Hemoglobin polymorphism and hematological profile of Geoffroy's side-necked turtle (Phrynops geoffroanus, Testudines) in the northwestern region of São Paulo State, Brazil

Complete blood counts and hemoglobin isoform data were gathered from 36 specimens of the turtle species Phrynops geoffroanus from the northwestern region of São Paulo State, Brazil. They were collected in an urban area. The hemoglobin profiles were obtained after red blood cell lysis and by electrophoretic migration in alkaline pH, acid pH, and neutral pH buffer. The hemoglobin components were confirmed using high-performance liquid chromatography (HPLC). Erythrogram analysis included hematocrit, total hemoglobin concentration, total red blood cell count, and red blood cell indices. The leukogram included a total white blood cell count and a calculation of the percent values of neutrophils, lymphocytes, monocytes, basophils, eosinophils, heterophils, and azurophils. HPLC analysis revealed three hemoglobin components; the first with a concentration of 5.5%, the second was a major component with an average concentration of 67.1%, and the third with a concentration of 28.5%. The hematological profile obtained for these specimens allowed us to establish a pattern for P. geoffroanus in São Paulo State Northwestern region. The average hematocrit values were 22.5% for females and 24.0% for males. For total hemoglobin, we found average values of 6.66 g/dL in females and 7.22 g/dL in males. The number of white blood cells was 2725 x 103/μL for females and 2775 x 103/μL for males. There was a predominance of heterophils, eosinophils, and monocytes in both sexes. No significant differences were found between males and females for hematological profile. The hematological results were compared to literature data for other Chelonia. They were similar to what is known for fresh water turtles. © FUNPEC-RP.

Hemoglobin and HFE gene polymorphisms in Brazilian populations in regions endemic for malaria

Our objective was to determine how the distribution of red blood cell diseases is related to malaria occurrence in north Brazil, a region endemic for malaria. We evaluated the incidence of two mutations in the HFE gene, H63D and C282Y, in two study groups: a control blood donor group, with no indication of malaria infection, and a group constituted of malaria patients of four states of the Amazonian region. The hemoglobin polymorphisms were obtained by HPLC and classical laboratory methodologies, and the two mutations in the HFE gene were assayed by PCR-RFLP. We found a high frequency of alpha thalassemia, but there were no significant differences between blood donors and malaria patients. There were also no significant differences in the frequencies of HbA(2); however, the frequency of HbF was significantly different in individuals with malaria from Para and Rondonia. The mean number of reticulocytes was significantly reduced in the blood donors from the northern region, suggesting an adaptive strategy of these populations to parasitic attack by Plasmodium. Most individuals were heterozygous for the H63D allele of the HFE gene in both study groups. In the blood donors group, the greatest frequency of the H63D allele was found in Caucasians of all the states. In the malaria patients group in Rondonia, there was a high frequency of the H63D allele among the non-Caucasians. In the other states, and in the malaria patients group, the H63D allele was the most frequent among the Caucasians. Based on our results, we suggest that the maintenance of polymorphism of the mutations in the gene HFE can be explained by selective factors other than malaria, or it is due to simple allelic oscillation and by the constant gene flow among the populations in Brazil.

Description of electrophoretic and chromatographic hemoglobin profile of Rhinoclemmys punctularia

Studies of the hemoglobin pattern in Brazilian reptiles are important for determining ecological and phylogenetic relationships, but they are scarce. Peripheral blood samples were obtained from 7 males and 18 females of Rhinoclemmys punctularia. The hematological profile was based on the total hemoglobin and hematocrit values. The hemoglobin profile was obtained using electrophoretic procedures at different pH, isoelectric focusing, globin chain electrophoresis, and HPLC. The hematocrit (31 ± 2%) and total hemoglobin (7.5 ± 0.2 g/dL) values did not indicate gender variations. Alkaline pH electrophoresis of the total blood samples treated with 1% saponin demonstrated the presence of four well-defined hemoglobin fractions, one major component (fraction I), showing cathodic migration and three others faster than fraction I with anodic migration. When the samples were precipitated with chloroform, only two hemoglobin fractions were observed, similar to fractions I and III from the first procedure. Isoelectric focusing and HPLC showed the same pattern. With acid and neutral pH electrophoresis, two fractions with anodic migration were observed. The globin chain identification at alkaline pH showed two fractions, but four fractions were observed at acidic pH, suggesting that different polypeptide chains are involved in the hemoglobin molecule. The chromatographic separation of the total blood sample demonstrated that the major fraction comprised 81.9% and the minor 18.1%. The results obtained demonstrated a similarity between these hemoglobin components and those of some Chelidae reported in the literature for both land and aquatic animals, reflecting the adaptation to environmental conditions. ©FUNPEC-RP.

