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1
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Tahir UA, Katz DH, Avila-Pachecho J, Bick AG, Pampana A, Robbins JM, Yu Z, Chen ZZ, Benson MD, Cruz DE, Ngo D, Deng S, Shi X, Zheng S, Eisman AS, Farrell L, Hall ME, Correa A, Tracy RP, Durda P, Taylor KD, Liu Y, Johnson WC, Guo X, Yao J, Chen YDI, Manichaikul AW, Ruberg FL, Blaner WS, Jain D, Bouchard C, Sarzynski MA, Rich SS, Rotter JI, Wang TJ, Wilson JG, Clish CB, Natarajan P, Gerszten RE. Whole Genome Association Study of the Plasma Metabolome Identifies Metabolites Linked to Cardiometabolic Disease in Black Individuals. Nat Commun 2022; 13:4923. [PMID: 35995766 PMCID: PMC9395431 DOI: 10.1038/s41467-022-32275-3] [Citation(s) in RCA: 32] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Accepted: 07/25/2022] [Indexed: 01/27/2023] Open
Abstract
Integrating genetic information with metabolomics has provided new insights into genes affecting human metabolism. However, gene-metabolite integration has been primarily studied in individuals of European Ancestry, limiting the opportunity to leverage genomic diversity for discovery. In addition, these analyses have principally involved known metabolites, with the majority of the profiled peaks left unannotated. Here, we perform a whole genome association study of 2,291 metabolite peaks (known and unknown features) in 2,466 Black individuals from the Jackson Heart Study. We identify 519 locus-metabolite associations for 427 metabolite peaks and validate our findings in two multi-ethnic cohorts. A significant proportion of these associations are in ancestry specific alleles including findings in APOE, TTR and CD36. We leverage tandem mass spectrometry to annotate unknown metabolites, providing new insight into hereditary diseases including transthyretin amyloidosis and sickle cell disease. Our integrative omics approach leverages genomic diversity to provide novel insights into diverse cardiometabolic diseases.
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Affiliation(s)
- Usman A Tahir
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Daniel H Katz
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | | | | | - Akhil Pampana
- Broad Institute of Harvard and MIT, Cambridge, MA, US
| | - Jeremy M Robbins
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Zhi Yu
- Broad Institute of Harvard and MIT, Cambridge, MA, US
| | - Zsu-Zsu Chen
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Mark D Benson
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Daniel E Cruz
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Debby Ngo
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Shuliang Deng
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Xu Shi
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Shuning Zheng
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Aaron S Eisman
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Laurie Farrell
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Michael E Hall
- University of Mississippi Medical Center, Jackson, MS, US
| | - Adolfo Correa
- University of Mississippi Medical Center, Jackson, MS, US
| | - Russell P Tracy
- Department of Pathology Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, VT, US
| | - Peter Durda
- Department of Pathology Laboratory Medicine, Larner College of Medicine, University of Vermont, Burlington, VT, US
| | - Kent D Taylor
- The Institute for Translational Genomics and Population Sciences, Department of Pediatrics, The Lundquist Institute for Biomedical Innovation at Harbor UCLA Medical Center, Torrance, CA, US
| | - Yongmei Liu
- Department of Medicine, Division of Cardiology, Duke Molecular Physiology Institute, Duke University Medical Center, Durham, NC, US
| | - W Craig Johnson
- Department of Biostatistics, University of Washington, Seattle, WA, US
| | - Xiuqing Guo
- The Institute for Translational Genomics and Population Sciences, Department of Pediatrics, The Lundquist Institute for Biomedical Innovation at Harbor UCLA Medical Center, Torrance, CA, US
| | - Jie Yao
- The Institute for Translational Genomics and Population Sciences, Department of Pediatrics, The Lundquist Institute for Biomedical Innovation at Harbor UCLA Medical Center, Torrance, CA, US
| | - Yii-Der Ida Chen
- The Institute for Translational Genomics and Population Sciences, Department of Pediatrics, The Lundquist Institute for Biomedical Innovation at Harbor UCLA Medical Center, Torrance, CA, US
| | - Ani W Manichaikul
- Center for Public Health Genomics, University of Virginia, Charlottesville, Virginia, US
- Division of Biostatistics and Epidemiology, Department of Public Health Sciences, University of Virginia, Charlottesville, Virginia, US
| | - Frederick L Ruberg
- Section of Cardiovascular Medicine, Boston University School of Medicine and Boston Medical Center, Boston, MA, US
| | | | - Deepti Jain
- University of Washington, Seattle, Washington, US
| | - Claude Bouchard
- Human Genomic Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA, US
| | - Mark A Sarzynski
- Department of Exercise Science, University of South Carolina, Columbia, SC, US
| | - Stephen S Rich
- Center for Public Health Genomics, University of Virginia, Charlottesville, Virginia, US
- Division of Biostatistics and Epidemiology, Department of Public Health Sciences, University of Virginia, Charlottesville, Virginia, US
| | - Jerome I Rotter
- The Institute for Translational Genomics and Population Sciences, Department of Pediatrics, The Lundquist Institute for Biomedical Innovation at Harbor UCLA Medical Center, Torrance, CA, US
| | - Thomas J Wang
- Department of Medicine, UT Southwestern Medical Center, Dallas, TX, US
| | - James G Wilson
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US
| | - Clary B Clish
- Broad Institute of Harvard and MIT, Cambridge, MA, US
| | - Pradeep Natarajan
- Broad Institute of Harvard and MIT, Cambridge, MA, US
- Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, US
| | - Robert E Gerszten
- Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, US.
- Broad Institute of Harvard and MIT, Cambridge, MA, US.
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Complement in sickle cell disease and targeted therapy: I know one thing, that I know nothing. Blood Rev 2021; 48:100805. [PMID: 33504459 DOI: 10.1016/j.blre.2021.100805] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2020] [Revised: 11/10/2020] [Accepted: 01/13/2021] [Indexed: 12/19/2022]
Abstract
Sickle cell disease (SCD) is a common inherited clinical syndrome, characterized by the presence of hemoglobin S. Anemia, susceptibility to infections and episodes of vaso-occlusive crisis (VOC) are among its features. Since SCD complications (VOC or delayed hemolytic transfusion reaction/DHTR) lead to significant morbidity and mortality, a number of studies have addressed their pathophysiology Although SCD pathophysiology has been mainly attributed to the interaction between sickle cells and neutrophils, platelets or endothelial cells in small vessels leading to hemolysis, the role of complement activation has been increasingly investigated. Importantly, complement inhibition with eculizumab has shown beneficial effects in DHTR. Given the unmet clinical need of novel therapeutics in SCD, our review summarizes current understanding of (a) complement system for the clinician, (b) complement activation in SCD both in asymptomatic state and severe clinical manifestations, (c) probable underlying mechanisms of complement activation in SCD, and (d) new therapeutic perspective of complement inhibition.
