RESUMEN
OBJECTIVE: Beta-thalassemia results from beta-globin gene mutations that lead to ineffective erythropoiesis, shortened red cell survival, and anemia. Patients with beta-thalassemia develop iron overload, despite which, hepcidin levels are low. This suggests that hepcidin regulation in beta-thalassemia is more sensitive to factors unrelated to iron state. Our preliminary data demonstrates that Hbb(th1/th1) mice, a model of beta-thalassemia intermedia, have lower bone marrow iron levels while levels in the liver and spleen are increased; the later account for the increased systemic iron burden in beta-thalassemia intermedia. We hypothesized that exogenous iron would improve anemia in beta-thalassemia intermedia despite systemic iron overload and further suppress hepcidin secondary to progressive expansion of erythroid precursors. MATERIALS AND METHODS: We investigate parameters involved in red cell production, precursor apoptosis, parenchymal iron distribution, and hepcidin expression in iron treated Hbb(th1/th1) mice. RESULTS: Exogenous iron results in an expansion of erythroid precursors in the liver and spleen, leading to an increase in the number of red cells, reticulocytes, and hemoglobin production. A decrease in hepcidin expression is also observed. CONCLUSIONS: These findings demonstrate for the first time that iron results in expansion of extramedullary erythropoiesis, which improves anemia and suggests that expansion of extramedullary erythropoiesis itself results in hepcidin suppression in beta-thalassemia intermedia.
Asunto(s)
Eritropoyesis/efectos de los fármacos , Hematínicos/farmacología , Hematopoyesis Extramedular/efectos de los fármacos , Hemoglobinas/biosíntesis , Complejo Hierro-Dextran/farmacología , Talasemia beta/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/genética , Médula Ósea/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Células Precursoras Eritroides/metabolismo , Eritropoyesis/genética , Hematopoyesis Extramedular/genética , Hemoglobinas/genética , Hepcidinas , Hierro/metabolismo , Ratones , Ratones Noqueados , Mutación , Talasemia beta/tratamiento farmacológico , Talasemia beta/genéticaRESUMEN
Sickle cell anemia is associated with the mutant hemoglobin HbS, which forms polymers in red blood cells of patients. The growth rate of the polymers is several micrometers per second, ensuring that a polymer fiber reaches the walls of an erythrocyte (which has a 7-microm diameter) within a few seconds after its nucleation. To understand the factors that determine this unusually fast rate, we analyze data on the growth rate of the polymer fibers. We show that the fiber growth follows a first-order Kramers-type kinetics model. The entropy of the transition state for incorporation into a fiber is 95 J mol(-1) K(-1), very close to the known entropy of polymerization. This agrees with a recent theoretical estimate for the hydrophobic interaction and suggests that the gain of entropy in the transition state is due to the release of the last layer of water molecules structured around contact sites on the surface of the HbS molecules. As a result of this entropy gain, the free-energy barrier for incorporation of HbS molecules into a fiber is negligible and fiber growth is unprecedentedly fast. This finding suggests that fiber growth can be slowed by components of the red cell cytosol, native or intentionally introduced, which restructure the hydration layer around the HbS molecules and thus lower the transition state entropy for incorporation of an incoming molecule into the growing fiber.
Asunto(s)
Anemia de Células Falciformes/metabolismo , Hemoglobina Falciforme/química , Biopolímeros/química , Entropía , Humanos , Cinética , Modelos MolecularesRESUMEN
Red blood cells with abnormal hemoglobins (Hb) are frequently associated with increased hemoglobin autoxidation, accumulation of iron in membranes, increased membrane damage and a shorter red cell life span. The mechanisms for many of these changes have not been elucidated. We have shown in our previous studies that hydrogen peroxide formed in association with hemoglobin autoxidation reacts with hemoglobin and initiates a cascade of reactions that results in heme degradation with the formation of two fluorescent emission bands and the release of iron. Heme degradation was assessed by measuring the fluorescent band at ex 321 nm. A 5.6 fold increase in fluorescence was found in red cells from sickle transgenic mice that expressed exclusively human globins when compared to red cells from control mice. When sickle transgenic mice co-express the gamma M transgene, that expresses HbF and inhibits polymerization, heme degradation is decreased. Mice expressing exclusively hemoglobin C had a 6.9 fold increase in fluorescence compared to control. Heme degradation was also increased 3.5 fold in beta-thalassemic mice generated by deletion of murine beta(major). Membrane bound IgG and red cell metHb were highly correlated with the intensity of the fluorescent heme degradation band. These results suggest that degradation of the heme moiety in intact hemoglobin and/or degradation of free heme by peroxides are higher in pathological RBCs. Concomitant release of iron appears to be responsible for the membrane damage that leads to IgG binding and the removal of red cells from circulation.
