RCAAP Repository

Bothrops snake venoms contain proteases that contribute to the local effects seen after envenoming. In this study, a non-hemorrhagic metalloprotease was isolated from the venom of B. moojeni. The enzyme was isolated by a combination of ion exchange and gel filtration chromatographies and named Moozincin. The enzyme was purified to homogeneity as judged by its migration profile in SDS PAGE stained with coomassie blue, and showed a molecular mass of about 30 kDa. Moozincin did not induce hemorrhage, blood clotting, defibrinating or phospholipase A2 activities, but displayed proteolytic activity on bovine fibrinogen. Moozincin cleaves the Aα-chain of fibrinogen first, followed by the Bβ-chain, and shows no effects on the γ-chain. The fibrinogenolytic activity of Moozincin was abolished after incubation with a chelating agent (EDTA, 1,10-phenantroline) β-mercaptoethanol, indicating that it is metal iondependent. In contrast, aprotinin and benzamidine did not affect these activities. Moozincin was active at pH 7 - 11 and was stable in solution at up to 50 °C; fibrinogenolytic activity was completely lost at ≥60 °C. Histological observations showed that Moozincin induces low myonecrosis upon intramuscular injection in mice evidenced by hyaline degeneration and leukocyte infiltrate. Moozincin induced cell alterations in lung, but not alter liver, kidney and heart cells.

The metabolomics is an area of science "omics" that characterizes the phenotypes of the metabolites and the connection of these with their corresponding genotypes, allowing the identification of the metabolome of an individual. This metabolomics research allows to view the rapid responses of the biochemical and biological analysis of metabolism in any situation, whether environmental change, pathological, genetic or in response to exercise stress. This study aims main to present the mass spectrometry together with the clinical laboratory as a research tool for obtaining the metabolome of an athlete's Olympic Windsurfing Class RS:X. During exercise, the energy demand is increased by requiring changes in metabolism, and thus the metabolism provides answers to these various metabolic modifications. Therefore, analytical techniques, such as laboratory tests and mass spectrometry may be used for the metabolic evaluation, allowing to investigate and intervene in a scientific manner, the exercise prescription, control and monitoring of athlete training, helping with interventions to adaptation of physical exercise in improving their athletic performance.

IQGAP1 stimulates branched actin filament nucleation by activating N-WASP, which in turn activates the Arp2/3 complex. N-WASP can be activated by other factors, including GTP-bound forms of Cdc42 and Rac1, which also bind IQGAP1. We report here the use of purified proteins for in vitro binding and actin polymerization assays, and of fluorescence resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions involving IQGAP1, N-WASP, and either Cdc42 or Rac1. In pyrene-actin assembly assays in the presence of N-WASP and the Arp2/3 complex, Cdc42 and IQGAP1 cooperatively stimulated actin filament nucleation, primarily by reducing the lag time before assembly Vmax was reached. Cooperativity reflected dose-dependent stimulation by Cdc42 of IQGAP1 binding to N-WASP. Rac1 and IQGAP1 behaved differently. At low Rac1, the two proteins cooperatively reduced the lag time before assembly Vmax was reached, but at high Rac1 Vmax was faster and reached more quickly for Rac1 alone than for either IQGAP1 alone, or the combination of Rac1 and IQGAP1. This negative cooperativity reflected dose-dependent inhibition by Rac1 of IQGAP1 binding to N-WASP. These results suggest that IQGAP1 interacts by distinct mechanisms with Cdc42 versus Rac1 to regulate actin filament assembly through N-WASP in vivo. To address this possibility, FRET microscopy was used to study interactions of GFP-IQGAP1 with mOrange-Cdc42 versus mOrange-Rac1 in live MDCK cells. Robust FRET was observed for both donor/acceptor pairs at F-actin rich cell-cell margins, but the average intermolecular FRET distances closer for IQGAP1-Cdc42 than for IQGAP1-Rac1. The distinct interactions of IQGAP1 with Cdc42 versus Rac1 observed in vitro were thus recapitulated in live cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Universidade Federal de Uberlândia