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Smith AS, Pal K, Nowak RB, Demenko A, Zaninetti C, Da Costa L, Favier R, Pecci A, Fowler VM. MYH9-related disease mutations cause abnormal red blood cell morphology through increased myosin-actin binding at the membrane. Am J Hematol 2019; 94:667-677. [PMID: 30916803 PMCID: PMC6510596 DOI: 10.1002/ajh.25472] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2018] [Revised: 03/21/2019] [Accepted: 03/25/2019] [Indexed: 01/29/2023]
Abstract
MYH9-related disease (MYH9-RD) is a rare, autosomal dominant disorder caused by mutations in MYH9, the gene encoding the actin-activated motor protein non-muscle myosin IIA (NMIIA). MYH9-RD patients suffer from bleeding syndromes, progressive kidney disease, deafness, and/or cataracts, but the impact of MYH9 mutations on other NMIIA-expressing tissues remains unknown. In human red blood cells (RBCs), NMIIA assembles into bipolar filaments and binds to actin filaments (F-actin) in the spectrin-F-actin membrane skeleton to control RBC biconcave disk shape and deformability. Here, we tested the effects of MYH9 mutations in different NMIIA domains (motor, coiled-coil rod, or non-helical tail) on RBC NMIIA function. We found that MYH9-RD does not cause clinically significant anemia and that patient RBCs have normal osmotic deformability as well as normal membrane skeleton composition and micron-scale distribution. However, analysis of complete blood count data and peripheral blood smears revealed reduced hemoglobin content and elongated shapes, respectively, of MYH9-RD RBCs. Patients with mutations in the NMIIA motor domain had the highest numbers of elongated RBCs. Patients with mutations in the motor domain also had elevated association of NMIIA with F-actin at the RBC membrane. Our findings support a central role for motor domain activity in NMIIA regulation of RBC shape and define a new sub-clinical phenotype of MYH9-RD.
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Affiliation(s)
- Alyson S. Smith
- Department of Molecular Medicine, The Scripps Research
Institute, La Jolla, CA 92037
| | - Kasturi Pal
- Department of Molecular Medicine, The Scripps Research
Institute, La Jolla, CA 92037
| | - Roberta B. Nowak
- Department of Molecular Medicine, The Scripps Research
Institute, La Jolla, CA 92037
| | - Anastasiya Demenko
- Department of Molecular Medicine, The Scripps Research
Institute, La Jolla, CA 92037
| | - Carlo Zaninetti
- Department of Internal Medicine, IRCCS Policlinico San
Matteo Foundation and University of Pavia, Pavia, Italy
| | - Lydie Da Costa
- AP-HP, Service d’Hématologie Biologique,
Hôpital R. Debré, Paris F-75019, France; Université Paris 7,
Sorbonne Paris Cité, Paris F-75010, France; INSERM U1134, INTS, F-75015,
France; Laboratoire d’Excellence GR-Ex, France
| | - Remi Favier
- Assistance Publique-Hôpitaux de Paris, Armand
Trousseau Children Hospital, French Reference Center for platelet disorders, Paris,
75012, France
| | - Alessandro Pecci
- Department of Internal Medicine, IRCCS Policlinico San
Matteo Foundation and University of Pavia, Pavia, Italy
| | - Velia M. Fowler
- Department of Molecular Medicine, The Scripps Research
Institute, La Jolla, CA 92037
- Department of Biological Sciences, University of Delaware, Newark, DE 19711
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Smith OS, Ajose OA, Adegoke SA, Adegoke OA, Adedeji TA, Oderinu KA. Plasma level of antioxidants is related to frequency of vaso-occlusive crises in children with sickle cell anaemia in steady state in Nigeria. PEDIATRIC HEMATOLOGY ONCOLOGY JOURNAL 2019. [DOI: 10.1016/j.phoj.2019.03.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
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Oztas Y, Yalcinkaya A. Oxidative alterations in sickle cell disease: Possible involvement in disease pathogenesis. World J Hematol 2017; 6:55-61. [DOI: 10.5315/wjh.v6.i3.55] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/08/2017] [Revised: 04/25/2017] [Accepted: 05/22/2017] [Indexed: 02/05/2023] Open
Abstract
Sickle cell disease (SCD) is the first molecular disease in the literature. Although the structural alteration and dysfunction of the sickle hemoglobin (HbS) are well understood, the many factors modifying the clinical signs and symptoms of the disease are under investigation. Besides having an abnormal electrophoretic mobility and solubility, HbS is unstable. The autooxidation rate of the abnormal HbS has been reported to be almost two times of the normal. There are two more components of the oxidative damage in SCD: Free radical induced oxidative damage during vaso-occlusion induced ischemia-reperfusion injury and decreased antioxidant capacity in the erythrocyte and in the circulation. We will discuss the effects of oxidative alterations in the erythrocyte and in the plasma of SCD patients in this review.
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Spector J, Kodippili GC, Ritchie K, Low PS. Single Molecule Studies of the Diffusion of Band 3 in Sickle Cell Erythrocytes. PLoS One 2016; 11:e0162514. [PMID: 27598991 PMCID: PMC5012561 DOI: 10.1371/journal.pone.0162514] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2016] [Accepted: 08/24/2016] [Indexed: 01/29/2023] Open
Abstract
Sickle cell disease (SCD) is caused by an inherited mutation in hemoglobin that leads to sickle hemoglobin (HbS) polymerization and premature HbS denaturation. Previous publications have shown that HbS denaturation is followed by binding of denatured HbS (a.k.a. hemichromes) to band 3, the consequent clustering of band 3 in the plane of the erythrocyte membrane that in turn promotes binding of autologous antibodies to the clustered band 3, and removal of the antibody-coated erythrocytes from circulation. Although each step of the above process has been individually demonstrated, the fraction of band 3 that is altered by association with denatured HbS has never been determined. For this purpose, we evaluated the lateral diffusion of band 3 in normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and hemoglobin SC erythrocytes (HbSC) in order to estimate the fraction of band 3 that was diffusing more slowly due to hemichrome-induced clustering. We labeled fewer than ten band 3 molecules per intact erythrocyte with a quantum dot to avoid perturbing membrane structure and we then monitored band 3 lateral diffusion by single particle tracking. We report here that the size of the slowly diffusing population of band 3 increases in the sequence: normal cells<HbSC<RSC<ISC. We also demonstrate that the size of the compartment in which band 3 is free to diffuse decreases roughly in the same order, with band 3 diffusing in two compartments of sizes 35 and 71 nm in normal cells, but only a single compartment in HbSC cells (58 nm), RSC (45 nm) and ISC (36 nm). These data suggest that the mobility of band 3 is increasingly constrained during SCD progression, suggesting a global impact of the mutated hemoglobin on erythrocyte membrane properties.