HB D Los Angeles in a Brazilian family

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Phenotypic expression of homozygous hemoglobin S in relation to β-globin haplotypes, glutathione S-transferase polymorphisms and detoxification enzymes

Sickle cell anemia (SCA) shows a pathophysiology that involves multiple changes in sickle cell erythrocytes, vaso-occlusive episodes, hemolysis, activation of inflammatory mediators, endothelial cell dysfunction, and oxidative stress. These events complicate treatment and culminate in the development of manifestations such as anemia, pain crises and multiorgan dysfunction. The aim of this study was to evaluate, in SCA patients, oxidative stress and antioxidant capacity markers, correlating them to treatment with hydroxyurea (HU), β-globin haplotypes and glutathione S-transferase polymorphisms (GSTT1, GSTM1 and GSTP1), in comparison to a control group (CG). The study groups were composed of 48 individuals without hemoglobinopathies (CG), SCA patients treated with HU [AF (+HU), N = 13] and untreated SCA patients [AF (-HU), N = 15], after informed consent. The groups were analyzed using cytological, electrophoretic, chromatographic and molecular methods and information from medical records. The GSTM1 and GSTT1 polymorphisms were determined by multiplex PCR, while the GSTP1 polymorphism by PCR-RFLP. Biochemical parameters were measured using spectrophotometric methods [TBARS, TEAC and catalase (CAT) and GST activities] and a chromatographic method [glutathione (GSH)]. The fetal Hb (Hb F) levels observed in the SCA (+HU) group (10.9%) confirmed the already well-described pharmacological effect of HU, but the SCA (-HU) group also had high Hb F levels (6.1%), which may have been influenced by genetic factors not targeted in this study. We found a higher frequency of the Bantu haplotype (48.2%), followed by the Benin (32.1%) and also Cameroon haplotypes, rare in our population, and 19.7% of atypical haplotypes. The presence of Bantu haplotype was related to higher lipid peroxidation levels in patients, but also, it conferred a differential response to HU treatment, raising Hb F levels in 52.6% (P = 0.03). The protective effect of Hb F was confirmed, because the increase in their levels resulted in a 41.3% decrease in lipid peroxidation levels (r = -0.74, P = 0.0156). The genotypic frequency of the GST polymorphisms observed was similar to that of other studies in the Brazilian population, and its association with biochemical markers revealed a significant difference only for the GSTP1 polymorphism, where patients with genotype V/V showed higher GSH and TEAC levels (P = 0.04 and P = 0.03, respectively) compared to patients with genotype I/I. The TBARS levels were about five to eight times higher in the SCA (+HU) and SCA (-HU) groups, respectively, compared to controls, and HU produced a 35.2% decrease in lipid peroxidation levels in the SCA (+HU) group (P < 0.0001). Moreover, the SCA (+HU) group showed higher TEAC levels when compared to CG (P = 0.002). We did not find any significant difference in GST activity between the groups studied (P = 0.76), but CAT activity was about 17 and 30% lower in SCA (+HU) and SCA (-HU) groups, respectively (P < 0.00001). Plasma GSH levels were ~2 times higher in SCA patients than in the control group (P = 0.0005) and showed a positive correlation with TBARS levels, confirming its antioxidant function. HU treatment contributed to higher CAT activity and TEAC levels and lower lipid peroxidation, and its pharmacological effect showed a “haplotype-dependent” response. These findings may contribute to elucidating the potential of HU in ameliorating oxidative stress in SCA subjects.