Asunto(s)
Anemia de Células Falciformes/metabolismo , Eritrocitos/metabolismo , Hemo/metabolismo , Enfermedad de la Hemoglobina C/metabolismo , Hemoglobinopatías/metabolismo , Estrés Oxidativo , Talasemia/metabolismo , Animales , Hemoglobina C/metabolismo , Inmunoglobulina G/metabolismo , Ratones , Ratones TransgénicosRESUMEN
BACKGROUND: There is evidence for increased risk of thrombosis in patients with beta-thalassemia intermedia and beta-thalassemia major. The present study investigated the prevalence of thromboembolic risk factors of prothrombin G20210A, factor V Leiden G1691A and methylentetrahydrofolate reductase (MTHFR) C677T, as well as the hematological and clinical profiles in beta-thalassemia major and intermedia patients from Western Iran. METHODS: Patients consisted of 158 beta-thalassemia patients, 151 beta-thalassemia major and 7 beta-thalassemia intermedia patients, including 82 males and 76 females aged 13.6 +/- 6.3 years. The control group were 180 healthy blood donors and school students, consisting of 103 males and 77 females aged 16.8 +/- 2.1. Genotyping was done by PCR-RFLP using Mnl I, Hind III and Hinf I for factor V Leiden and prothrombin G20210A and MTHFR, respectively. RESULTS: The prevalence of prothrombin G20210A variant in patients and healthy individuals were 1.3 and 3.3%, respectively. Factor V Leiden G1691A was insignificantly higher in beta-thalassemia patients (prevalence 5.7% and allele frequency 3.2%) compared to healthy individuals (2.8%). This mutation was found in eight beta-thalassemia major (5.3%) and one beta-thalassemia intermedia (14.3%) patients. The prevalence of MTHFR C677T polymorphism was slightly higher in patients (50%) compared to healthy individuals (48.3%). Around 71% of beta-thalassemia intermedia and 38.4% of beta-thalassemia major patients had undergone splenectomy. In beta-thalassemia major patients, 5.3% had insulin dependent diabetes mellitus (IDDM) and 6.6% had HCV antibodies. All patients with IDDM were splenectomized and in one of them the prothrombin G20210A variant was found. Two patients, a 7-year-old boy with beta-thalassemia intermedia receiving regularly blood transfusion and a beta-thalassemia major patient (a 22-year-old splenectomized female), were found to be homozygous for MTHFR 677TT and heterozygous for factor V Leiden G1691A. Double heterozygosity for factor V Leiden G1691A and MTHFR C677T and also homozygous factor V Leiden 1691AA were found in two beta-thalassemia major patients. No thromboembolic event has been recorded in the files of patients. CONCLUSIONS: The results of present study establish the prevalence of biological risk factors of thrombosis in beta-thalassemia patients from Western Iran. It seems that thrombophilic mutations may not be associated with thrombotic events in thalassemic patients, which needs to be confirmed by the study of larger sample sizes.