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MESH Headings
- Anemia, Sickle Cell/blood
- Anemia, Sickle Cell/pathology
- Anion Exchange Protein 1, Erythrocyte/chemistry
- Anion Exchange Protein 1, Erythrocyte/metabolism
- Cells, Cultured
- Diffusion
- Erythrocyte Membrane/chemistry
- Erythrocyte Membrane/metabolism
- Erythrocyte Membrane/ultrastructure
- Erythrocytes, Abnormal/chemistry
- Erythrocytes, Abnormal/metabolism
- Erythrocytes, Abnormal/ultrastructure
- Hemeproteins/chemistry
- Hemeproteins/metabolism
- Hemoglobin, Sickle/chemistry
- Hemoglobin, Sickle/metabolism
- Humans
- Molecular Probes/chemistry
- Quantum Dots/chemistry
- Single Molecule Imaging/methods
- Staining and Labeling/methods
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Affiliation(s)
- Jeff Spector
- Department of Physics, Purdue University, West Lafayette, IN, 47907, United States of America
| | - Gayani C. Kodippili
- Department of Chemistry, Purdue University, West Lafayette, IN, 47907, United States of America
| | - Ken Ritchie
- Department of Physics, Purdue University, West Lafayette, IN, 47907, United States of America
| | - Philip S. Low
- Department of Chemistry, Purdue University, West Lafayette, IN, 47907, United States of America
- * E-mail:
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Al-Naama LM, Hassan MK, Mehdi JK. Association of erythrocytes antioxidant enzymes and their cofactors with markers of oxidative stress in patients with sickle cell anemia. Qatar Med J 2016; 2015:14. [PMID: 26835411 PMCID: PMC4719435 DOI: 10.5339/qmj.2015.14] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2015] [Accepted: 11/19/2015] [Indexed: 01/29/2023] Open
Abstract
BACKGROUND Sickle cell anemia (SCA) is an inherited blood disease with known complications as a result of certain pathophysiological dysfunctions. It has been suggested that an increase in oxidative stress contributes to the incidence of these changes. OBJECTIVES This study investigated the oxidant/antioxidant status of patients with SCA, and evaluated the effect of SCA on antioxidant enzymes and their cofactors. METHODS The study included 42 patients with SCA (in steady state), and a control group of 50 age-matched individuals without SCA. Serum malondialdehyde (MDA), copper, zinc, ferritin and iron levels, red blood cell (RBC) superoxide dismutase (SOD) and catalase levels were measured for the SCA and control groups. RESULTS Significantly lower levels of antioxidant enzymes (RBC SOD and catalase) and higher serum MDA levels (biomarker of oxidative stress) were found in SCA patients compared to the control group (all p < 0.001). Increased levels of serum ferritin, iron and copper and decreased zinc concentrations were also found in the SCA patients compared to the control group (all p < 0.001). In the SCA group, there were significant negative correlations between MDA levels and RBC SOD, RBC catalase, and serum zinc levels (p < 0.01), while a significant positive correlation between MDA with serum copper and iron levels (p < 0.01) was observed. CONCLUSION SCA is associated with alterations in markers of oxidative stress including an increased MDA level, decreased antioxidant enzyme levels, and altered levels of enzyme cofactors (zinc, copper, and iron). This suggests that these antioxidant enzymes could be used as effective therapeutic targets for the treatment of this disease and supplementation of patients with substances with antioxidant properties may reduce the complications of this disease.
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Affiliation(s)
| | | | - Jawad K. Mehdi
- Department of Biochemistry, Health and Medical Technical College, Basrah, Iraq
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Singh P, Pande R. DNA Folding Transition in Presence of Naphthylhydroxamic Acids as Revealed by Fluorescence Microscopic Single Molecule Observation Method. J Fluoresc 2015; 26:67-73. [DOI: 10.1007/s10895-015-1661-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2015] [Accepted: 09/14/2015] [Indexed: 10/22/2022]
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Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease. Blood 2015; 125:3805-14. [PMID: 25827830 DOI: 10.1182/blood-2014-07-589283] [Citation(s) in RCA: 208] [Impact Index Per Article: 20.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2014] [Accepted: 03/12/2015] [Indexed: 01/26/2023] Open
Abstract
Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD.
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Frumence E, Genetet S, Ripoche P, Iolascon A, Andolfo I, Le Van Kim C, Colin Y, Mouro-Chanteloup I, Lopez C. Rapid Cl−/HCO3−exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells. Am J Physiol Cell Physiol 2013; 305:C654-62. [DOI: 10.1152/ajpcell.00142.2013] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.
