RESUMO
The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Glicoforinas/deficiência , Células-Tronco Embrionárias Murinas/citologia , Plasmodium berghei/fisiologia , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Diferenciação Celular , Linhagem Celular , Eritropoese , Glicoforinas/metabolismo , Interações Hospedeiro-Parasita , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/parasitologiaRESUMO
Band 3 protein accounts for the largest percentage of whole erythrocyte membrane proteins. Abnormalities in this protein are closely associated with pathologies including hereditary spherocytosis (HS), Southeast Asian ovalocytosis and distant renal tubular acidosis. Currently, EMA binding capacity measurement in erythrocytes is the most useful screening test for diagnosing HS. We have also demonstrated reduced EMA binding capacity in patients with HS who have deficiencies of membrane proteins such as ankyrin not directly binding to EMA and who have as yet undetectable abnormalities of membrane proteins. However, even patients with hereditary elliptocytosis, who have a partial spectrin deficiency, were found to show reduced EMA binding capacity. Six of 7 had spherocytic elliptocytosis. Therefore, it is necessary to meticulously diagnose HS by ruling out all other possibilities.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Anquirinas/deficiência , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Esferocitose Hereditária/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Proteína 1 de Troca de Ânion do Eritrócito/genética , Anquirinas/química , Anquirinas/genética , Anquirinas/metabolismo , Humanos , Mutação , Ligação Proteica , Esferocitose Hereditária/genéticaRESUMO
The present study was conducted to develop a simple and rapid procedure to determine the genotype of band 3 deficiency in bovine embryos by a novel real-time PCR method using a fluorescent quenching-based probe (QProbe-PCR). QProbe-PCR successfully distinguished wild type and R664X mutant alleles by melting curve analysis. Minimal amounts of DNA template were required for the detection of wild type/wild type alleles, mutant/mutant alleles, and wild type/mutant alleles; their amounts were 10 pg, 25 pg, and 50 pg, respectively. When 10 blastomeres were used as a DNA sample, accuracies of genotyping by QProbe-PCR were 100% and 89% in embryos homozygous for the wild type allele and heterozygous for the wild type and mutant alleles, respectively. QProbe-PCR takes approximately 2 h for genotyping and requires lesser time than the conventional method using PCR-RFLP, which requires digestion with a restriction enzyme and electrophoresis. Our data showed that QProbe-PCR is a useful method for rapid analysis of the genetic deficiency in preimplantation embryos. Reduction in the time required for genotyping enabled the transfer of genetically selected embryos to recipient cows on the day of embryo collection. These results suggest that determination of the genotype for the genetic deficiency in embryos is useful to select animals free from the genetic disease, and it also makes it possible to produce an animal model homozygous for the mutation.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/genética , Blastocisto/citologia , Técnicas de Genotipagem/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Blastocisto/metabolismo , Bovinos , Genótipo , Mutação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos TestesRESUMO
Anion exchanger-1 (AE1) mediates chloride-bicarbonate exchange across the plasma membranes of erythrocytes and, via a slightly shorter transcript, kidney epithelial cells. On an omnivorous human diet, kidney AE1 is mainly active basolaterally in α-intercalated cells of the collecting duct, where it is functionally coupled with apical proton pumps to maintain normal acid-base homeostasis. The C-terminal tail of AE1 has an important role in its polarized membrane residency. We have identified the ß1 subunit of Na(+),K(+)-ATPase (sodium pump) as a binding partner for AE1 in the human kidney. Kidney AE1 and ß1 colocalized in renal α-intercalated cells and coimmunoprecipitated (together with the catalytic α1 subunit of the sodium pump) from human kidney membrane fractions. ELISA and fluorescence titration assays confirmed that AE1 and ß1 interact directly, with a Kd value of 0.81 µM. GST-AE1 pull-down assays using human kidney membrane proteins showed that the last 11 residues of AE1 are important for ß1 binding. siRNA-induced knockdown of ß1 in cell culture resulted in a significant reduction in kidney AE1 levels at the cell membrane, whereas overexpression of kidney AE1 increased cell surface sodium pump levels. Notably, membrane staining of ß1 was reduced throughout collecting ducts of AE1-null mouse kidney, where increased fractional excretion of sodium has been reported. These data suggest a requirement of ß1 for proper kidney AE1 membrane residency, and that activities of AE1 and the sodium pump are coregulated in kidney.