Cardiovascular complications of diabetes mellitus are associated with oxidative stress that occurs as a result of the disarrangement between the production of reactive oxygen species (ROS) and their neutralization by antioxidant systems under hyperglycemia. The exacerbated production of ROS may contribute to increased cardiac collagen content, collagen glycation in consequence of the formation of advanced glycation end products (AGEs). These mechanisms are proposed to explain the increase in cross-linking and increased stiffness of the myocardium in patients with diabetes. Phaseolamin inhibits pancreatic alpha-amylase, which leads to a reduction on hyperglycemia. The aim of this study was to evaluate the protective effects of Phaseolamin in the cardiac tissue against damage of streptozotocin (STZ)-induced diabetic rats under oxidative stress levels and collagen type I deposition in vivo. Animals were distributed in six groups: non-diabetic (ND); non-treated diabetic (NTD); groups treated with commercial Phaseolamin at 100, 500 and 1500 mg/kg (D100, D500 and D1500 respectively); group treated with acarbose at 25 mg/kg (DACA). Treatments were given once daily by gavage during 20 days.The total antioxidant activity measured in NTD group was lower (p<0.001) when compared with ND animals and greater in diabetic patients who received treatment with acarbose and Phaseolamin (p<0.001).The enzymatic defense system represented by catalase (CAT) and superoxide dismutase (SOD) increased its activity in NTD group (p<0.001). The activity of SOD and CAT decreased after treatment with Phaseolamin (D100, D500, D1500 and DACA). The tissue damage caused by lipid peroxidation was reduced in D1500 (p<0.05), although it was increased to other groups (p<0.001). Phaseolamin treatment reduced the hyperglycemic state (16-42%) and decreases the formation of ROS, preventing the exacerbation of the antioxidant system in this tissue. Our study showed significantly increased deposition of collagen in all diabetic groups compared to ND rats. Groups D1500 and DACA showed reduced values of type I collagen compared to NTD group (p<0.001). In our experimental model, treatment with Phaseolamin prevents the development of ROS and myocardial collagen changes in diabetic rats. We suggest that Phaseolamin can be a complementary treatment for diabetes reducing the appearance of heart complications.

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Neurotransmitter secretion and subsequent release, shares many features with constitutive membrane trafficking. Intracellular membrane fusion in eukaryotic cells involves a several family&#146;s proteins, including SNARES, Rabs, Sec1/Munc-18 (SMproteins), and molecular motors in an evolutionary conserved machinery. Honeybee brain has been successfully used as a neurobiology model to investigate memory, learning and behavior. In the current work, through the techniques Western blot and immunohistochemistry, SNAREs proteins, myosin-V, and, CaMKII are identified in protein fractions and tissue of honeybee worker&#146;s brain (Apis mellifera). By immunoblotting, using specific antibodies: syntaxin and CaMKII was probed in the casts drone and queen; for the brain fractions H and S1 of worker bee, rabbit and rat, the CaMKII antibody was tested; the brain fractions H, S1, P1, S2, P2, and enriched membrane fraction P2TX, of worker bee, chicken, rabbit and rat was probed with antibody against myosin-Vfor comparison betweenyours respective immunoreactivity. In addition, honeybee worker&#146;s brain regions fractions were tested with antibodies Clathrin and CaMKII. Indeed, the immunoreactivity for the antibodies raised against vertebrates, showed conserved regions in hymenoptera. This orthology suggests that many of the proteins important for transmitter release have homologs involved in intracellular vesicle transport, and all forms of vesicle trafficking share common basic principles with others organisms.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