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Affiliation(s)
- Etienne Frumence
- Inserm U665, Paris, France
- Université Paris Diderot, Sorbonne Paris Cité, UMR-S665, Paris, France
- Institut National de la Transfusion Sanguine, Paris, France
- Laboratoire d'Excellence GR-Ex., Paris, France
- Université de la Réunion, Saint-Denis, France; and
| | - Sandrine Genetet
- Inserm U665, Paris, France
- Université Paris Diderot, Sorbonne Paris Cité, UMR-S665, Paris, France
- Institut National de la Transfusion Sanguine, Paris, France
- Laboratoire d'Excellence GR-Ex., Paris, France
| | - Pierre Ripoche
- Inserm U665, Paris, France
- Université Paris Diderot, Sorbonne Paris Cité, UMR-S665, Paris, France
- Institut National de la Transfusion Sanguine, Paris, France
- Laboratoire d'Excellence GR-Ex., Paris, France
| | - Achille Iolascon
- Chair of Medical Genetics, Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, and CEINGE-Advanced Biotechnologies, Naples, Italy
| | - Immacolata Andolfo
- Chair of Medical Genetics, Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, and CEINGE-Advanced Biotechnologies, Naples, Italy
| | - Caroline Le Van Kim
- Inserm U665, Paris, France
- Université Paris Diderot, Sorbonne Paris Cité, UMR-S665, Paris, France
- Institut National de la Transfusion Sanguine, Paris, France
- Laboratoire d'Excellence GR-Ex., Paris, France
| | - Yves Colin
- Inserm U665, Paris, France
- Université Paris Diderot, Sorbonne Paris Cité, UMR-S665, Paris, France
- Institut National de la Transfusion Sanguine, Paris, France
- Laboratoire d'Excellence GR-Ex., Paris, France
| | - Isabelle Mouro-Chanteloup
- Inserm U665, Paris, France
- Université Paris Diderot, Sorbonne Paris Cité, UMR-S665, Paris, France
- Institut National de la Transfusion Sanguine, Paris, France
- Laboratoire d'Excellence GR-Ex., Paris, France
| | - Claude Lopez
- Inserm U665, Paris, France
- Université Paris Diderot, Sorbonne Paris Cité, UMR-S665, Paris, France
- Institut National de la Transfusion Sanguine, Paris, France
- Laboratoire d'Excellence GR-Ex., Paris, France
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Good MF, Reiman JM, Rodriguez IB, Ito K, Yanow SK, El-Deeb IM, Batzloff MR, Stanisic DI, Engwerda C, Spithill T, Hoffman SL, Lee M, McPhun V. Cross-species malaria immunity induced by chemically attenuated parasites. J Clin Invest 2013; 123:66634. [PMID: 23863622 PMCID: PMC4011145 DOI: 10.1172/jci66634] [Citation(s) in RCA: 68] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2012] [Accepted: 04/26/2013] [Indexed: 01/29/2023] Open
Abstract
Vaccine development for the blood stages of malaria has focused on the induction of antibodies to parasite surface antigens, most of which are highly polymorphic. An alternate strategy has evolved from observations that low-density infections can induce antibody-independent immunity to different strains. To test this strategy, we treated parasitized red blood cells from the rodent parasite Plasmodium chabaudi with seco-cyclopropyl pyrrolo indole analogs. These drugs irreversibly alkylate parasite DNA, blocking their ability to replicate. After administration in mice, DNA from the vaccine could be detected in the blood for over 110 days and a single vaccination induced profound immunity to different malaria parasite species. Immunity was mediated by CD4+ T cells and was dependent on the red blood cell membrane remaining intact. The human parasite, Plasmodium falciparum, could also be attenuated by treatment with seco-cyclopropyl pyrrolo indole analogs. These data demonstrate that vaccination with chemically attenuated parasites induces protective immunity and provide a compelling rationale for testing a blood-stage parasite-based vaccine targeting human Plasmodium species.
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Stuart MJ, Yamaja Setty BN. Hemostatic Alterations in Sickle Cell Disease: Relationships to Disease Pathophysiology. ACTA ACUST UNITED AC 2010. [DOI: 10.1080/15513810109168816] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
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Rotter MA, Chu H, Low PS, Ferrone FA. Band 3 catalyzes sickle hemoglobin polymerization. Biophys Chem 2009; 146:55-9. [PMID: 19880238 DOI: 10.1016/j.bpc.2009.10.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2009] [Revised: 10/13/2009] [Accepted: 10/13/2009] [Indexed: 12/27/2022]
Abstract
We have measured homogeneous and heterogeneous nucleation rates of sickle hemoglobin (HbS) in the presence of a strongly binding deletion mutant of the cytoplasmic domain of band 3 (cdb3), a membrane protein known to form dimers and to bind 2 HbS molecules to such a dimer, and we find that it accelerated both rates by a factor of 2. A weakly binding mutant, in contrast showed no impact on nucleation rates, contrary to naïve expectations of a slight enhancement based on the molecular crowding of the solution by the mutant. We find we can explain these phenomena by a model of HbS-cdb3 interaction in which the strong binding mutant, by stabilizing an HbS dimer, catalyzes the nucleation process, while the weak mutant binds only 1 HbS molecule, effectively inactivating it and thereby compensating for the crowding of the solution by the cdb3. The catalytic behavior we observe could play a role in intracellular processes.
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Affiliation(s)
- Maria A Rotter
- Department of Physics, Drexel University, Philadelphia, PA 19104, United States
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Balzan S, Del Carratore R, Nicolini G, Forini F, Lubrano V, Simili M, Benedetti PA, Iervasi G. TSH induces co-localization of TSH receptor and Na/K-ATPase in human erythrocytes. Cell Biochem Funct 2009; 27:259-63. [PMID: 19466745 DOI: 10.1002/cbf.1567] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Thyroid stimulating hormone (TSH) binds to a specific TSH receptor (TSHR) which activates adenylate cyclase and increases cAMP levels in thyroidal cells. Recent studies have reported the presence of TSH receptor in several extra-thyroidal cell types, including erythrocytes. We have previously suggested that TSH is able to influence the erythrocyte Na/K-ATPase ouabain binding properties through a receptor mediated mechanism. The direct interaction of TSH receptor with the Na/K-pump and a functional role of TSHR in erythrocytes was not demonstrated. The interaction of TSH receptor with Na/K-pump and a TSHR functional role are not yet demonstrated in erythrocytes. In this study, we examined the interaction between the two receptors after TSH treatment using immunofluorescence coupled to confocal microscopy and a co-immunoprecipitation technique. The cAMP dependent signalling after TSH treatment was measured to verify TSHR functionality. We found that TSH receptor and Na/K-ATPase are localized on the membranes of both erythrocytes and erythrocyte ghosts; TSH receptor responds to TSH treatment by increasing intracellular cAMP levels from two to tenfold. In ghost membranes TSH treatment enhances up to three fold co-localization of TSHR with Na/K-ATPase and co-immunoprecipitation confirms their direct physical interaction. In conclusion our results are compatible with the existence, in erythrocytes, of a functional TSHR that interacts with Na/K-ATPase after TSH treatment, thus suggesting a novel cell signalling pathway, potentially active in local circulatory control.
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15
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Sen G, Biswas D, Ray M, Biswas T. Albumin–quercetin combination offers a therapeutic advantage in the prevention of reduced survival of erythrocytes in visceral leishmaniasis. Blood Cells Mol Dis 2007; 39:245-54. [PMID: 17644434 DOI: 10.1016/j.bcmd.2007.06.008] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2007] [Revised: 06/18/2007] [Accepted: 06/18/2007] [Indexed: 10/23/2022]
Abstract
Visceral leishmaniasis is associated with the reduced survival of erythrocytes, the cause of which remains to be fully explored. Here, we described the mechanism underlying the shortened lifespan of erythrocytes in visceral leishmaniasis and proposed a combination therapy with quercetin and hamster serum albumin towards its rectification. Decreased redox potential in erythrocytes followed by oxidative denaturation of hemoglobin and pathologic association of iron with the cell membrane facilitated premature hemolysis during leishmanial infection. Recently, we have reported the therapeutic efficacy of quercetin in arresting the enhanced destruction of erythrocytes in visceral leishmaniasis. Since serum albumin, the principal carrier protein for quercetin gets depleted in visceral leishmaniasis, the situation may compromise the efficacy of quercetin in this disease. We now report the use of quercetin-hamster serum albumin combination to increase the bioavailability of quercetin. The combination targeted hemoglobin oxidation and produced an effective attenuation of heme degradation. This led to decreased iron decompartmentalization, thereby increasing the life span of erythrocytes during leishmanial infection. Thus, we speculate that suppression of iron decompartmentalization, with the combination of quercetin and serum albumin might be a new approach in the prevention of reduced survival of erythrocytes in visceral leishmaniasis.