Asunto(s)
Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Polimorfismo de Nucleótido Simple/genética , Trombofilia/complicaciones , Talasemia beta/complicaciones , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Factor V/genética , Femenino , Frecuencia de los Genes , Humanos , Lactante , Irán/epidemiología , Masculino , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Prevalencia , Protrombina/genética , Trombofilia/epidemiología , Trombofilia/genética , Adulto Joven , Talasemia beta/genéticaRESUMEN
BACKGROUND: A hypercoagulable state in sickle cell disease (SCD) and beta thalassemia has been established and thrombosis is an important aspect of the clinical spectrum of sickle cell disease. In a case-control study, the prevalence of factor V Leiden and prothrombin G20210A mutations were investigated among SCD patients from Southern Iran. METHODS: Patients comprised 60 individuals with SCD; of them 35 were with sickle cell anemia (SS) including 21 males and 14 females aged 17.2+/-8.3 years, 15 were sickle cell trait (AS) consisted of nine males and six females aged 30+/-15.4 years and 10 were sickle/beta thalassemia (S/Thal) (three males and seven females) aged 24.6+/-10.4 years. The control group were 126 apparently healthy individuals (50 males and 76 females) aged 20.1+/-9.8 years. Genotyping was done by polymerase chain reaction restriction fragment-length polymorphism (PCR-RFLP) using Mnl I and Hind III for factor V Leiden and prothrombin G20210A, respectively. RESULTS: Heterozygous factor V Leiden mutation was found in five of 35 (14.3%) SS patients, two of 15 (13.3%) AS individuals, one (a sickle/beta-zero thalassemia patient with IVSII.1 G-->A mutation) of 10 S/Thal patients (10%), and two of 126 (1.6%) control subjects (P<0.05). However, only one AS individual (6.7%) was found to be a carrier for prothrombin G20210A compared to five of 126 (4%) healthy individuals. Adjusted logistic regression analysis for the effects of age and sex was performed and a significant association was found between factor V Leiden mutation and sickle cell anemia with odds ratios (OR) of 6.5 (95% confidence intervals [CI] 1.19-35.33, P=0.03) in SS patients. However, increased prevalence of the factor V Leiden in AS individuals and S/Thal patients was not statistically significant compared to controls (OR 3.84, 95% CI 0.49-29.9, P=0.19 and OR 3.77, 95% CI 0.31-45.9, P=0.29, respectively). CONCLUSIONS: Our findings indicate a significant correlation between factor V Leiden and sickle cell anemia among Iranian patients. Association between venous thrombophilia and factor V Leiden mutation in Iranians with sickle cell anemia should be further studied.
Asunto(s)
Anemia de Células Falciformes/genética , Coagulación Sanguínea/genética , Factor V/genética , Mutación , Trombofilia/genética , Adolescente , Adulto , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/etnología , Estudios de Casos y Controles , Niño , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Irán/epidemiología , Modelos Logísticos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Polimorfismo de Longitud del Fragmento de Restricción , Protrombina/genética , Medición de Riesgo , Rasgo Drepanocítico/sangre , Rasgo Drepanocítico/genética , Trombofilia/sangre , Trombofilia/etnología , Talasemia beta/sangre , Talasemia beta/genéticaRESUMEN
BACKGROUND: The mutation in factor V (FV) G1691A, known as factor V Leiden, and prothrombin (FII) gene G20210A are the two most prevalent causes of inherited thrombophilia. The present study reports the prevalence of factor V Leiden and the prothrombin G20210A gene mutations among healthy individuals of Kurdish ethnic background in Western Iran. METHODS: Four hundred thirty-four healthy unrelated individuals, 255 male and 179 female, with a mean age of 28.7+/-15.5 from the Kermanshah Province of Iran were studied for prothrombin G20210A mutation. The factor V Leiden mutation was studied in 404 healthy individuals, of whom 232 were male and 172 were female. The factor V Leiden and prothrombin G20210A were detected by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method using Mnl I and Hind III restriction enzymes, respectively. RESULTS: Among 434 individuals studied for prothrombin G20210A mutation seven carried this mutation as heterozygous (four female subjects and three male), giving a prevalence of 1.6% [95% confidence intervals (CI) 0.5-2.7) and an allele frequency of 0.8%. No homozygous prothrombin 20210AA was found. Factor V G1691A mutation was detected as heterozygous in 11 of 404 healthy individuals (five female and six male) and as homozygous in one male indicating a prevalence of 2.97% (95% CI 1.3-4.6) and allele frequency of 1.6%. CONCLUSIONS: Our results indicated that the factor V Leiden and prothrombin G20210A mutations are not rare among populations of Western Iran and that the relationship between venous thrombophilia and these mutations have to be further studied in Western Iran population, which, in turn, may suggest a causal effect.
