Characterization and oxygen binding properties of des-Arg human hemoglobin

The role of chloride in the stabilization of the deoxy conformation of hemoglobin (Hb), the low oxygen affinity state, has been studied in order to identify the nature of this binding. Previous studies have shown that arginines 141α could be involved in the binding of this ion to the protein. Thus, des-Arg Hb, human hemoglobin modified by removal of the α-chain C-terminal residue Arg141α, is a possible model for studies of these interactions. The loss of Arg141α and all the salt bridges in which it participates is associated with subtle structural perturbations of the α-chains, which include an increase in the conformational flexibility and further shift to the oxy state, increasing oxygen affinity. Thus, this Hb has been the target of many studies of structural and functional behavior along with medical applications. In the present study, we describe the biochemical characterization of des-Arg Hb by electrophoresis, high-performance liquid chromatography and mass spectroscopy. The effects of chloride binding on the oxygen affinity and on the cooperativity to des-Arg Hb and to native human hemoglobin, HbA, were measured and compared. We confirm that des-Arg Hb presents high oxygen affinity and low cooperativity in the presence of bound chloride and show that the binding of chloride to des-Arg does not change its functional characteristics as observed with HbA. These results indicate that Arg141α may be involved in the chloride effect on Hb oxygenation. Moreover, they show that these residues contribute to lower Hb oxygen affinity to a level compatible with its biological function.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Thesis Abstract Geoffroy's side-necked turtle [Phrynops geoffroanus (Schweigger, 1812), Testudines: Chelidae] as a model for evolutionary ecotoxicology: relationship between environmental contamination, conditions and genetic variability

The objective of this study was to evaluate the role of human activity factors, such as environmental contamination and habitat changes, as drivers for changing the physiological, biochemical, and genetic diversity of Geoffroy’s side-necked turtle populations in one of the most impacted watersheds in southeastern Brazil. The impact of chemical and organic contamination was determined by ecotoxicological analyses to assess the action of some of the major components involved in protection against oxidative stress, phase I and II detoxification metabolism, and antioxidant capacity. The results indicated the influence of domestic and industrial effluents on detoxification metabolism and oxidative stress. However, in spite of increased activity and effect of EROD (CYP1A1) and glutathione S-transferase (GST) activity, GST average values in the urban area agreed with those expected for hypoxic conditions according to the literature. This observation suggests that increased GST in response to ROS production due to the presence of pollutants increases the antioxidant defense network, controlling the oxidative damage caused by hypoxia and reperfusion. To determine the conditions that are reflected in individual ability (fitness), we evaluated the mathematical relationship between weight and length, and found that changes in body shape and weight increase, allowing inferences about animal health and welfare. The data obtained indicate differences in conditions that are associated with the area, but also with sex and reproductive period, and contamination gradient, indicating a strong influence of environmental stressors on the physiology of the specimens. The evaluation of genetic structure among populations of Preto River and Felicidade Stream, based on microsatellites, demonstrated that there was no genetic differentiation, due to extensive gene flow between the areas and high genetic diversity. However, after analysis of intrapopulation structure, we observed the existence of five genetic groups that reflected changes in habitat created by damming and siltation, which initiate separation processes (barriers) between sub-populations. The relationship between the data obtained for biochemical parameters, condition factors and genetic diversity was analyzed by heterozygosity-fitness correlation. The negative relationship observed may be explained by the profile of structural and ecological changes in the populations studied, indicating the important influence of humans on the biology of natural populations. Therefore, Phrynops geoffroanus shows adaptation to environmental contamination, and ecological changes and possible loss of habitat are altering the genetic diversity of the populations studied. This is the first study evaluating all these aspects of P. geoffroanus simultaneously in natural populations in Brazil, using this species as a model.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Biologia, Instituto de Biociências Letras e Ciências Exatas de São José do Rio Preto, Sao Jose do Rio Preto, Rua Cristóvão Colombo, 2265, Jardim Nazareth, CEP 15054-000, SP, BrasilUniversidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Biologia, Instituto de Biociências Letras e Ciências Exatas de São José do Rio Preto, Sao Jose do Rio Preto, Rua Cristóvão Colombo, 2265, Jardim Nazareth, CEP 15054-000, SP, BrasilCNPq: 143419/2008-

Serum ferritin and transferrin saturation levels in β0 and β+ thalassemia patients

There have been few studies on the mutations that cause heterozygous beta-thalassemia and how they affect the iron profile. One hundred and thirty-eight individuals were analyzed, 90 thalasemic β0 and 48 thalasemic β+, identified by classical and molecular methods. Mutations in the hemochromatosis (HFE) gene, detected using PCR-RFLP, were found in 30.4% of these beta-thalassemic patients; heterozygosity for H63D (20.3%) was the most frequent. Ferritin levels and transferrin saturation were similar in beta-thalassemics with and without mutations in the HFE gene. Ferritin concentrations were significantly higher in men and in individuals over 40 years of age. Transferrin saturation also was significantly higher in men, but only in those without HFE gene mutations. There was no significant difference in the iron profile among the β0 and β+ thalassemics, with and without HFE gene mutations. The frequency of ferritin values above 200 ng/mL in women and 300 ng/mL in men was also similar in β0 and β+ thalassemics (P > 0.72). Our conclusion is that ferritin levels are variable in the beta-thalassemia, trait regardless of the type of beta-globin mutation. Furthermore, HFE gene polymorphisms do not change the iron profile in these individuals. ©FUNPEC-RP www.funpecrp.com.br.Departamento de Biologia Universidade Estadual Paulista Júlio de Mesquita Filho, São José do Rio Preto, SPDepartamento de Biologia Universidade Estadual Paulista Júlio de Mesquita Filho, São José do Rio Preto, S

Frequencies of -308G/A (TNFA) and -509C/T (TGFB1) polymorphisms in sickle cell anemia patients from Brazil

Sickle cell anemia is an affection that causes chronic inflammation, with consequences for vaso-occlusion, oxidative stress and cytokine production. Genetic polymorphisms in markers involved in this process can modulate the inflammatory response, including polymorphism -308G/A of TNFA (tumor necrosis factor alpha) and -509C/T of TGFB1 (transforming growth factor beta 1), reported to increase TNF-alpha and TGF-beta 1 production, respectively. Changes in the cytokine balance are important risk Factors for clinical events; consequently, we examined the frequencies of these polymorphisms in 240 Brazilian sickle cell anemia patients from southeast Brazil. PCR-RFLP was used to detect these polymorphism. The -509C/T (TGFB1) polymorphism was more frequent than -308G/A (TNFA), with allelic frequency of 0.3 for the mutant allele T (TGFB) agaist 0.1 for the mutant allele A (TNFA). These allelic frequencies are similar to those known from populations with ethnicity similar to the Brazilian population. Inheritance of these polymorphisms does not seem to be associated with that of the Hb S mutation; however, this information could be useful in analyses of specific clinical characteristics of sickle cell anemia