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Affiliation(s)
- Gargi Sen
- Indian Association for Cultivation of Science, Kolkata 700032, India
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16
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Ramot Y, Lewis DA, Ortel TL, Streicker M, Moser G, Elmore S, Ward SM, Peddada S, Nyska A. Age and dose sensitivities in the 2-butoxyethanol F344 rat model of hemolytic anemia and disseminated thrombosis. ACTA ACUST UNITED AC 2007; 58:311-22. [PMID: 17261363 DOI: 10.1016/j.etp.2006.11.003] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2006] [Accepted: 11/01/2006] [Indexed: 11/18/2022]
Abstract
In hemolytic disorders, such as sickle cell disease and beta-thalassemia, the mechanisms of thrombosis are poorly understood. Appropriate animal models would increase the understanding of the pathophysiology of thrombosis. We previously reported that rats exposed to 2-butoxyethanol (2-BE) developed hemolytic anemia and disseminated thrombosis resembling sickle cell disease and beta-thalassemia. To characterize our model further, we investigated age- and dose-related differences in sensitivity to 2-BE. We exposed groups of 6- and 12-week-old F344 rats (5 animals/group) to 62.5, 125, and 250 mg/kg/day of 2-BE for up to 4 days. Blood was collected on days 2-4 for complete blood count and measurement of intracellular adhesion molecule-1 (ICAM-1). Histopathological evaluation was performed to find evidence of disseminated thrombosis. The maximum hemolytic response, resulting in decreased erythrocyte count and higher mean cell volume (MCV) occurred in the 12-week-old rats treated with the highest dose of 2-BE (250 mg/kg, p<0.0001). The highest increase in ICAM-1 levels occurred in the 12-week-old rats treated with 125 and 250 mg/kg 2-BE (p<0.0001). No intravascular thrombi were noted in the 6-week-old 2-BE-treated animals. The majority of intravascular thrombi occurred in the 12-week-old rats treated with 250 mg/kg 2-BE. Because our findings show age- and dose-related sensitivities, we suggest that 12-week-old rats and doses of 250 mg/kg be used in the 2-BE model.
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Affiliation(s)
- Yuval Ramot
- Hadassah Medical Center, Hebrew University, Jerusalem, Israel
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17
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Gifford SC, Derganc J, Shevkoplyas SS, Yoshida T, Bitensky MW. A detailed study of time-dependent changes in human red blood cells: from reticulocyte maturation to erythrocyte senescence. Br J Haematol 2006; 135:395-404. [PMID: 16989660 DOI: 10.1111/j.1365-2141.2006.06279.x] [Citation(s) in RCA: 101] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The use of microfabrication technology in the study of biological systems continues to grow rapidly in both prevalence and ascendancy. Customised microdevices that provide superior results than traditional macroscopic methods can be designed in order to investigate specific cell types and cellular processes. This study showed the benefit of this approach in precisely characterising the progressive losses of surface area and haemoglobin (Hb) content by the human red blood cell (RBC), from newborn reticulocyte to senescent erythrocyte. The high-throughput, multiparametric measurements made on individual cells with a specialised microdevice enabled, for the first time, delineation and quantification of the losses that occur during the two stages of the human RBC lifespan. Data acquired on tens of thousands of red cells showed that nearly as much membrane area is lost during the 1-2 d of reticulocyte maturation (c. 10-14%) as in the subsequent 4 months of erythrocyte ageing (c. 16-17%). The total decrease in Hb over the red cell lifespan is also estimated (c. 15%) and a model describing the complete time-course of diminishing mean RBC area and Hb is proposed. The relationship between the losses of Hb and area, and their possible influence on red cell lifespan, are discussed.
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Affiliation(s)
- Sean C Gifford
- Visual and Circulatory Biophysics Laboratory, Department of Biomedical Engineering, Boston University, Boston, MA, USA
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18
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Wandersee NJ, Punzalan RC, Rettig MP, Kennedy MD, Pajewski NM, Sabina RL, Paul Scott J, Low PS, Hillery CA. Erythrocyte adhesion is modified by alterations in cellular tonicity and volume. Br J Haematol 2006; 131:366-77. [PMID: 16225657 DOI: 10.1111/j.1365-2141.2005.05767.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
We tested the hypothesis that dehydration-induced alterations in red blood cell (RBC) membrane organisation or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilised TSP under flow conditions. Sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (P<0.005), suggesting that cellular dehydration may contribute to TSP adhesion. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (P<0.01). Furthermore, increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers. Finally, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides, chondroitin sulphate A and high molecular weight dextran sulphate, but not by competitors of CD47-, band 3-, or RBC phosphatidylserine-mediated adhesion. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs to kidney capillaries following re-infusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, causing normal erythrocytes to display adhesive properties similar to those of sickle cells and vice versa.
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Affiliation(s)
- Nancy J Wandersee
- Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
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19
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Aprelev A, Rotter MA, Etzion Z, Bookchin RM, Briehl RW, Ferrone FA. The effects of erythrocyte membranes on the nucleation of sickle hemoglobin. Biophys J 2005; 88:2815-22. [PMID: 15653736 PMCID: PMC1305376 DOI: 10.1529/biophysj.104.051086] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Pathology in sickle cell disease begins with nucleation-dependent polymerization of deoxyhemoglobin S into stiff, rodlike fibers that deform and rigidify red cells. We have measured the effect of erythrocyte membranes on the rate of homogeneous nucleation in sickle hemoglobin, using preparations of open ghosts (OGs) with intact cytoskeletons from sickle (SS) and normal adult (AA) red cells. Nucleation rates were measured by inducing polymerization by laser photolysis of carboxy sickle hemoglobin and observing stochastic variation of replicate experiments of the time for the scattering signals to reach 10% of their respective maxima. By optical imaging of membrane fragments added to a hemoglobin solution we contrast the rate of nucleation immediately adjacent to membrane fragments with nucleation in a region of the same solution but devoid of membranes. From analysis of 29,272 kinetic curves obtained, we conclude that the effect of AA OGs is negligible (10% enhancement of nucleation rates +/-20%), whereas SS OGs caused 80% enhancement (+/-20%). In red cells, where more membrane surface is available to Hb, this implies enhancement of nucleation by a factor of 6. These experiments represent a 10-fold improvement in precision over previous approaches and are the first direct, quantitative measure of the impact of erythrocyte membranes on the homogeneous nucleation process that is responsible for polymer initiation in sickle cell disease.