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/fisiologia , Membrana Celular/metabolismo , Rim/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Proteína 1 de Troca de Ânion do Eritrócito/genética , Linhagem Celular , Membrana Celular/patologia , Células Cultivadas , Homeostase/fisiologia , Humanos , Rim/patologia , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Ligação Proteica , RNA Interferente Pequeno/farmacologia , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacosRESUMO
In a family with mild dominant spherocytosis, affected members showed partial band 3 deficiency. The index patient showed more severe clinical symptoms than his relatives, and his red blood cells displayed concomitant low pyruvate kinase activity. We investigated the contribution of partial PK deficiency to the phenotypic expression of mutant band 3 in this family. Pyruvate kinase deficiency and band 3 deficiency were characterized by DNA analysis. Results of red cell osmotic fragility testing, the results of cell deformability obtained by the Automated Rheoscope and Cell Analyzer and the results obtained by Osmotic Gradient Ektacytometry, which is a combination of these tests, were related to the red cell ATP content. Spherocytosis in this family was due to a novel heterozygous mutation in SLC4A1, the gene for band 3. Reduced PK activity of the index patient was attributed to a novel mutation in PKLR inherited from his mother, who was without clinical symptoms. Partial PK deficiency was associated with decreased red cell ATP content and markedly increased osmotic fragility. This suggests an aggravating effect of low ATP levels on the phenotypic expression of band 3 deficiency.
Assuntos
Anemia Hemolítica Congênita não Esferocítica/genética , Proteína 1 de Troca de Ânion do Eritrócito/genética , Anquirinas/deficiência , Mutação , Fenótipo , Piruvato Quinase/deficiência , Piruvato Quinase/genética , Erros Inatos do Metabolismo dos Piruvatos/genética , Esferocitose Hereditária/genética , Trifosfato de Adenosina/metabolismo , Adulto , Idoso , Anemia Hemolítica Congênita não Esferocítica/complicações , Anemia Hemolítica Congênita não Esferocítica/metabolismo , Anemia Hemolítica Congênita não Esferocítica/patologia , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Anquirinas/genética , Anquirinas/metabolismo , Deformação Eritrocítica , Eritrócitos/metabolismo , Eritrócitos/patologia , Feminino , Expressão Gênica , Genótipo , Heterozigoto , Humanos , Padrões de Herança , Masculino , Pessoa de Meia-Idade , Fragilidade Osmótica , Linhagem , Piruvato Quinase/metabolismo , Erros Inatos do Metabolismo dos Piruvatos/complicações , Erros Inatos do Metabolismo dos Piruvatos/metabolismo , Erros Inatos do Metabolismo dos Piruvatos/patologia , Esferocitose Hereditária/complicações , Esferocitose Hereditária/metabolismo , Esferocitose Hereditária/patologiaRESUMO
Hereditary Spherocytosis (HS) is a haemolytic anaemia caused by erythrocyte protein membrane defects - spectrin, ankyrin, band 3 or protein 4.2 - that lead to membrane destabilization. This study aimed to evaluate the prevalence of protein deficiencies and the role of membrane proteins or membrane-linked proteins in membrane disturbance and in HS clinical outcome. A total of 215 Portuguese individuals were studied - 203 from 71 families plus 12 individual unrelated subjects; 160 of them were diagnosed with HS. They were classified as presenting mild, moderate or severe forms of HS according to the degree of haemolytic anaemia. Standardized electrophoretic erythrocyte membrane protein analysis was used to identify and quantify protein deficiencies. Band 3 and ankyrin were found to account for the majority of the erythrocyte protein defects underlying HS. Increasing isolated protein deficiency or increasing imbalance between combined protein deficiencies seemed to underlie HS severity, by increasing membrane destabilization. There was an increased membrane linkage of the cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and peroxiredoxin 2, and of denatured haemoglobin, suggesting that this linkage could interfere with membrane structure. Our data suggest that the quantification and the analysis of RBC membrane proteins may be helpful in predicting the clinical outcome of HS.