ABSTRACT - CHAPTER I With the aim of guarantee the stability of the biological organization complexes, nature uses several mechanisms, as the control of pH, temperature and concentration of solutes in the internal medium. The control of the solute concentration contributes to what we can call as osmostabilization. This paper tries to apply the known theories about the stabilization of proteins now on the biological membranes. The osmostabilizing solutes, also called as osmolytes, increase the free energy of the native state (N), but increase much more the free energy of the unfolded state (D) of a protein, in such a way that the osmolytes stabilize the N state of a protein by the increase in free energy barrier between the states, which makes less probable the unfolding and the loss of the protein function. The reason of this difference would be in the preferential interaction of the protein with the water. The protein prefers binds to water than to the osmolyte, which is then excluded from the inner hydration shell of the protein surface, according an effect that was designated as solvophobic or osmophobic effect. The larger contact surface of the D state requires a higher investment of free energy for its hydration, beyond a better organization of the water molecules around the external surface, in such a way that the conformational entropy of the solvent is lower around the D state than it is around the N state. This means that the osmolytes stabilize the native state in relation to the unfolded state of the protein. The biological membranes have an important aspect of their structures in common with water soluble proteins. They also hide their hydrophobic groups in their anhydrous interior, constituted by a phospholipids bilayer, in which the polar heads of these lipids make contact with the aqueous surrounding in the external and internal media of the biological structure encompassed by the membrane. The erythrocytes constitute a very valid model to study the behavior of the membranes. The utilization of osmolytes increases the stability of erythrocytes preparations and permits even their cryopreservation. In the presence of osmolytes the erythrocytes suffer reversible morphological alterations with volume decrease. Thus, the erythrocytes will exist in an equilibrium process between two states, an expanded (R) and a compact one (T), where T is the more stable state. In this study, we tentatively explained this larger stability of the T state of the erythrocytes with basis in the preferential hydration theory of Timasheff. ABSTRACT - CHAPTER II The erythrocyte constitutes a very adequate model to study the stability of biological membranes, since the rupture of its membrane promotes release of hemoglobin, which capacity to adsorb light in the visible region of the spectra permits the monitoration of the membrane denaturation. In this work, we studied the effect of the presence of sorbitol on the thermal dependence of the stability of human erythrocytes against the denaturant action of ethanol in 0,9% NaCl. The membrane denaturation was monitored by the measurement of the absorbance at 540 nm (A540 nm). The dependence of A540 nm with the ethanol concentration in 0.9% NaCl, in the absence and presence of 1 mol.L-1 sorbitol, was studied at 27, 32, 37 and 42 °C. After complete denaturation of the erythrocytes, the A540 nm values were converted in percentage of hemolysis. All the dependencies of the % of hemolysis with the concentration of ethanol followed sigmoidal transition lines, which were adjusted to the Boltzman equation, in order to determine the concentration of ethanol able to promote 50% of hemolysis (D50). The incorporation of 1 mol.L-1 sorbitol promoted statistically significant decreases (P<0.01) in the D50 values, for all considered temperatures. The increase in the temperature also promoted statistically significant decreases (P<0.01) in the D50 values in the absence and in the presence of sorbitol. The values of D50 presented a linear dependence with the temperature. The slope of that line in the presence of sorbitol was significantly smaller (P<0.01) than it was in absence of that solute. This means that the presence of 1 mol.L-1 sorbitol increases the chaotropic action of ethanol, at the same time that it presents a stabilizing action, which increase with an increase in the temperature. If we assume linearity beyond the interval of 27 and 42 °C, those regression lines will intercept around 68.8 °C, where the stabilizing effect of sorbitol would neutralize its synergism with the chaotropic action of ethanol. These effects were explained with basis in a two state equilibrium model for the erythrocyte, a less stable expanded state and a more stable contracted state. The rationality of the model is discussed. Whatever is the adopted explanation, our results permit conclude that 1 mol.L-1 sorbitol, in the presence of 0.9% NaCl, increases the chaotropic action of ethanol and temperature on the erythrocyte membrane, although it does not present any chaotropic action itself between 0 and 1.5 mol.L-1, by the same time that it also presents a stabilizing action on the membrane that increases with the increase in the temperature.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Dengue fever is caused by an arbovirus of the Flaviridae family, transmitted from person to another through an intermediate fly vector, Aedes aegypti. It is a tropical and subtropical infectious disease characterized by fever and strong pain in joints, which could also lead to bleeding in its hemorrhagic form. In this investigation, Phage Display technology was used to identify recombinant peptides with affinity to polyclonal antibodies (IgY) raised in immunized chickens with total proteins of the DENV-3 culture. Animal sera was purified in a HiTrap column and concentrated through dialysis. The IgY s were immunoreactive against DENV cultures, but were not type specific. Phage clones were selected from a random peptide library (PhD-7) in four cycles of biopanning against IgY. Selected phages were amplified in deepwell microtiter plates and submitted to ELISA tests. Immunoreactive clones against IgY were sequenced, translated and analysed through bioinformatics. Fourteen distinct clones were selected and aligned against viral proteome sequences and among themselves. Three consensus sequences among phage clones were detected: VLRN, APP and LPP. The peptide search in BLASTp showed similarity to the following viral proteins: polyprotein, envelop, and the nonstructural proteins NS1, NS2a, NS3 and NS5. All of them have matched with DENV-1, -2, -3, and/or -4 sequences, corroborating with the lack of type specificity of the raised IgY. Considering the VLRN motif, the analyses of antigenicity indexes of similar peptides demonstrated that its antigenicity is highly influenced by neighboring residues. Three-dimensional analysis of the DENV-2 capsid protein, with the alignment of the VLRN motif, have identified two target sequences, NRVSTVQQL and EIGRMLNILNRR, that are present in the polyprotein of all four viral types, which may contain the two possible domains VxRN and LRN, respectively. Six selected phages have presented known protein domains, and five of them presented specific phosphorylation and glycosylation sites, similar to known eukaryotic viruses; however, they may not be physiologically active sites in the dengue virus. Finally, the peptides were used to detect human IgG and IgM. ELISA tests were performed in two patients with isolated infections of DENV-1 or DENV-3. The reactivity of the 14 clones was superior to total antigens obtained from cultures, but they were not type specific.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Several molecular markers have been studied in order to differentiate prostate cancer (PCa) and benign prostatic hyperplasia (BPH). They tried to help the traditional methods in the patient evaluation (PSA and digital rectal exam). This study analyzed the LSP1 gene expression by RT-PCR in BPH and prostate cancer patients. We tried to identify the specific pattern of each pathology related to the gene expression. Fifty patients more than forty years old were selected based on PSA levels > 4ng/ml or altered digital rectal exam. Twenty five healthy patients with less than thirty years old were selected as a control group. Blood sample analyses were performed as prostatic core biopsies. The final pathology was the gold standard for diagnosis defining three study groups: PCa, 24 patients; BPH, 26 patients and control, 25 patients. The study analysis was performed based on intervals of LSP1 RT-PCR expression, defined as &#8805; 1, from 0.5 to 1 and < 0.5. A second study phase analyzed LSP1 expression in prostatic tissues from specimens of twenty three patients. The results in blood sample showed a clear difference in intervals of LSP1 expression for the study groups. The BPH patients presented the higher expression, prostate cancer patients with median expression and control group with the lower one. The relative risk of BPH in LSP1 expressions &#8805; 1 is eleven times greater than prostate cancer. Although, it is not a specific marker for prostate cancer or benign hyperplasia. The LSP1 analysis represents a promising complementary purpose to PSA.