Asunto(s)
Factor V/genética , Mutación , Protrombina/genética , Trombofilia/genética , Adolescente , Adulto , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Heterocigoto , Homocigoto , Humanos , Irán/epidemiología , Masculino , Polimorfismo de Longitud del Fragmento de Restricción , Trombofilia/etnologíaRESUMEN
Recombinant alpha-Savaria globin (alpha(S49R)) was assembled with beta(S) chains by the alloplex intermediate pathway to generate tetrameric rHbS-Sarvaria (alpha (2) (S49R) beta (2) (E6V) ) that exhibited normal O(2) affinity and co-operatively at pH 7.4. Allosteric effectors, 2,3-DPG, L35, and NaCl increased O(2) affinity by 15%. Bohr effects were similar for rHbS-Savaria and HbS (0.38 +/- 0.025 vs. 0.46 +/- 0.03, respectively). The C(SAT) of HbS increased from 16.7 +/- 0.8 to 27.0 +/- 1.0 g/dL. Co-polymerization demonstrated inhibition predominantly by the Cis-dimer. Molecular modeling indicated that the positive charge at alpha-49 generated a strong anion-binding site and reduced flexibility of the CD-region by restricting movement in the E and F helices. The molecular distance between Arg-49 and Asn-78 in the neighboring double strand decreased, and electrostatic repulsion between the inter-double strands increased, resulting in inhibition of polymerization. The Savaria mutation may be useful for the design of super-inhibitory alpha-chains and gene therapy of sickle cell anemia.
Asunto(s)
Regulación Alostérica , Hemoglobinas Anormales/genética , Hemoglobinas Anormales/metabolismo , Mutación/genética , Animales , Sitios de Unión , Hemo/química , Hemo/metabolismo , Hemoglobinas Anormales/química , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Oocitos/citología , Oocitos/metabolismo , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Hemoglobin Q (HbQ)-Iran [alpha75 (EF4) Asp-->His] is an alpha-chain variant that in the heterozygous state has normal hematology and has not been reported in association with a thalassemic phenotype. Here, for the first time, we described the hematologic characteristics of a 5-year-old boy with HbQ-Iran/-alpha3.7 kb trans to HbQ-Iran mutation/beta0-thalassemia (IVSII.1.G-->A) living in the Kermanshah province of Iran. OBSERVATIONS: The level of HbQ-Iran was found to be 22.4%. However, a significant reduction in mean corpuscular volume (59.3 fL) and mean corpuscular hemoglobin (19.6 pg) and an elevation of hemoglobin F (6.3%) was observed. CONCLUSIONS: This report indicates that HbQ-Iran to be a benign structural variant of Hb, that in combination with -alpha3.7 kb gene and beta0-thalassemia, presents a minor beta-thalassemia picture with moderate anemia.
Asunto(s)
Hemoglobinas Anormales/genética , Talasemia alfa/genética , Talasemia beta/genética , Sustitución de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Preescolar , Histidina/química , Histidina/genética , Humanos , Masculino , MutaciónRESUMEN
Patients with sickle cell anemia exhibit mild to moderate renal and liver damage. Glutathione S-transferase A1-1 is produced during kidney and liver damage. We hypothesized that cellular damage in sickle transgenic mice would lead to increased serum and urine murine glutathione S-transferase A1-1 levels. Levels of murine glutathione S-transferase A1-1 in the serum and urine of S+S-Antilles, NY1DD, and control mice were measured by ELISA, which revealed that the serum of S+S-Antilles mice, relative to controls, had elevated levels of murine glutathione S-transferase A1-1 (P = 0.005) as did NY1DD mice (P = 0.02, baseline vs. 2-day hypoxia). Serum liver enzymes, such as aspartate amino transferase and alanine amino transferase, as well as lactate dehydrogenase were increased in S+S-Antilles mice relative to controls (P = 0.000006, P = 0.0003, and P = 0.029, respectively). Urine murine glutathione S-transferase A1-1 of S+S-Antilles mice, as well as NY1DD mice under hypoxic stress, was not significantly different from controls. Murine glutathione S-transferase class-mu was measured by ELISA in the urine of sickle transgenic mice and control mice to define the location of tubular damage at the proximal convoluted tubule; murine Glutathione S-transferase class-mu was below the limit of detection. These findings suggest that elevated levels of murine glutathione S-transferase A1-1 in the serum reflect release during liver damage and that proximal tubular damage does not lead to appreciable urinary murine glutathione S-transferase A1-1.