Assuntos
Membrana Eritrocítica/metabolismo , Proteínas de Membrana/deficiência , Esferocitose Hereditária/sangue , Adulto , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Anquirinas/deficiência , Criança , Eletroforese em Gel de Poliacrilamida , Hemoglobinas/metabolismo , Proteínas de Homeodomínio/sangue , Humanos , Prognóstico , Índice de Gravidade de Doença , Espectrina/deficiênciaRESUMO
Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteínas de Ligação a Calmodulina/deficiência , Proteínas de Ligação a Calmodulina/genética , Mutação , Espectrina/deficiência , Espectrina/genética , Esferocitose Hereditária/sangue , Esferocitose Hereditária/genética , Alelos , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação a Calmodulina/sangue , Códon sem Sentido , Primers do DNA/genética , Membrana Eritrocítica/metabolismo , Eritrócitos Anormais/metabolismo , Eritrócitos Anormais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Mutantes , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Estabilidade de RNA/genética , Homologia de Sequência de Aminoácidos , Esferocitose Hereditária/patologiaRESUMO
The hereditary stomatocytoses (HSt) are a diverse group of conditions. Common features include hemolytic anemia, a red cell cation leak and morphological changes, but the severity of the condition can vary enormously. We have previously shown that one form of HSt (cryohydrocytosis), where the monovalent cation leak is increased at low temperature, results from amino acid substitutions in the membrane domain of band 3 (anion exchanger 1, SLC4A1). These substitutions appear to convert band 3 from an anion exchanger into a cation channel. More recently we found that over-hydrated hereditary stomatocytosis (OHSt) results from amino acid substitutions in Rh-associated glycoprotein (RhAG), a putative gas channel protein. Both band 3 and RhAG associate in the red cell membrane to form a macrocomplex that is thought to be involved in red cell gas exchange. In this paper I will review the data that has been published so far on the molecular basis of HSt. I will mention other similar conditions that cause either a cation leak or stomatocytosis or both, and consider the mechanisms of red cell shape change and permeability.
Assuntos
Anemia Hemolítica Congênita/sangue , Cátions/sangue , Membrana Eritrocítica/metabolismo , Acidose Tubular Renal/sangue , Acidose Tubular Renal/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Anemia Hemolítica Congênita/classificação , Anemia Hemolítica Congênita/genética , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/fisiologia , Ânions/metabolismo , Proteínas Sanguíneas/química , Proteínas Sanguíneas/deficiência , Proteínas Sanguíneas/fisiologia , Água Corporal , Permeabilidade da Membrana Celular , Temperatura Baixa/efeitos adversos , Eritrócitos Anormais/metabolismo , Heterogeneidade Genética , Humanos , Transporte de Íons/genética , Transporte de Íons/fisiologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Mutação de Sentido Incorreto , Mutação PuntualRESUMO
Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking alpha-spectrin, ankyrin, protein 4.2, protein 4.1, beta-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Membrana Eritrocítica/metabolismo , Glicólise/fisiologia , Sequência de Aminoácidos , Animais , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Proteína 1 de Troca de Ânion do Eritrócito/genética , Sequência de Bases , DNA Complementar/genética , Frutose-Bifosfato Aldolase/sangue , Gliceraldeído-3-Fosfato Desidrogenases/sangue , L-Lactato Desidrogenase/sangue , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Complexos Multienzimáticos/sangue , Oxigênio/sangue , Fosfofrutoquinases/sangue , Piruvato Quinase/sangue , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND/AIMS: Slc26a7 is a member of a family of anion transport proteins, Solute-Linked Carrier 26 (Slc26). Slc26a7, which can mediate Cl-/HCO3- exchange, is expressed in the acid-secreting, A-intercalated cells of the kidney collecting duct. On the basolateral side of the A-intercalated cells, Slc26a7 co-localizes with the anion exchanger 1 (AE1), a Cl-/HCO3- exchanger that mediates bicarbonate reabsorption in the collecting duct. METHODS: To test if Slc26a7 is involved in acid-base regulation, as its localization and function suggest, we examined the effect of acid loading and deletion of AE1 on Slc26a7 expression with quantitative real-time RT-PCR and Western blotting. RESULTS: Four days of acid loading increased Slc26a7 mRNA expression in the kidney inner medulla by 57% (n = 6 acid loaded vs. n = 6 control rats; p < 0.001), whereas mRNA expression in the outer medulla and the cortex did not change. Western blotting analysis demonstrated increased Slc26a7 protein expression in both outer (140%) and inner medulla (50%) in acid-loaded animals (n = 3) compared to controls (n = 3; p < 0.05). The expression of Slc26a7 mRNA was increased by 66% in the kidneys of AE1 knockout mice (n = 5) compared to the wild types (n = 5, p < 0.001). The increase in Slc26a7 mRNA correlated with a twofold increase in protein expression (p < 0.05). CONCLUSION: We suggest that the increase in Slc26a7 expression caused by acid challenge and deletion of AE1 represents an adaptive response, indicating that Slc26a7 contributes to the regulation of acid-base balance by the kidney.