ABSTRACT - CHAPTER I - Pachymerus nucleorum (Fabricius, 1792) is a beetle specie of the Bruchinae sub-family, family Chysomelidae, that principal host of this insect is a palm babassu (Orbinya spp). The objective of this work was identify and to characterize the ATPase activity in P. Nucleorum larvae. The P. nucleorum larvae guts were discartated and the greasy body was homogenized in 50 mM phosphate buffer, pH 7,5 containing protease inhibitor (0,2 ug/mL Aprotinine, 1 mM Benzamidine, 250 mM sucrose and 1 mM EDTA), after that the samples were centrifugated at 15000xg for 30 minutes, 4 º C. Amonium sulfate was added to the supernatant fraction (S1) and centrifugated at 45000xg for 30 minutes, 4 º C. The precipitated fraction (P2) was ressuspended in Imidazol buffer, pH 8,0 containing 1 mM EDTA and 0,2 mM &#946;-mercaptoetanol and centrifugated at 45000xg for 30 minutes, 4 º C. Final concentrations of 10 mM ATP and 5 mM MgCl2 was added to the P3 fraction and centrifugated. The fraction P4 presented Mg-ATPase e Co- ATPase activity and main polipeptides with 180, 54, 43 and 17 kDa. The Mg- ATPase activity was not estimulated by calcium or calcium/calmoduline, and KEDTA ATPase activity was absent. The Mg-ATPase was not estimulated by other cátions and was slightly inhibited by vanadate. The Mg-GTPase activity was about 50% of Mg-ATPase activity. ABSTRACT - CHAPTER II - Pachymerus nucleorum (Fabricius, 1792) is a beetle specie of the Bruchinae sub-family, family Chysomelidae and principal host is coconut of the palm babassu (Orbinya spp), typical of the varzea zones and river valleys. The objective in this work was identify and to characterize the amylolytic activity in P. nucleorum larvae guts. The guts were immediately homogenized in phosphatate buffer 50 mM pH 7,5 containing protease inhibitor (Aprotinine 0,2 ug/mL and Benzamidine 1 mM), sucrose 250 mM and EDTA 1 mM, after that the samples were centrifugated at 15000xg for 30 minutes, 4 º C. The supernatant fraction (S1) presented higher amylolytic activity in relation to the precipitated fraction (P1). Analyses in SDS-PAGE revealed polipeptides of 62 kDa, in fraction P1 and in fraction S1 polipetides of 12 and 40 kDa. The fraction S1 presented maximum amylolytic activity in 50°C, and estimulation of the activity in 20% and 45% in presence of calcium 0,5 mM or sodium chloride 14 mM, respectively. The preparation containing sucrose and EDTA presented higher amylolytic activity in relation to the preparation without sucrose and EDTA. An increase in the amylolytic activity was observed in both preparations when stored in refrigerator, however the preparation without sucrose and EDTA presented higher increase in this activity.