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Affiliation(s)
- Alexey Aprelev
- Department of Physics, Drexel University, Philadelphia, Pennsylvania 19104, USA
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20
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Murakami K, Mawatari S. Oxidation of hemoglobin to methemoglobin in intact erythrocyte by a hydroperoxide induces formation of glutathionyl hemoglobin and binding of alpha-hemoglobin to membrane. Arch Biochem Biophys 2003; 417:244-50. [PMID: 12941307 DOI: 10.1016/s0003-9861(03)00389-8] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Biochemical consequences of oxidation of hemoglobin (Hb) in intact human erythrocytes were studied. The incubation of washed erythrocyte with 1mM tert-butylhydroperoxide induced an increase in glutathionyl-Hb (G-Hb). The formation of G-Hb occurred linearly until 10 min in parallel with the formation of methemoglobin (metHb) after exhaustion of reduced glutathione. The results show that metHb, but not normal Hb, reacts with oxidized glutathione to form G-Hb. G-Hb was confirmed by immunoblotting with anti-glutathione antibody and the formation of G-Hb was accompanied by parallel decrease in beta-globin detected with a reversed phase HPLC. Electrophoretic studies showed time-dependent increase in membrane-associated alpha-Hb until 10 min, indicating that a part of unpaired alpha-Hb bound to the membrane. Pre-beta-globin increased despite the decrease in beta-globin and a part of the increase was independent of the decrease in beta-globin. Pre-beta-globin reacted with anti-glutathione antibody, but it differs from G-Hb in many features.
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Affiliation(s)
- Kaori Murakami
- Department of Nutrition and Health Science, Faculty of Human Environmental Science, Fukuoka Women's University, 1-1-1 Kasumigaoka, Higashi-ku, Fukuoka, Japan
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21
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Tokumasu F, Dvorak J. Development and application of quantum dots for immunocytochemistry of human erythrocytes. J Microsc 2003; 211:256-61. [PMID: 12950474 DOI: 10.1046/j.1365-2818.2003.01219.x] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Recent developments in quantum dot technology have resulted in the introduction of new fluorescence immunocytochemical probes. In contrast to organic fluorophores, which are not photostable, the high quantum yield and remarkable photostability of quantum dots solve major problems associated with immunocytochemical studies of erythrocytes. We report here the first application of quantum dots to immunocytochemical studies of human erythrocytes capable of being used in high-magnification, three-dimensional erythrocyte reconstruction techniques. The procedure consists of stabilizing human erythrocytes with a homofunctional imidoester cross-linker to minimize fixative-induced autofluorescence followed by reacting with a quantum dot - monoclonal antibody complex to label band 3 protein. Our new procedure clearly showed a non-homogeneous, raft-like distribution of band 3 protein in the erythrocyte membrane. We also demonstrate the applicability of our technique to studies of erythrocyte membrane modifications occurring during the invasion of a malaria parasite.
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Affiliation(s)
- F Tokumasu
- Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425, USA
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22
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Condon MR, Kim JE, Deitch EA, Machiedo GW, Spolarics Z. Appearance of an erythrocyte population with decreased deformability and hemoglobin content following sepsis. Am J Physiol Heart Circ Physiol 2003; 284:H2177-84. [PMID: 12742829 DOI: 10.1152/ajpheart.01069.2002] [Citation(s) in RCA: 64] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
With the use of the cecal ligation and puncture model in mice, this study tested whether sepsis-induced decreased erythrocyte deformability is restricted to a subpopulation of cells. Erythrocyte subpopulations were isolated by centrifugal elutriation. Lineweaver-Burk conversion of deformability-response curves to shear stress was used to determine the shear stress at half-maximal cell elongation (K(EI)) and maximal cell elongation (EI(max)). Sepsis decreased erythrocyte deformability in whole blood. K(EI) values were elevated (2.7 vs. 2.1 Pa) and EI(max) values decreased (0.56 vs. 0.50) in sepsis compared with sham mice. K(EI) values for cells eluted at 7 ml/min (smallest and oldest cells) were similar; however, K(EI) values for cells eluted at 8 ml/min were greater in septic than sham animals (2.50 vs. 2.10). Younger and larger subpopulations of erythrocytes (eluted at 9, 10, and 11 ml/min) also showed a tendency of decreased deformability in sepsis. Mean corpuscular hemoglobin content was decreased in cells eluted at 7 and 8 ml/min in sepsis (4.5 and 10.2 pg) compared to sham (7.4 and 11.4 pg) mice. This study indicates that an erythrocyte subpopulation that represents 20% of circulating cells shows the most pronounced decrease in cell deformability during sepsis. Increased rigidity together with decreased corpuscular hemoglobin content in these cells may contribute to microcirculatory dysfunction and immune modulation during sepsis.
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Affiliation(s)
- Michael R Condon
- Department of Surgery, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark 07103, USA
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23
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Couto FD, De Albuquerque ABL, Adorno EV, De Moura Neto JP, De Freitas Abbehusen L, De Oliveira JLB, Dos Reis MG, De Souza Gonçalves M. alpha-Thalassemia 2, 3.7 kb deletion and hemoglobin AC heterozygosity in pregnancy: a molecular and hematological analysis. CLINICAL AND LABORATORY HAEMATOLOGY 2003; 25:29-34. [PMID: 12542439 DOI: 10.1046/j.1365-2257.2003.00487.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
alpha-Thalassemia is a synthesis hemoglobinopathy with a worldwide distribution. alpha-thalassemia-23.7kb (alpha-Thal23.7kb) was investigated by PCR and standard hematologic analysis techniques in 106 pregnant women - 53 heterozygous for hemoglobin (Hb) A and C (AC) and 53 homozygous for the normal Hb A (AA) with similar ages and race ancestry. Eleven (21%) of AC women were alpha-Thal23.7kb heterozygous and 1 (2%) was homozygous, while 12 AA women (23%) were heterozygous. In the AA group, the MCV differed among those with normal alpha genes and those with alpha-Thal23.7kb (P = 0.031). Statistical analysis of AC group patients with normal alpha genes and alpha-Thal23.7kb carriers showed differences in MCV (P = 0.001); MCH (P = 0.003) and Hb C concentrations (P = 0.011). Analysis of AA and AC group patients with normal alpha genes showed differences in RBC (P = 0.033), Hb concentration (P = 0.003) and MCHC (P < 0.0001). There were no statistically significant differences for any hematologic parameters between AC and AA group patients with the alpha-Thal23.7kb genotype. The AC alpha-Thal23.7kb homozygous women had low hematologic parameters. Serum ferritin levels were normal among the groups studied. These results emphasize the importance of diagnosis and follow-up of patients with hemoglobinopathy carriers during pregnancy in order to administer adequate therapy and avoid further complications for mothers and newborns.