Assuntos
Equilíbrio Ácido-Base , Acidose/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Antiporters/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Medula Renal/metabolismo , Acidose/induzido quimicamente , Cloreto de Amônio , Animais , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Proteína 1 de Troca de Ânion do Eritrócito/genética , Antiporters/genética , Western Blotting , Antiportadores de Cloreto-Bicarbonato/genética , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transportadores de Sulfato , Regulação para CimaRESUMO
The mechanisms underlying apparently spontaneous autoimmune diseases, such as autoimmune hemolytic anemia (AIHA) in New Zealand Black (NZB) mice, are unknown. Here, we determine the contribution of the dominant red blood cell (RBC) autoantigen, the anion exchanger protein Band 3, to the development of NZB autoimmune responses. The approach was to prevent Band 3 expression in NZB mice by disrupting the AE1 gene. AE1(-/-) NZB mice produced RBC autoantibodies at the same levels as the wild-type strain, but they differed in recognizing antigens that correspond to glycophorins, rather than Band 3. Splenic T-helper (Th) cells from wild-type NZB mice proliferated strongly against multiple Band 3 peptides, particularly the dominant epitope within aa861-874. This helper response was severely attenuated in AE1(-/-) animals, leaving only weak proliferation to peptide aa861-874. The results demonstrate that the defect in self-tolerance in NZB AIHA is directed to the RBC type, and is not specific for, or dependent on, Band 3. However, the predisposition to RBC autoimmunity may be focused onto Band 3 by weak Th cell cross-reactivity between the helper dominant epitope and an exogenous antigen. The redundancy of the major autoantigen illustrates the requirement for specific therapy to induce dominant forms of tolerance, such as T-cell regulation.
Assuntos
Anemia Hemolítica Autoimune/imunologia , Proteína 1 de Troca de Ânion do Eritrócito/imunologia , Autoanticorpos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Autoantígenos , Proliferação de Células , Reações Cruzadas/imunologia , Eritrócitos Anormais/imunologia , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos NZB , Camundongos Knockout , Tolerância a Antígenos Próprios/imunologia , Baço/citologia , Linfócitos T Auxiliares-Indutores/citologiaRESUMO
Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl(-) removal, and energy depletion activate Ca(2+)-permeable cation channels with Ca(2+)-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 (AE1(-/-) mice) than in their wild-type littermates (AE1(+/+) mice) despite increased percentages of reticulocytes (AE1(-/-): 49%, AE1(+/+): 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1(-/-)erythrocytes/reticulocytes ( approximately 10%) than in AE1(+/+) erythrocytes ( approximately 1%). Osmotic shock (addition of 400 mM sucrose), Cl(-) removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1(-/-) erythrocytes/reticulocytes than in AE1(+/+) erythrocytes. The increase of annexin binding following exposure to the Ca(2+) ionophore ionomycin (1 muM) was, however, similar in AE1(-/-) and in AE1(+/+) erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca(2+) permeability in AE1(-/-) erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1(-/-) mice than in AE1(+/+) mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca(2+) entry and subsequent scrambling of cell membrane phospholipids.