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fluorescent amplified fragment length polymorphism genotyping of human and animal Staphylococcus aureus isolates from dairy farms with manual milking

Fluorescence amplified fragment length polymorphism (fAFLP) was used to assess the genetic relatedness of 40 Staphylococcus aureus strains isolated from human and animal skin samples in seven dairy farms with manual milking. S. aureus was isolated from 11 out of 30 (36%) human skin samples and from 29 out of 100 (29%) teat skin samples from apparently healthy cows. Genomic DNA from each isolate was double-digested with EcoRI and MseI and complementary oligonucleotide adaptors were ligated to the restriction fragments. Pre-selective and selective, amplification reactions were performed, the amplified fragments were separated by electrophoresis in an ABI377 sequencer and analysed using GeneScan 3.1 and Genotyper 2.5. Three single isolates (a-c), a predominant cluster with 35 isolates (d) and another cluster with two isolates (e) were identified. Both clusters d and e included human and animal isolates genetically related, because the profiles had 90-100% homology. Since no cluster was comprised uniquely of human or animal isolates and given the close genetic relatedness among human and animal samples in the farms, the present findings support the. hypothesis that dairy workers can spread S. aureus through manual milking. (C) 2005 Elsevier B.V. All rights reserved.

Characterization and polymorphism screening of IGF-I and prolactin genes in Nelore heifers

Insulin growth factor I (IGF-I) and prolactin (PRL) are peptide hormones that exert complementary effects on reproductive traits by acting on folliculogenesis. In view of the lack of information about the IGF-I and PRL genes in Bos indicus, the objective of this study was to partially characterize the promoter regions of these genes and to screen animals of different ages at first pregnancy for the presence of polymorphisms in these regions. In addition, we determined whether polymorphisms influence the regulation of the two hormone genes, evaluating their association with sexual precocity.The animals were divided into three groups according to age at first pregnancy: 1) 100 heifers considered to be sexually precocious that became pregnant at 15-16 months of age, 2) 100 heifers that became pregnant during the normal breeding season at 24 months of age, and 3) 100 heifers that did not become pregnant until 24 months of age. For the IGF-I gene, PCR-RFLP-SnaBI analysis showed the presence of genotypes AB and BB at frequencies of 0.02 and 0.98, respectively. Sequencing of the IGF-I gene fragment revealed a single nitrogen base change from cytosine to thymine, corresponding to the restriction site of SnaBI. The polymorphisms identified in the 5'-flanking region of the IGF-I gene may serve as a basis for future studies of molecular markers in cattle. For the PRL gene, PCR-RFLP-HaeIII analysis showed the presence of only one migration pattern, a finding characterizing the region studied as monomorphic. The study of other regions in the IGF-I and PRL genes might provide molecular data that can be used in the future for the selection of sexually precocious animals.

Polymorphism analysis of the hsp70 stress gene in Broiler chickens (Gallus gallus) of different breeds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Restriction fragment length polymorphism (RFLP) in Exon 2 of the BoLA-DRB3 gene in South American cattle

The Bola-DRB3 gene participates in the development of the immune response and is highly polymorphic. For these reasons, it has been a candidate gene in studies of the genetic basis of disease resistance and in population genetic analysis. South American native cattle breeds have been widely replaced by improved exotic breeds leading to a loss of genetic resources. In particular South American native breeds have high levels of fertility and disease resistance. This work describes genetic variability in the BoLA-DRB3 gene in native (Caracu, Pantaneiro, Argentinean Creole) and exotic (Holstein, Jersey, Nelore, Gir) cattle breeds in Brazil and Argentina. PCR-RFLP alleles were identified by combining the restriction patterns for the BoLA-DRB3.2 locus obtained with RsaI, BstY, and HaeIII restriction enzymes. Allelic frequencies and deviations from the Hardy-Weinberg equilibrium were also calculated. Analysis of the 24 BoLA-DRB3 PCR-RFLP alleles identified showed differences in the allele distributions among breeds.

Chromosome polymorphism in the Brazilian dwarf brocket deer, Mazama nana (Mammalia, Cervidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MUC1 gene polymorphism in three Nelore lines selected for growth and its association with growth and carcass traits

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Association between MUC1 gene polymorphism and expected progeny differences in Nelore cattle (Bos primigenius indicus)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)