Abstract

The objective of this project is to test the hypothesis that interaction of mutant and unpaired Hb chains with specific membrane components affects KCI cotransport and deformability of red cells (RBC).
Two Specific Aims will be pursued: 1) to determine the extent to which mutant unpaired Hb chains modify KCI co-transport, membrane deformability and mechanical strength, and 2) to identify membrane components that interact with Hb to mediate these effects. To define (IOV) and resealed membranes in the presence of normal Hb vs mutant Hb and excess alpha and beta globin chains. Differential extraction and readdition of selected membrane skeletal components will test for medication of Hb effects via the membrane skeleton, and maneuvers to block association of Hb chains with integral membrane proteins will test for medication by B3 and glycophrin. Differential effects of various type of Hb on membrane deformability and mechanical strength will be assessed by comparative assays of resealed membranes containing various concentrations and types of Hb, using the ektacytometer, a diffraction viscometer. To identify specific membrane components that interact with Hb to modulate these functions, RBC will be treated with variety of cross-linking agents at low concentrations to stabilize Hb/membrane associations in the intact cell and prevent their disruption during analysis. Hb cross-linking to specific membrane components will be detected by separating membrane proteins by polyacrylamide gel electrophoresis, preparing immunoblots and probing them with anti-Hb and antibodies against membrane components. Apparent complexes between Hb and membrane components will be confirmed by immunoprecipitation with antibody against the membrane component, and probing of immunoblots of the immunoprecipitate using anti-Hb. To probe for interaction of Hb with the protein that mediates KCI cotransport, rabbit polyclonal antibodies against the protein will be prepared, making use of the differential expression of the antigen in high K and low K sheep RBC. It is expected that information derived from these studies will provide new information about the effects of HbS and HbC on membrane function that may be useful in understanding the variable pathophysiologic manifestations in the sickling disorders.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Comprehensive Center (P60)
Project #
5P60HL020985-21
Application #
2781707
Study Section
Project Start
Project End
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
21
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
University of California San Francisco
Department
Type
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143

  Publications

Goodman, Jessica; Hassell, Kathryn; Irwin, David et al. (2014) The splenic syndrome in individuals with sickle cell trait. High Alt Med Biol 15:468-71
James, Ellen Butensky; Vreman, Hendrik J; Wong, Ronald J et al. (2010) Elevated exhaled carbon monoxide concentration in hemoglobinopathies and its relation to red blood cell transfusion therapy. Pediatr Hematol Oncol 27:112-21
Jenkins, Zandra A; Hagar, Ward; Bowlus, Christopher L et al. (2007) Iron homeostasis during transfusional iron overload in beta-thalassemia and sickle cell disease: changes in iron regulatory protein, hepcidin, and ferritin expression. Pediatr Hematol Oncol 24:237-43
Styles, Lori A; Abboud, Miguel; Larkin, Sandra et al. (2007) Transfusion prevents acute chest syndrome predicted by elevated secretory phospholipase A2. Br J Haematol 136:343-4
Kuypers, Frans A; Larkin, Sandra K; Emeis, Jef J et al. (2007) Interaction of an annexin V homodimer (Diannexin) with phosphatidylserine on cell surfaces and consequent antithrombotic activity. Thromb Haemost 97:478-86
Neumayr, Lynne D; Aguilar, Christine; Earles, Ann N et al. (2006) Physical therapy alone compared with core decompression and physical therapy for femoral head osteonecrosis in sickle cell disease. Results of a multicenter study at a mean of three years after treatment. J Bone Joint Surg Am 88:2573-82
Wilson, Leslie S; Moskowitz, Judith Tedlie; Acree, Michael et al. (2005) The economic burden of home care for children with HIV and other chronic illnesses. Am J Public Health 95:1445-52
Vichinsky, Elliott; Butensky, Ellen; Fung, Ellen et al. (2005) Comparison of organ dysfunction in transfused patients with SCD or beta thalassemia. Am J Hematol 80:70-4
Pakbaz, Zahra; Fischer, Roland; Treadwell, Marsha et al. (2005) A simple model to assess and improve adherence to iron chelation therapy with deferoxamine in patients with thalassemia. Ann N Y Acad Sci 1054:486-91
Banerjee, Tinku; Kuypers, Frans A (2004) Reactive oxygen species and phosphatidylserine externalization in murine sickle red cells. Br J Haematol 124:391-402

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