Assuntos
Anemia Hemolítica/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Apoptose , Cálcio/metabolismo , Eritrócitos/metabolismo , Anemia Hemolítica/sangue , Anemia Hemolítica/genética , Anemia Hemolítica/patologia , Anemia Hemolítica/fisiopatologia , Animais , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Proteína 1 de Troca de Ânion do Eritrócito/genética , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Cloretos/metabolismo , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/patologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Glucose/deficiência , Hemólise , Soluções Hipertônicas , Ionomicina/farmacologia , Ionóforos/farmacologia , Camundongos , Camundongos Knockout , Pressão Osmótica , Fosfatidilserinas/metabolismo , Contagem de Plaquetas , Contagem de Reticulócitos , Reticulócitos/metabolismo , Reticulócitos/patologia , Reticulocitose , Sacarose/metabolismo , Fatores de TempoRESUMO
Anion exchanger 1 (AE1; SLC4A1), the plasma membrane Cl(-)/HCO(3)(-) exchanger of erythrocytes, is also expressed in heart. The aim of this study was to assess the role of AE1 in heart function through study of AE1-null (AE1(-/-)) mice, which manifest severe hemolytic anemia resulting from erythrocyte fragility. Heart weight-to-body weight ratios were significantly higher in the AE1(-/-) mice than in wild-type (AE1(+/+)) littermates at both 1-3 days postnatal (3.01 +/- 0.38 vs. 1.45 +/- 0.04) and at 7 days postnatal (9.45 +/- 0.53 vs. 4.13 +/- 0.41), indicating that loss of AE1 led to cardiac hypertrophy. Heterozygous (AE1(+/-)) mice had no signs of cardiac hypertrophy. Morphology of the adult AE1(-/-) mutant heart revealed an increased left ventricular mass, accompanied by increased collagen deposition and fibrosis. M-mode echocardiography revealed dysfunction of the AE1(-/-) hearts, including dilated left ventricle end diastole and systole and expanded left ventricular mass compared with AE1(+/+) hearts. Expression of intracellular pH-regulatory mechanisms in the hypertrophic myocardium of neonate AE1(-/-) mutant mice was indistinguishable from AE1(+/-) and AE1(+/+) mice, as assessed by quantitative real-time RT-PCR. Confocal immunofluorescence revealed that, in normal mouse myocardium, AE1 is sarcolemmal, whereas AE3 and slc26a6 are found both at the sarcolemma and in internal membranes (T tubules and sarcoplasmic reticulum). These results indicate that AE1(-/-) mice, which suffer from severe hemolytic anemia and spherocytosis, display cardiac hypertrophy and impaired cardiac function, reminiscent of findings in patients with hereditary abnormalities of red blood cells. No essential role for AE1 in heart function was found.
Assuntos
Anemia Hemolítica/genética , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Cardiomegalia/genética , Anemia Hemolítica/complicações , Animais , Peso Corporal , Cardiomegalia/induzido quimicamente , Cardiomegalia/patologia , DNA/genética , DNA/isolamento & purificação , Primers do DNA , Ecocardiografia , Membrana Eritrocítica/fisiologia , Coração/anatomia & histologia , Cardiopatias/genética , Camundongos , Camundongos Knockout , Tamanho do Órgão , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fresh human blood samples were collected from healthy controls and splenectomized and unsplenectomized patients with hereditary spherocytosis due to band 3 or ankyrin and spectrin deficiency. The erythrocytes were separated into age-related fractions using self-forming Percoll density gradients. Membrane proteins were analysed by 2D electrophoresis and identified by mass spectrometry. Annexin VII was present in reticulocytes but was then lost as the cells matured. A different pattern was found in band 3-deficient samples: annexin VII was in fact present in both mature and immature red cell membranes. Cytoskeletal anomalies may then influence the turn-over of annexin VII during erythrocyte maturation.
Assuntos
Anexina A7/metabolismo , Membrana Eritrocítica/química , Eritrócitos/citologia , Proteínas de Membrana/análise , Esferocitose Hereditária/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Anquirinas/deficiência , Contagem de Células , Eletroforese em Gel Bidimensional , Envelhecimento Eritrocítico , Humanos , Proteínas de Membrana/química , Transporte Proteico , Espectrina/deficiênciaAssuntos
Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Esferocitose Hereditária/genética , Adulto , Substituição de Aminoácidos , Sequência de Bases , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , Esferocitose Hereditária/etiologiaRESUMO
Band 3 deficiency with hereditary spherocytosis and hemolytic anemia in Japanese black cattle, band 3(Bov.Yamagata), is caused by a total lack of band 3 protein with an autosomal dominant inheritance. Genotyping for band 3 deficiency and sexing were successfully achieved in biopsied embryo cells with efficiencies of 98.4% and 97.4%, respectively. Transfer of the embryo that was determined as homozygous for the mutant allele into a recipient cow resulted in the production of a fetus exhibiting the genotype and red cell phenotypes characteristic of band 3(Bov.Yamagata). These results demonstrate that our procedure is reliable and applicable to produce animals free from or homozygous for the mutant allele by breeding carrier animals.