Asunto(s)
Anemia de Células Falciformes/enzimología , Globinas/genética , Glutatión Transferasa/biosíntesis , Hemoglobina Falciforme/genética , Enfermedad Veno-Oclusiva Hepática/enzimología , Isoenzimas/biosíntesis , Enfermedades Renales/enzimología , Alanina Transaminasa/sangre , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/genética , Animales , Aspartato Aminotransferasas/sangre , Inducción Enzimática , Femenino , Perfilación de la Expresión Génica , Glutatión Transferasa/sangre , Glutatión Transferasa/genética , Glutatión Transferasa/orina , Enfermedad Veno-Oclusiva Hepática/etiología , Humanos , Hipoxia/enzimología , Isoenzimas/sangre , Isoenzimas/genética , Isoenzimas/orina , Enfermedades Renales/etiología , Túbulos Renales/enzimología , Túbulos Renales/patología , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Sickle cell anemia is a debilitating genetic disease that affects hundreds of thousands of babies born each year worldwide. Its primary pathogenic event is the polymerization of a mutant, sickle cell, hemoglobin (HbS); and this is one of a line of diseases (Alzheimer's, Huntington's, prion, etc.) in which nucleation initiates pathophysiology. We show that the homogeneous nucleation of HbS polymers follows a two-step mechanism with metastable dense liquid clusters serving as precursor to the ordered nuclei of the HbS polymer. The evidence comes from data on the rates of fiber nucleation and growth and nucleation delay times, the interaction of fibers with polarized light, and mesoscopic metastable HbS clusters in solution. The presence of a precursor in the HbS nucleation mechanism potentially allows low-concentration solution components to strongly affect the nucleation kinetics. The variations of these concentrations in patients might account for the high variability of the disease in genetically identical patients. In addition, these components can potentially be utilized for control of HbS polymerization and treatment of the disease.
Asunto(s)
Hemoglobina Falciforme/química , Anemia de Células Falciformes/sangre , Eritrocitos/química , Hemoglobina Falciforme/aislamiento & purificación , Humanos , Cinética , Sustancias Macromoleculares/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Renal disease is common in sickle cell anemia. In this exploratory work, we used data from a longitudinal study of the natural history of sickle cell disease to examine the hypothesis that polymorphisms (SNPs) in selected candidate genes are associated with glomerular filtration rate (GFR). DNA samples and clinical and laboratory data were available for 1,140 patients with sickle cell anemia. GFR was estimated using the Cockcroft-Gault and Schwartz formulas for adults and children, respectively. We examined approximately 175 haplotype tagging (ht) SNPs in about 70 genes of the TGFbeta/BMP pathway for their association with GFR using linear regression. Four SNPs in BMPR1B, a bone morphogenetic protein (BMP) receptor gene, yielded statistically significant associations (P values ranging from 0.015 to 0.046). Three haplotypes in this gene were also associated with GFR. The TGF-beta/BMP pathway has been associated with the development of diabetic nephropathy, which has some features in common with sickle cell nephropathy. Our results suggest that, as with other subphenotypes of sickle cell disease, renal function may be genetically modulated.
