Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human alpha-tropomyosin

Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+. However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.

Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immunocytochemical and biochemical study of myogenic regulatory protein expression

There has been persistent controversy regarding the nature of cell differentiation in alveolar soft-part sarcoma (ASPS) since its first description in 1952. Some studies suggest that ASPS might represent an unusual variant of skeletal muscle tumor, Given the availability of new monoclonal antibodies to probe for skeletal muscle differentiation and the rapid advance in immunocytochemical techniques for deparaffinized, formalin-fixed tissue sections, we wished to test the proposed hypothesis that ASPS might represent a new type of rhabdomyosarcoma Twelve archival samples of ASPS were retrieved, and we investigated the expression of two myogenic regulatory proteins, MyoD1 and myogenin, as rvell as other muscle-associated proteins, using sensitive immunocytochemical techniques. Despite the presence of desmin immunostaining in six ASPSs, no tumors were positive for either muscle actin or myoglobin Most importantly, no specimen showed nuclear expression of MyoD1 or myogenin, In 11 tumors, however, there was considerable granular immunostaining in the tumor cell cytoplasm with the anti-MyoD1 monoclonal antibody 5.8A, a phenomenon observed in various nonmuscle normal and neoplastic tissues with this antibody, To analyze the exact nature of immunostaining of MyoD1 and desmin in ASPS, biochemical analyses using available fresh frozen tumor tissue were performed, Although a 53-kDa band was noted with antidesmin antibody on Western blot analysis, no specific protein band that corresponds to the 45-kDa MyoD1 was detected with antibody 5.8A. These results confirm the presence of desmin in ASPS but argue against authentic expression of MyoD1, They also suggest that the cytoplasmic immunostaining observed with anti-MyoD1 antibody 5.8A most likely represents a nonspecific cross-reaction with an unknown cytoplasmic antigen, Considering the master role that MyoD1 and myogenin play in skeletal muscle commitment and differentiation and the lack of expression of these two proteins in ASPS as determined immunocytochemically and biochemically, we think that the histogenesis of ASPS remains unknown.

Conservation genetics of the endangered Pampas deer (Ozotoceros bezoarticus)

The Pampas deer (Ozotoceros bezoarticus L. 1758) is the most endangered neotropical cervid, and in the past occupied a wide range of open habitats including grassland, pampas, savanna, and cerrado (Brazil) from 5 degrees to 41 degrees S. To better understand the effect of habitat fragmentation on gene flow and genetic variation, and to uncover genetic units for conservation, we examined DNA sequences from the mitochondrial control region of 54 individuals from six localities distributed throughout the present geographical range of the Pampas deer. Our results suggest that the control region of the Pampas deer is one of the most polymorphic of any mammal. This remarkably high variability probably reflects large historic population sizes of millions of individuals in contrast to numbers of fewer than 80 000 today. Gene flow between populations is generally close to one migrant per generation and, with the exception of two populations from Argentina, all populations are significantly differentiated. The degree of gene flow was correlated with geographical distance between populations, a result consistent with limited dispersal being the primary determinant of genetic differentiation between populations. The molecular genetic results provide a mandate for habitat restoration and reintroduction of Pampas deer so that levels of genetic variation can be preserved and historic patterns of abundance can be reconstructed. However, the source of individuals for reintroduction generally should be from populations geographically closest to those now in danger of extinction.

Early cytotoxic and genotoxic effects of atrazine on Wistar rat liver: A morphological, immunohistochemical, biochemical, and molecular study

Risk assessments suggest that intermediate and long-term exposure to triazine herbicides and its metabolites through water can cause severe damage to human health. The objective of this study was to investigate the possible effects of atrazine on Wistar rats submitted to subacute treatment. For this purpose, the activity of catalase and alanine aminotransferase was quantified, and the effect of the herbicide on cell membranes was examined based on the measurement of lipid peroxidation and consequent formation of malondialdehyde and on the mRNA expression of antioxidant enzymes (Mn-superoxide dismutase [SOD] and GSTM1) and connexins. In addition, we evaluated histopathological alterations in the liver, cellular expression of SOD and glutathione (GST), activation of heat shock proteins (HSPs) by immunohistochemistry, and the induction of apoptosis. The genotoxic potential of the herbicide was investigated by the micronucleus test in bone marrow smears. Adult male Wistar rats were treated with an aqueous solution of atrazine at a concentration of 400 mg/kg/day, by gavage, for 14 consecutive days. Control groups were also included. The results showed an increase of catalase levels and maintenance of the expression of antioxidant enzymes (SOD and GST). In addition, lipid peroxidation, hepatic tissue degeneration, activation of HSP90, increased levels of connexin mRNA, and genotoxicity were observed. In conclusion, atrazine induced early hepatic oxidative stress that triggered defense mechanisms to maintain the morphophysiological integrity of the liver. Further studies are needed to better understand the effects of this herbicide on human health. (C) 2011 Elsevier B.V. All rights reserved.

The application of biochemical responses to assess environmental quality of tropical estuaries: field surveys

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Perioperative administration of vedaprofen, tramadol or their combination does not interfere with platelet aggregation, bleeding time and biochemical variables in cats

A randomized double blind and placebo controlled design was used to investigate the hemostatic, biochemical, gastrointestinal and behavioral effects of pre- and postoperative administration of vedaprofen 0.5 mg/kg PO (V), tramadol 2 mg/kg SC (T), their association (VT) or placebo (P) in 40 adult female cats (3.0 +/- 0.32 kg; 1.8 +/- 0.7 years) distributed in groups of 10. Platelet aggregation and bleeding time were measured before and 52 11 after ovariohysterectomy. Serum urea, creatinine, alanine aminotransferase, alkaline phosphatase and gamma-glutamyl transferase concentrations were measured before and 7 days postoperatively. The occurrence of vomiting, frequency and consistency of feces, and behavior were observed for 7 days postoperatively. Morphine (0.5 mg/kg, IM) was used as rescue analgesic. Laboratory variables did not change. Vomiting was observed only after morphine administration. Mild euphoria was observed in T and VT. The perioperative use of vedaprofen and/or tramadol did not modify the hemostatic, biochemical and gastrointestinal function in cats. (C) 2009 ESFM and AAFP. published by Elsevier Ltd. All rights reserved.

Epidemiology and genetics of endemic goiter. II. Genetic aspects

Complex genetic models and segregation analysis were applied to family data obtained in a hyperendemic goiter area in Brazil. The single locus and Falconer's models did not fit the data. Edward's model showed convergency, but statistical concordance has not been obtained. Although the genetic load model explains statistically the family data, it would be hard to imagine that endemic goiter could be explained by a model where synergism among genetic and environmental factors is not assumed.

Epidemiology and genetics of endemic goiter. I. Epidemiological aspects

This study dealt with approximately 2,000 children and their parents living in a hyperendemic goiter area in Central Brazil, which is bounded by the jungle to the north and by a large plain to the south. The determination of goiter was made according to the methods and classification adopted by WHO. Conspicuous forms of goiter were found in 41% of the children examined. Multiple linear regression analysis showed an increase with age in the frequency of goiter in both sexes. Although data from the literature show that Mulattoes and Negroes have statistically higher frequencies of goiter than do Whites, our multiple linear regression analysis revealed no evidence for an effect of race on the endemism. There was no significant association with socioeconomic level. The presence of goiter in parents was shown to be statistically associated with its occurrence in the children.