Assuntos
Anemia Hemolítica/veterinária , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Blastocisto/metabolismo , Doenças dos Bovinos/embriologia , Doenças dos Bovinos/genética , Análise para Determinação do Sexo/veterinária , Esferocitose Hereditária/veterinária , Anemia Hemolítica/diagnóstico , Anemia Hemolítica/embriologia , Anemia Hemolítica/genética , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Bovinos , Doenças dos Bovinos/diagnóstico , Transferência Embrionária/veterinária , Eritrócitos , Feminino , Genótipo , Masculino , Mutação , Esferocitose Hereditária/diagnóstico , Esferocitose Hereditária/embriologia , Esferocitose Hereditária/genéticaRESUMO
Vertical and horizontal interactions between membrane constituents account for integrity, strength and deformability of the erythrocyte. Disruption of vertical interactions caused by membrane protein deficiencies in hereditary spherocytosis (HS), favor membrane vesiculation with development of spherocytic cells. Our aim was to evaluate the hematological and clinical presentation of HS according to the type and amount of protein deficiency. We studied 81 Portuguese individuals, 71 belonging to 21 families plus 10 unrelated subjects, and found that 51 of them were HS patients. Patients were classified as presenting mild, typical or severe HS, according to laboratory results and clinical follow-up. We performed screening tests and the standardized electrophoretic membrane protein analysis to identify and quantify protein deficiencies. We found band 3 and ankyrin deficiencies as the major causes for HS. The ratios between the value of the primary and/or secondary protein deficiencies showed significantly different values according to the severity of HS, and a significant inverse correlation with the severity of HS was observed. In mild HS, the ratios between protein deficiencies reflected equivalent protein deficiencies, while an unbalance was observed in typical HS, which was enhanced in severe HS. Our data suggest that the relative quantification of each major membrane protein and of the ratios between the values of protein deficiencies may be helpful in providing additional data about the clinical outcome of HS.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Anquirinas/deficiência , Proteínas de Membrana/sangue , Proteínas de Membrana/deficiência , Esferocitose Hereditária/terapia , Membrana Eritrocítica/química , Humanos , Portugal , Valor Preditivo dos Testes , Esferocitose Hereditária/sangue , Resultado do TratamentoRESUMO
The authors report the case of a 9-year-old Caucasian girl, born in northern Portugal, with chronic nonspherocytic haemolytic anaemia and without family history of anaemia. The aethiological study of this anaemia revealed pyruvate kinase deficiency (PKD), because of two previously described mutations (426Arg-->Trp and 510Arg-->Gln). Since the blood smear revealed features not fully compatible with PKD diagnosis, additional tests were performed for the propositus and her parents, namely red blood cell membrane protein analysis. A decrease in proteins band 3 (15%) and 4.2 (18%) was found in the propositus. Her father presented only a decrease in band 3 (11%). Coexistence of PKD and erythrocyte membrane proteins deficiency in the same patient is very uncommon. Our findings suggest that a careful blood smear observation may lead to the identification of a combined deficiency in erythrocyte membrane proteins and enzymopathies.
Assuntos
Anemia Hemolítica Congênita/etiologia , Proteína 1 de Troca de Ânion do Eritrócito/deficiência , Eritrócitos/metabolismo , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/complicações , Anemia Hemolítica Congênita/genética , Anemia Hemolítica Congênita/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Criança , Análise Mutacional de DNA , Eritrócitos/enzimologia , Éxons , Saúde da Família , Feminino , Testes Hematológicos , Heterozigoto , Humanos , Mutação Puntual , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Erros Inatos do Metabolismo dos Piruvatos/genética , Erros Inatos do Metabolismo dos Piruvatos/metabolismoRESUMO
Defects in red blood cell (RBC) membrane skeleton components cause hereditary spherocytosis (HS). Clinically, HS varies significantly even among individuals with identical gene defects, illustrating the profound effects of genetic background on disease severity. We exploited a new spontaneous mouse model, wan, which arose on the inbred C3H/HeJ strain, to identify quantitative trait loci (QTL) that modify the HS phenotype. Homozygous wan mice have severe HS due to a complete deficiency of erythroid band 3. A QTL analysis of RBC count, hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin content (MCHC) was performed in wan/wan mice from an F2 intercross between C3H/HeJ(+/wan) and CAST/Ei(+/+) F1 hybrids. Hematologic and survival data from C3H, CAST/Ei F2 wan homozygotes support the hypothesis that genetic modifiers significantly influence the band-3 null HS phenotype. Significant QTL were identified for the MCV trait only, suggesting that RBC membrane characteristics are a target for modifier gene action. The most significant quantitative trait locus, Hsm1 (hereditary spherocytosis modifier 1), localizes to mouse Chromosome 12 and is dominant. The peak LOD score was obtained with a marker for Spnb1 encoding erythroid beta-spectrin, an obvious candidate gene.