Asunto(s)
Anemia de Células Falciformes/fisiopatología , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Tasa de Filtración Glomerular/genética , Polimorfismo de Nucleótido Simple , Adulto , Anemia de Células Falciformes/genética , ADN/análisis , Femenino , Haplotipos , Humanos , Modelos Lineales , Desequilibrio de Ligamiento , Estudios Longitudinales , MasculinoRESUMEN
Sickle cell hemoglobin (HbS) is a mutant, whose polymerization while in deoxy state in the venous circulation underlies the debilitating sickle cell anemia. It has been suggested that the nucleation of the HbS polymers occurs within clusters of dense liquid, existing in HbS solutions. We use dynamic light scattering with solutions of deoxy-HbS, and, for comparison, of oxy-HbS and oxy-normal adult hemoglobin, HbA. We show that solutions of all three Hb variants contain clusters of dense liquid, several hundred nanometers in size, which are metastable with respect to the Hb solutions. The clusters form within a few seconds after solution preparation and their sizes and numbers remain relatively steady for up to 3 h. The lower bound of the cluster lifetime is 15 ms. The clusters exist in broad temperature and Hb concentration ranges, and occupy 10(-5)-10(-2) of the solution volume. The results on the cluster properties can serve as test data for a potential future microscopic theory of cluster stability and kinetics. More importantly, if the clusters are a part of the nucleation mechanism of HbS polymers, the rate of HbS polymerization can be controlled by varying the cluster properties.
Asunto(s)
Biofisica/métodos , Hemoglobina Falciforme/química , Hemoglobina Falciforme/metabolismo , Relación Dosis-Respuesta a Droga , Hemoglobinas/química , Humanos , Cinética , Luz , Conformación Molecular , Mutación , Polímeros/química , Conformación Proteica , Dispersión de Radiación , Temperatura , Termodinámica , Factores de TiempoRESUMEN
Polymerization of sickle cell hemoglobin (HbS) in deoxy state is one of the basic events in the pathophysiology of sickle cell anemia. For insight into the polymerization process, we monitor the kinetics of nucleation and growth of the HbS polymer fibers. We define a technique for the determination of the rates J and delay times theta of nucleation and the fiber growth rates R of deoxy-HbS fibers, based on photolysis of CO-HbS by laser illumination. We solve numerically time-dependent equations of heat conductance and CO transport, coupled with respective photo-chemical processes, during kinetics experiments under continuous illumination. After calibration with experimentally determined values, we define a regime of illumination ensuring uniform temperature and deoxy-HbS concentration, and fast (within <1 s) egress to steady conditions. With these procedures, data on the nucleation and growth kinetics have relative errors of <5% and are reproducible within 10% in independent experiments. The nucleation rates and delay times have steep, exponential dependencies on temperature. In contrast, the average fiber growth rates only weakly depend on temperature. The individual growth rates vary by up to 40% under identical conditions. These variations are attributed to instability of the coupled kinetics and diffusion towards the growing end of a fiber. The activation energy for incorporation of HbS molecules into a polymer is E(A)=50 kJ mol(-1), a low value indicating the significance of the hydrophobic contacts in the HbS polymer. More importantly, the contrast between the strong theta(T) and weak R(T) dependencies suggests that the homogenous nucleation of HbS polymers occurs within clusters of a precursor phase. This conclusion may have significant consequences for the understanding of the pathophysiology of sickle cell anemia and should be tested in further work.
Asunto(s)
Hemoglobina Falciforme/química , Hemoglobina Falciforme/metabolismo , Cinética , Polímeros/química , Automatización , Hemoglobina Falciforme/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Metionina/análisis , Métodos , Fotólisis , Sulfatos/farmacología , TemperaturaRESUMEN
The linkage of pair-wise interactions of contact site mutations of HbS has been studied using Le Lamentin [His-20 (alpha)-->Gln], Hoshida [Glu-43 (beta)-->Gln] and alpha(2)beta (2) (T87Q) mutations as the prototype of three distinct classes of contact sites of deoxy HbS fiber. Binary mixture experiments established that beta(A)-chain with the Thr-87 (beta)-->Gln mutation is as potent as the gamma-chain of HbF (alpha(2)gamma(2)) in inhibiting polymerization. On combining the influence of Le Lamentin mutation with that of beta (2) (T87Q) mutations; the net influence is only partial additivity. On the other hand, in binary mixture studies, combined influence of Hoshida mutation with that of beta (2) (T87Q) mutations is synergistic. Besides, a significant level of synergistic complementation is also seen when the Le Lamentin and Hoshida mutations are combined in HbS (symmetrical tetramers). Le Lamentin and Hoshida mutation introduced into the cis-dimer of the asymmetric hybrid tetramer completely neutralizes the Val-6 (beta) dependent polymerization. Accordingly, we propose that combining the perturbation of intra-double strand contact site with that of an inter-double strand contact site exhibit synergy when they are present in two different chains of the alphabeta dimer. A comparison of the present results with that of the earlier studies suggest that when the two contact site perturbations are from the same sub-unit of the alphabeta dimer only partial additivity is observed. The map of interaction linkage of the contact site mutations exposes new strategies in the design of novel anti-sickling Hbs for the gene therapy of sickle cell disease.
