This proposal is designed to study the complex interplay of the components of the human red blood cell (RBC) membrane that result in its unique functional capacities. Abnormalities of these capabilities are very well exemplified by the pathophysiology of the human thalassemias, disorders of world wide importance, that cause anemia sufficient to produce morbidity and even mortality. While these inherited disorders may eventually be managed by the tools of molecular biology, we propose that they can be ameliorated by applying what we plan to learn in this project. The pathophysiology of alpha and beta thalassemia is different, and it is hypothesized that each is caused by excess beta or alpha globin chains binding to the RBC membrane and its skeleton, producing misshapen RBC with altered cellular hydration and deranged membrane material properties, leading to intramedullary hemolysis (ineffective erythropoiesis) and peripheral red cell destruction. Experiments are proposed to determine the mechanism underlying the apoptosis contributes to intramedullary lysis in beta thalassemia major and to contrast alpha vs. beta thalassemia and discover: how much of the excess unmatched globin chains bind; what specific derangements of membrane function are produced and how; which proteins show evidence of oxidant attack, whether the RBC abnormalities are preconditioned by events occurring in the marrow during erythroid maturation (beta thalassemia) perhaps altering the orderly assembly of membrane proteins, or whether senescence is particularly damaging (alpha thalassemia). Methods to be used include: analysis of membrane proteins by gel electrophoresis and Western blotting; isolation and purification of populations of thalassemic reticulocytes and beta thalassemic marrow erythroblasts for analysis; Northern blot studies to detect mRNA's mediating apoptotic programs; immunofluorescent confocal laser microscopy to identify protein distribution in marrow erythroblasts and circulating reticulocytes and RBC; ektacytometry and micropipette analysis to provide correlative data on cellular and membrane material properties; methods to identify protein thiol oxidation and tyrosine oxidation. Because we will compare marrow erythroblasts with reticulocytes and mature RBC, we can detect the site and stage of the alterations. The results should allow us to pinpoint key elements in the pathophysiology and perhaps clarify which of the current therapeutic clinical protocols is likely to produce cellular and thus clinical benefit.
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