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Affiliation(s)
- F D Couto
- Centro de Pesquisas Gonçalo Moniz/FIOCRUZ-Pathology and Molecular Biology Laboratory, Bahia, Brazil
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24
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Kennedy JR. Malaria: a vaccine concept based on sickle haemoglobin's augmentation of an innate autoimmune process to band 3. Int J Parasitol 2001; 31:1275-7. [PMID: 11513898 DOI: 10.1016/s0020-7519(01)00236-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The protection from malaria afforded by sickle haemoglobin (and certain other haemoglobinopathies) suggests that it may be possible to utilise a common property that their erythrocytes share with both malaria-infected erythrocytes and senescent erythrocytes to develop a vaccine. All three conditions cause clustering of a specific protein molecule, band 3, on their erythrocyte's surface and this protein, when present on senescent erythrocytes at least, results in the immune recognition and removal of these by naturally occurring antibodies. It is hypothesised that if an up-regulated immune response to this protein on sickle cells is responsible for the benefit afforded to malaria patients then a vaccine using antigenic band 3 peptides may provide similar protection.
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25
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Fetal hemoglobin in sickle cell disease: relationship to erythrocyte phosphatidylserine exposure and coagulation activation. Blood 2000. [DOI: 10.1182/blood.v96.3.1119.015k53_1119_1124] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
In sickle cell disease (SCD), loss of erythrocyte membrane phospholipid asymmetry occurs with the exposure of phosphatidylserine (PS), which provides a docking site for coagulation proteins. In vivo sickling/desickling, with resulting red cell membrane changes and microvesicle formation, appears to be one of the factors responsible for PS exposure. We evaluated children with SCD homozygous for sickle hemoglobin (SS disease) and controls (n = 65) and demonstrate that high levels of fetal hemoglobin (assessed as F cells) are associated with decreased microvesicle formation, PS exposure, and thrombin generation. F cells correlated inversely with both microvesicles and PS positivity (P < .000001) in SS disease. Multiple regression analyses using various hematologic parameters as independent variables, and either microvesicles or PS positivity as the dependent variable, showed a strong relationship only with F cells. Additionally, plasma prothrombin fragment F1.2 levels (a marker for thrombin generation) correlated with both PS positivity (P < .001) and F cells (P < .01). An F-cell level of approximately 70% was associated with normal levels of prothrombin fragment F1.2 and with microvesicle formation indistinguishable from control values. We suggest that the use of such surrogate biologic markers in conjunction with F-cell numbers may provide valuable insights into the biology and consequences of in vivo sickling.
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26
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Fetal hemoglobin in sickle cell disease: relationship to erythrocyte phosphatidylserine exposure and coagulation activation. Blood 2000. [DOI: 10.1182/blood.v96.3.1119] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
AbstractIn sickle cell disease (SCD), loss of erythrocyte membrane phospholipid asymmetry occurs with the exposure of phosphatidylserine (PS), which provides a docking site for coagulation proteins. In vivo sickling/desickling, with resulting red cell membrane changes and microvesicle formation, appears to be one of the factors responsible for PS exposure. We evaluated children with SCD homozygous for sickle hemoglobin (SS disease) and controls (n = 65) and demonstrate that high levels of fetal hemoglobin (assessed as F cells) are associated with decreased microvesicle formation, PS exposure, and thrombin generation. F cells correlated inversely with both microvesicles and PS positivity (P < .000001) in SS disease. Multiple regression analyses using various hematologic parameters as independent variables, and either microvesicles or PS positivity as the dependent variable, showed a strong relationship only with F cells. Additionally, plasma prothrombin fragment F1.2 levels (a marker for thrombin generation) correlated with both PS positivity (P < .001) and F cells (P < .01). An F-cell level of approximately 70% was associated with normal levels of prothrombin fragment F1.2 and with microvesicle formation indistinguishable from control values. We suggest that the use of such surrogate biologic markers in conjunction with F-cell numbers may provide valuable insights into the biology and consequences of in vivo sickling.
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27
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Chu L, Ebersole JL, Holt SC. Hemoxidation and binding of the 46-kDa cystalysin of Treponema denticola leads to a cysteine-dependent hemolysis of human erythrocytes. ORAL MICROBIOLOGY AND IMMUNOLOGY 1999; 14:293-303. [PMID: 10551156 DOI: 10.1034/j.1399-302x.1999.140505.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Cystalysin, a 46-kDa protein isolated from the cytosol of Treponema denticola, was capable of both cysteine dependent hemoxidation and hemolysis of human and sheep red blood cells. The activities were characteristic of a cysteine desulfhydrase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting analysis of the interaction of cystalysin with the red blood cells revealed an interaction of the protein with the red blood cell membrane. Substrates for the enzyme (including L-cysteine and beta-chloroalanine) enhanced the interaction, which occurred with both whole red blood cells as well as with isolated and purified red blood cell ghosts. SDS-PAGE and western immunoblotting employing anti-hemoglobin serum revealed that, during the hemoxidative events, the hemoglobin molecule associated with the red blood cell membrane, forming putative Heinz bodies. Spectrophotometric analysis of the hemoxidative events (cystalysin + cysteine + red blood cells) revealed a chemical modification of the native hemoglobin to sulfhemoglobin and methemoglobin. Hemoxidation also resulted in the degradation of both the red blood cell alpha- and beta-spectrin. The results presented suggest that the interaction of cystalysin with the red blood cell membrane results in the chemical oxidation of the hemoglobin molecule as well as an alteration in the red blood cell membrane itself.
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Affiliation(s)
- L Chu
- Department of Microbiology, University of Texas Health Science Center at San Antonio 78284, USA
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28
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Cibert C, Prulière G, Lacombe C, Deprette C, Cassoly R. Calculation of a Gap restoration in the membrane skeleton of the red blood cell: possible role for myosin II in local repair. Biophys J 1999; 76:1153-65. [PMID: 10049301 PMCID: PMC1300097 DOI: 10.1016/s0006-3495(99)77280-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Human red blood cells contain all of the elements involved in the formation of nonmuscle actomyosin II complexes (V. M. Fowler. 1986. J. Cell. Biochem. 31:1-9; 1996. Curr. Opin. Cell Biol. 8:86-96). No clear function has yet been attributed to these complexes. Using a mathematical model for the structure of the red blood cell spectrin skeleton (M. J. Saxton. 1992. J. Theor. Biol. 155:517-536), we have explored a possible role for myosin II bipolar minifilaments in the restoration of the membrane skeleton, which may be locally damaged by major mechanical or chemical stress. We propose that the establishment of stable links between distant antiparallel actin protofilaments after a local myosin II activation may initiate the repair of the disrupted area. We show that it is possible to define conditions in which the calculated number of myosin II minifilaments bound to actin protofilaments is consistent with the estimated number of myosin II minifilaments present in the red blood cells. A clear restoration effect can be observed when more than 50% of the spectrin polymers of a defined area are disrupted. It corresponds to a significant increase in the spectrin density in the protein free region of the membrane. This may be involved in a more complex repair process of the red blood cell membrane, which includes the vesiculation of the bilayer and the compaction of the disassembled spectrin network.