Asunto(s)
Hemoglobina Falciforme/química , Animales , Biopolímeros , Hemoglobina Falciforme/genética , Hemoglobinas Anormales/química , Hemoglobinas Anormales/genética , Ratones , Ratones Transgénicos , MutaciónRESUMEN
A total of 1,000 school boys ages 14-18 years, were randomly selected from six schools of Kermanshah and screened for G6PD deficiency. Fifty-three out of 1,000 were found to be severely G6PD deficient giving a frequency of 5.3% for G6PD deficiency in males in Kermanshah. We performed DNA analysis on 68 G6PD deficient children, 52 school boys and 16 children with the history of favism and hemolytic anemia using a combination of PCR-RFLP technique, single strand conformation polymorphism (SSCP) analysis and DNA sequencing. Three polymorphic G6PD mutations were identified: G6PD Mediterranean, Chatham and Cosenza. The commonest allele was found to be the G6PD Mediterranean (91.2%) followed by the G6PD Chatham (7.3%) and the G6PD Cosenza (1.5%). Haplotype analysis revealed that 56 out of 62 (90%) G6PD Mediterranean was linked to haplotype BclI(+) (1311T). In contrast, all G6PD Chatham and Cosenza had haplotype BclI(-) (1311C). The polymorphism IVS11-93 (T-->C) was present in 88.5% of the subjects studied. Only 4/55 (7.3%) of the Mediterranean alleles were associated with the T form and were always related to the nt 1311C. Our findings indicate that the allele frequency of G6PD Mediterranean mutation in Kermanshah is higher than those from two Fars ethnic groups living in Northern and Southern Iran. Nevertheless, they are in strict accordance with a previous report of the prevalence of the G6PD Mediterranean in Kurdish and Middle East population. Also, of strong association of the G6PD Mediterranean mutation and the presence of the polymorphism nt 1311C-->T in the Kermanshah population demonstrate that the presence of this mutation may be the result of migrations that have taken place through the history of Iran.
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Etnicidad/genética , Deficiencia de Glucosafosfato Deshidrogenasa/etnología , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/metabolismo , Adolescente , Alelos , Humanos , Irán/epidemiología , Masculino , Mutación Puntual , Polimorfismo Genético , Análisis de Secuencia de ADN/métodosRESUMEN
Transgenic mouse models of hemoglobinopathies unravel pathophysiological mechanisms; yet the validity of the red blood cell (RBC) model of human hemoglobin (hHb) enveloped by a mouse (m) membrane has been questioned. Isoelectric focusing of hHb and mHb from transgenic mRBC shows a greater association of mHb to the mouse membrane compared to normal hHbA, supporting a species-specific Hb-mRBC membrane interaction. Enhanced hmutant Hb (HbE, HbS and HbC)-mRBC membrane affinities correlates with enhanced membrane lipid peroxidation and parallel those reported in hRBC, lending support to transgenic mRBC as models of hemoglobinopathies. Species-specific Hb-membrane interaction may be overridden by Hb charge and conformational alterations.
