STUDY OF CHANGES IN THE CALCIUM CONCENTRATION OF ERYTHROCYTES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 大肠杆菌的溶血素（HlyA）被认为是一个称为RTX（毒素重复）的毒素家族的原型，RTX是一组具有遗传和结构特征的蛋白质。HlyA是大肠杆菌的重要毒力因子。大肠杆菌肠外感染，如脑膜炎、败血症和泌尿系感染。高浓度的毒素导致几种细胞溶解，如不同物种的红细胞、粒细胞、单核细胞、内皮细胞和肾上皮细胞。在低浓度下，它诱导细胞因子和细胞凋亡的产生。由于已报告其他细胞中的许多亚细胞溶解效应是由细胞内钙的增加触发的，因此在先前对LFD的访问中，我们在与亚溶解浓度的HlyA孵育时跟踪了红细胞内的钙浓度。使用钙指示剂绿色1，2-光子激发和荧光寿命成像显微镜（FLIM）监测钙浓度。在这个机会中，我们提出的证据表明，在sublytic浓度，HlyA诱导兔红细胞中的钙浓度增加，在第一个10秒。 本次访视LFD的目的是研究钙是否通过毒素形成的孔流入红细胞，或者毒素是否激活红细胞中预先存在的阳离子通道。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ¿-hemolysin (HlyA) from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin), a group of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin cause the lysis of several cells as erythrocytes, granulocytes, monocytes, endothelial and renal epithelial cells of different species. At low concentrations it induces the production of cytokines and apoptosis. Since many of the subcytolytic effects in other cells have been reported to be triggered by the increase of intracellular calcium, in a previous visit to the LFD we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA. Calcium concentration was monitored using the calcium indicator Green 1, 2-photon excitation and fluorescence lifetime imaging microscopy (FLIM). In that opportunity we present evidences that, at sublytic concentrations, HlyA induces an increase of calcium concentration in rabbit erythrocytes in the first 10 s. The aim of the present visit to the LFD is to study if the influx of calcium to the erythrocytes is through the pore that the toxin forms or if the toxin activates preexisting cation channels in the erythrocyte.