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Affiliation(s)
- C Cibert
- Laboratoire de Biologie du Développement, Institut Jacques Monod, UMR 7592, CNRS, Universités Paris VI et Paris VII, F-75005 Paris, France.
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29
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Lahiri T, Kar NS, Chakrabarti A, Dasgupta AK. Onset of Percolation and Fractal Classification Scheme for Multilamellar Lipid Vesicles. J Colloid Interface Sci 1999; 211:89-95. [PMID: 9929438 DOI: 10.1006/jcis.1998.5934] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Percolation, like phase transition, expresses a critical property of an assembly. The paper provides a brief description of the onset of percolation and the fractal behavior of clusters formed as a result of spontaneous assembly of multilamellar lipid vesicles (MLVs). The onset of percolation was studied using a series of mixed lipid systems. The percolation threshold showed a strong linear dependence on the net charge of the constituent lipids. Second, the vesicular clusters were characterized using a canonical coordinate frame spanned by the fractal dimension and the porosity of the cluster images. MLVs made of different lipid constituents formed different domains on such representations. The location of the individual domains relative to a simulated random cluster seemed to provide a measure of order-element present in such clusters. The specificity of the domain locations and their sensitivity to alteration in a microenvironment was studied in some detail. Copyright 1999 Academic Press.
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Affiliation(s)
- T Lahiri
- Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, Nadia-741 235, India
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30
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Lahiri T, Chakrabarti A, Dasgupta AK. Multilamellar vesicular clusters of phosphatidylcholine and their sensitivity to spectrin: a study by fractal analysis. J Struct Biol 1998; 123:179-86. [PMID: 9878573 DOI: 10.1006/jsbi.1998.4030] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The cluster patterns of multilamellar vesicles (MLV) of dimyristoylphosphatidylcholine (DMPC) were analyzed using a combination of fractal analysis and lattice simulation. Self-assembly of DMPC MLVs resulted in two types of microscopically observable clusters. The clusters were classified on the basis of their mass fractal dimension, two-dimensional porosity, and the light scattering properties. Spectrin, a cytoskeletal protein, well known for its role in determining the cellular morphology, was used to perturb such spontaneously formed clusters. The fragmentation of the clusters by hydrodynamic perturbation followed a power law, implying again a fractal behavior. A lattice-based simulation was performed generating different class of cluster patterns. The observed correspondence between the cluster patterns and their stability was discussed in the framework of the proposed lattice simulation.
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Affiliation(s)
- T Lahiri
- Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, 741235, India
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Abstract
Phospholipid asymmetry in the red blood cell (RBC) lipid bilayer is well maintained during the life of the cell, with phosphatidylserine (PS) virtually exclusively located in the inner monolayer. Loss of phospholipid asymmetry, and consequently exposure of PS, is thought to play an important role in red cell pathology. The anemia in the human thalassemias is caused by a combination of ineffective erythropoiesis (intramedullary hemolysis) and a decreased survival of adult RBCs in the peripheral blood. This premature destruction of the thalassemic RBC could in part be due to a loss of phospholipid asymmetry, because cells that expose PS are recognized and removed by macrophages. In addition, PS exposure can play a role in the hypercoagulable state reported to exist in severe β-thalassemia intermedia. We describe PS exposure in RBCs of 56 comparably anemic patients with different genetic backgrounds of the α- or β-thalassemia phenotype. The use of fluorescently labeled annexin V allowed us to determine loss of phospholipid asymmetry in individual cells. Our data indicate that in a number of thalassemic patients, subpopulations of red cells circulate that expose PS on their outer surface. The number of such cells can vary dramatically from patient to patient, from as low as that found in normal controls (less than 0.2%) up to 20%. Analysis by fluorescent microscopy of β-thalassemic RBCs indicates that PS on the outer leaflet is distributed either over the entire membrane or localized in areas possibly related to regions rich in membrane-bound α-globin chains. We hypothesize that these membrane sites in which iron carrying globin chains accumulate and cause oxidative damage, could be important in the loss of membrane lipid organization. In conclusion, we report the presence of PS-exposing subpopulations of thalassemic RBC that are most likely physiologically important, because they could provide a surface for enhancing hemostasis as recently reported, and because such exposure may mediate the rapid removal of these RBCs from the circulation, thereby contributing to the anemia.
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LaBrake CC, Fung LW. Sickle hemoglobin is more fusogenic than normal hemoglobin at physiological pH and ionic strength conditions. BIOCHIMICA ET BIOPHYSICA ACTA 1998; 1406:152-61. [PMID: 9573351 DOI: 10.1016/s0925-4439(97)00093-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
We used electron microscopy, quasi-elastic light scattering and static light scattering to show that human hemoglobin (Hb) interacts with bovine brain phosphatidylserine lipid vesicles and promotes vesicle fusion in an isotonic buffer at pH 7.4. The fusogenic properties of Hb were observed in both small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs). A simple turbidity measurement method was used to follow increases in vesicle size (scattering diameter) as a function of time. For the first 3 h, upon incubation with oxygenated Hb, the scattering diameters of vesicles increased at a rate of 7.8 nm/h for LUVs. Continuous incubation with Hb led to complicated vesicle fusion, probably due to the oxidation products of Hb and lipid molecules. In the absence of both Hb and lipid oxidation, using Hb liganded with carbon monoxide, we obtained, for the entire 20 h incubation period, a fusion rate of 2.9 nm/h for LUVs. We also studied interactions between sickle Hb and vesicles under the same conditions and found that the vesicle fusion rates for sickle Hb were about 2 times faster than those for normal Hb. These results showed that sickle Hb exhibited more extensive interactions with lipid bilayer than normal Hb at physiological pH and ionic strength conditions, and provide insights toward understanding the molecular mechanisms in sickle cell abnormalities.
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Affiliation(s)
- C C LaBrake
- Department of Chemistry, Loyola University of Chicago, Chicago, IL 60626, USA
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