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Membrana Eritrocítica/metabolismo , Hemoglobinas/metabolismo , Animales , Hemoglobina A/metabolismo , Hemoglobinas/genética , Humanos , Peroxidación de Lípido , Ratones , Ratones Transgénicos , Mutación , Especificidad de la EspecieRESUMEN
Hb D-Los Angeles is characterized by the substitution of glutamine for glutamic acid at position 121 of the beta-globin chain. The present investigation is the first study on the beta-globin gene haplotypes associated with beta-D-Los Angeles in Western Iran. Thirty two individuals from 11 unrelated families from Western Iran were studied. The Hb D-Los Angeles status of all cases was confirmed using polymerase chain reaction (PCR) followed by digestion with EcoRl. The haplotype of the beta-globin gene cluster was determined by a PCR-RFLP (restriction fragment length polymorphism) procedure. The haplotype background of the betaA chromosomes was also determined in 35 normal subjects from the same geographic region. The beta-globin gene haplotype analysis demonstrated that all beta-D-Los Angeles genes (23 genes) were in linkage disequilibrium with haplotype I [+----++]. Among the 70 betaA chromosomes, 30 chromosomes (42.9%) were associated with haplotype I. The present study indicates the unicentric origin of the beta-D-Los Angeles gene in Western Iran. It seems that this mutation may have occurred at the same chromosomal background common in the local population.
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Globinas/genética , Haplotipos , Hemoglobinas Anormales/genética , Mutación Puntual , Adolescente , Adulto , Niño , Preescolar , ADN/análisis , Femenino , Globinas/análisis , Hemoglobinas Anormales/análisis , Humanos , Lactante , Irán/epidemiología , Masculino , Reacción en Cadena de la Polimerasa/métodosRESUMEN
An actively detunable planar quadrature surface coil for human body imaging at 4 T has been constructed and compared with a conventional linear surface coil. The coil could be used as a transmit/receive or a receive-only device in combination with a volume transmit coil. Transmission, reception profiles and the corresponding images acquired with each coil, as well as with both individual modes of the quadrature coil, are presented. Data collected using a tissue equivalent loaded phantom recorded with the linear surface coil demonstrated significant intensity distortions due to RF penetration artifact. The quadrature surface coil, on the other hand, provided compensation of the artifact, separately in its transmission and reception profiles as well as in the resultant images. Substantial sensitivity gain was also observed for the quadrature coil compared to the linear device. Significant advantages of using the quadrature surface coil over the linear device at 4 T have, therefore, been demonstrated.
Asunto(s)
Aumento de la Imagen/métodos , Imagen por Resonancia Magnética/métodos , Artefactos , Diseño de Equipo , Humanos , Imagen por Resonancia Magnética/instrumentación , Fantasmas de Imagen , Ondas de Radio , Sensibilidad y EspecificidadRESUMEN
In sickle cell anemia (SCA), inflammatory (i.e., intravascular sickling and transient vasoocclusive) events result in chronic endothelial activation. In addition to sickling behavior, sickle (SS) red blood cells exhibit abnormal interaction with the vascular endothelium, which is considered to have an important role in initiation of vasoocclusion. Upregulation of endothelial adhesion molecules caused by oxidants (and cytokines) may lead to increased SS red cell adhesion. We hypothesize that endothelial activation is indispensable in SS red cell adhesion to the endothelium and that antioxidants will have an inhibitory effect on this interaction. We examined the effect of selected antioxidants in ex vivo mesocecum vasculature, a well-established model that allows measurement of hemodynamic parameters and, by intravital microscopy, can allow quantification of adhesion. We tested antioxidant enzymes (SOD and catalase) and an intravascular SOD mimetic, polynitroxyl albumin (PNA), in the presence of platelet-activating factor (PAF); the latter causes endothelial oxidant generation and endothelial activation, which characterize SCA. In ex vivo preparations, PAF not only induced marked endothelial oxidant generation, it also enhanced SS red cell adhesion, resulting in frequent blockage of small-diameter venules. The adhesion, inversely related to venular diameter, and vasoocclusion were markedly inhibited by antioxidants, resulting in improved hemodynamics. PNA, the most effective antioxidant, also abolished SS red cell adhesion in non-PAF-activated preparations. Thus SS red cell adhesion and related vasoocclusion may be ameliorated by antioxidant therapy with a stable and long-acting molecule (e.g., PNA).