STUDIES OF SICKLING MECHANISMS & RED CELL MEMBRANES

本项目涉及镰刀的分子和细胞病理生理学。 细胞疾病和相关的红细胞疾病，主要集中在 循环中致密、脱水的SS细胞的起源，并强调 与其他红细胞疾病和细胞生理学有关的基本问题：I. 镰刀如何影响SS网织红细胞异质性的产生 不同的成熟红细胞亚群？Ii.这是什么性质的 镰刀诱导的通透途径及其如何产生观察到的 离子传输和含量的异常？三、队形如何形成， 脱氧-Hb S聚合物在固体悬浮液中的击穿和可能的结构变化 细胞直接改变细胞体积、离子分布和新陈代谢？ 为了达到这些目的，研究将：一、进一步发展我们的假设 最密集SS细胞的Rtic起源：使用我们新的模拟 预测条件的非稳态红细胞和RETIC模型 具有不同传输特性的不同SS retic；标识其 运输异质性，钙离子和镁离子代谢，以及我们的新发现 Ca2=-敏感的氯离子渗透性；并评估“应力阻力”在 血管闭塞症的脱水过程和致密细胞的变化 镰刀危机；II.镰刀诱导的通透途径研究 (“镰刀”)和SS的离子、pH和容量异常的机制 网状细胞和较老的细胞；描述了我们新发现的一种 在无钙条件下放大的镰状Na/K，用于运输和 泄漏的超微结构研究；单细胞荧光成像 含钙离子螯合剂，定位钙离子渗漏及分布 正常细胞和镰状细胞的Pca和稳态[Ca~(2+)]；测量PO2‘S 需要聚合物组分才能渗透不同密度的S细胞， 检验致密SS细胞可能在大多数情况下通透性的假设 循环中的时间.由内向外的囊泡中K：C1共转运的试验 来自网状细胞和成熟的红细胞，如果它能被暴露在 体外培养Hb、S或C型；检测我们新发现的肌苷是否升高 SS细胞中的单磷酸反映了它们对[Ca+]的暴露；和研究 用电子顺磁法研究SS组分中MetHb和半铬的形成 共鸣。应用新的准确方法来(I)估计 Hb在聚合物中的浓度Cp；(Ii)测试Cp是否随 影响聚合物溶解度的晶胞因素(C)；(Iii)估计 将HBs a、F和C加入到聚合物中(作为杂化物，四聚体， 以及在T或R构象中)，以及掺入低和中等分子量 进入聚合物缔合水室(PWC)的物质，源自 CP)，不包括可溶大分子。然后我们可以：预测 聚合的渗透效应作为细胞MCHC的函数，并测试 直接预测；CP和非S Hb掺入测量，研究 用电子显微镜观察聚合物的超微结构，并评价糖酵解作用 在致密的SS细胞中由于聚合而导致的酶-底物重分布。

This project concerns the molecular and cellular pathophysiology of sickle cell disease and related red cell disorders, with a major focus on the origin of dense, dehydrated SS cells in the circulation, and emphasis on basic issues relevant to other red cell disorders and cell physiology: I. How does sickling act on SS reticulocyte (retic) heterogeneity to generate different mature red cell subpopulations? II. What is the nature of the sickling-induced permeability pathway and how does it produce the observed abnormalities of ion transport and content? III. How does the formation, breakdown and possible structural variations of deoxy-Hb S polymers in SS cells directly alter cell volume, ion distribution, and metabolism? Studies to these ends will: I. Develop further our hypothesis of a direct retic origin of most dense SS cells: use simulations of our new non-steady-state red cell and retic models to predict conditions to separate SS retics with different transport properties; identify their transport heterogeneities, Ca2= and Mg2= metabolism, and our newly found Ca2=-sensitive Cl permeability; and assess the role of "stress retics" in the dehydration process, and in dense cell variations in vasoocclusive sickle crises; II. Study the sickling-induced permeability pathway ("Psickle") and the mechanisms of ion, pH and volume abnormalities in SS retics and older cells; characterize our newly found heparin effect of a magnified Psickle-Na/K in the absence of Ca2+, for transport and ultrastructural studies of the leak; fluorescence-image single cells containing Ca2+ chelators, to locate the Ca2+ leaks and the distribution of Pca and steady state [Ca2+] in normal and sickle cells; measure what pO2's and polymer fractions are needed to permeabilize different density S cells, testing the hypothesis that dense SS cells may be permeabilized most of the time in the circulation; test for K:C1 cotransport in inside-out vesicles from retics and mature red cells, and if it can be activated by exposure to Hb S or C in vitro; test whether our newly found increased inosine monophosphate in SS cells reflects their exposure to [Ca2+]; and study metHb and hemichrome formation in SS fractions using electron paramagnetic resonance. III. Apply new accurate methods to (i) estimate the concentration of Hb in the polymer, Cp; (ii) test whether Cp changes with cell factors affecting polymer solubility (C); (iii) estimate the incorporation of Hbs a, F and C into the polymer (as hybrids, tetramers, and in T or R conformations), and incorporation of low and intermediate MW substances into the polymer-associates water compartment (PWC, derived from Cp) which excludes soluble macromolecules. We can then: predict the osmotic effects of polymerization as a function of cell MCHC, and test the predictions directly; with Cp and non-S Hb incorporation measured, study polymer ultrastructure by electronmicroscopy, and assess glycolytic effects of enzyme-substrate redistribution due to polymerization in dense SS cells.