-09-20
[Abstract] Ca2+ is an essential signaling messenger in all eukariotic cells, playing a pivotal role in many cellular functions as cell growth control (differentiation, fertilization and apoptosis), secretion, gene expression, enzyme regulation, among many others. This basic premise includes trypanosomatids as Trypanosoma cruzi and various species of Leishmania, the causative agents of Chagas disease and leishmaniasis respectively, where intracellular Ca2+ concentration ([Ca2+]i) has been demonstrated to be finely regulated. Nevertheless [Ca2+]i has been difficult to measure because of its very low cytoplasmic concentration (typically around 50-100 nM), when compared to the large concentration in the outside milieu ...
[摘要] [摘要] Ca 2+ 是所有真核细胞中必不可少的信号信使，在许多细胞功能中起着关键作用，例如细胞生长控制（分化，受精和凋亡），分泌，基因表达，酶调控等。这个基本前提包括锥虫如锥虫克氏锥虫和各种物种利什曼原虫，分别南美锥虫病和利什曼病的病原体，其中细胞内Ca 2+ 浓度（[钙2 + ] 我）已被证明被精细地调节。然而，[Ca 2 + ] i 与外界环境中的高浓度（血液中约2 mM）相比，由于其细胞质浓度非常低（通常约为50-100 nM ）而难以测量。细胞内荧光Ca 2+ 敏感指示剂的开发对于实现该目标至关重要。成功的基础是乙酰氧甲基化衍生物前体的合成，该合成物使通常由许多亲水性羧基组成的荧光分子（其非常亲和的Ca 2+ 结合（因此非常亲水））容易穿过质膜。一旦进入细胞内部，非特异性酯酶会将疏水部分从荧光骨架结构中分离出来，释放出羧基，然后将其转化为分子的酸形式，该分子保留在细胞质中并恢复其在Ca中发荧光的能力。2+ 依赖方式。其中，的Fura-2是迄今为止最常用的，因为它是一个比例（两个不同的波长激发和一个发射）的Ca 2+ 与钙指示剂2+ 与内[Ca亲和力兼容2 + ] 我。该协议主要包括用Fura-2加载指数相寄生虫，并通过双波长荧光分光光度计记录[Ca 2 + ] i 中的变化。这种技术允许有价值的信息AB的获取出的[Ca 2 + ] 我Ç ...
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| Aggregation Prevention Assay for Chaperone Activity of Proteins Using Spectroflurometry
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Author:
Date:
2017-01-20
[Abstract] The ability to stabilize other proteins against thermal aggregation is one of the major characteristics of chaperone proteins. Molecular chaperones bind to nonnative conformations of proteins. Folding of the substrate is triggered by a dynamic association and dissociation cycles which keep the substrate protein on track of the folding pathway (Figure 1). Usually molecular chaperones exhibit differential affinities with different conformations of the substrate. With the exception of the sHsp family of molecular chaperones, the shift from a high-affinity binding state to the low-affinity release state is triggered by ATP binding and hydrolysis (Haselback and Buchner, 2015). Aggregation prevention assay is a simple, yet definitive assay to determine the chaperone activity of heat labile ...
[摘要] 稳定其他蛋白质抵抗热聚集的能力是伴侣蛋白质的主要特征之一。分子伴侣与蛋白质的非构象构象结合。通过动态关联和解离循环来触发底物的折叠，这些循环使底物蛋白保持在折叠通路上（图1）。通常分子伴侣表现出与底物不同构象的差异亲和力。除了分子伴侣的sHsp家族之外，从高亲和力结合状态向低亲和力释放状态的转变由ATP结合和水解引发（Haselback和Buchner，2015）。聚集预防测定是一种简单但尚未确定的测定法，用于确定热不稳定蛋白质如麦芽糖糊精葡糖苷酶（MalZ），柠檬酸盐合酶（CS）和Nde I的伴侣活性。这是基于具有伴侣相似活性的蛋白质预防来自热聚集的蛋白质底物（MalZ，CS和Nde I）的前提。在这里，我们描述了使用我们实验室鉴定的两种不同伴侣蛋白，抵抗素和MoxR1的聚合预防测定的详细方案。分析抗性蛋白（hRes）和MoxR1 a结核分枝杆菌蛋白质对预防MalZ /柠檬酸合酶（CS）/ Nde I聚集的影响。
背景 ![]()
图1.分子伴侣的作用机制。柠檬酸合酶通过越来越多的结构化中间体（I <1>，2 ...
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| Loading of Cells with Fluorescent Probe to Study Intracellular Acid-base Homeostasis in Lactic Acid Bacteria
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Author:
Date:
2015-01-20
[Abstract] Here we describe a protocol which we have used to study the homeostasis intracellular in vivo in lactic acid bacteria (LAB) using a fluorescent probe. This type of probes can be used for determining changes in the pH of cytoplasm with high sensitivity, temporal resolution and technical simplicity as well as accessing the rate of change of intracellular pH in response to a stimulus from kinetic measurements on short time scales (Breeuwer et al., 1996; Molenaar et al., 1991). This protocol has been designed to measure the intracellular pH using the pH-sensitive fluorescent probe 2´,7´-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) in LAB, Enterococcus faecalis (E. faecalis), Lactococcus lactis (L. lactis) and Lactobacillus ...
[摘要] 在这里我们描述了一个协议，我们已经用来研究乳酸细菌（LAB）使用荧光探针在体内细胞内体内平衡。 这种类型的探针可用于以高灵敏度，时间分辨率和技术简单性确定细胞质pH的变化，以及响应于来自短时间尺度上的动力学测量的刺激，获得细胞内pH的变化速率（Breeuwer& et al。，1996; Molenaar et al。，1991）。 该方案已经设计为使用pH敏感性荧光探针在LAB中的2'-，7'-双 - （2-羧乙基）-5（和-6） - 羧基荧光素（BCECF），粪肠球菌 （ E.cfaecalis ），乳酸乳球菌（<乳酸乳球菌）和干酪乳杆菌 em=""> L。casei ）。
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