Leucurogin, a new recombinant disintegrin cloned from Bothrops leucurus (white-tailed-jararaca) with potent activity upon platelet aggregation and tumor growth

Disintegrins and disintegrins-like proteins are able to inhibit platelet aggregation and integrin-mediated cell adhesion. the aim of this study was to produce one disintegrin-like cloned from Bothrops leucurus venom gland and to characterize it regarding biological activity. the recombinant protein was purified by one step procedure involving anion-exchange chromatography (DEAE-cellulose) and presented a molecular mass of 10.4 kDa. the purified protein was able to inhibit platelet aggregation induced by collagen (IC(50) = 0.65 mu M) and to inhibit growth of Ehrlich tumor implanted in mice by more than 50% after 7 days administration of 10 mu g/day. No effects were observed upon adenosine 5'diphosphate (ADP)-and arachidonic acid (AA)-induced platelet aggregation. the recombinant protein was recognized by an antibody specific for jararhagin one metalloproteinase isolated from Bothrops jararaca venom, and therefore it was named leucurogin. Anti-angiogenesis effect of leucurogin was evaluated by the sponge implant model. After 7 days administration leucurogin inhibited, in a dose dependent way, the vascularization process in the sponge. Leucurogin represents a new biotechnological tool to understand biological processes where disintegrins-like are involved and may help to characterize integrins that can be involved in development and progression of malignant cells. (C) 2011 Elsevier B.V. All rights reserved.FAEPFundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed Minas Gerais, Inst Ciencias Biol, BR-31270901 Belo Horizonte, MG, BrazilUniv Mogi das Cruzes, Ctr Civ, BR-08780911 São Paulo, BrazilProteobras Desenvolvimento Biotecnol Ltda, BR-13069310 São Paulo, BrazilFundacao Ezequiel Dias, BR-30510010 Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04023062 São Paulo, BrazilWeb of Scienc

Isometric handgrip does not elicit cardiovascular overload or post-exercise hypotension in hypertensive older women

Background: Arterial hypertension is a serious health problem affecting mainly the elderly population. Recent studies have considered both aerobic and resistance exercises as a non-pharmacological aid for arterial hypertension treatment. However, the cardiovascular responses of the elderly to isometric resistance exercise (eg, isometric handgrip [IHG]) have not yet been documented. Objective: The purpose of this study was to investigate cardiovascular responses to different intensities of isometric exercise, as well as the occurrence of post-isometric exercise hypotension in hypertensive elderly people under antihypertensive medication treatment. Patients and methods: Twelve women volunteered to participate in the study after a maximal voluntary contraction test (MVC) and standardization of the intervention workload consisting of two sessions of IHG exercise performed in four sets of five contractions of a 10-second duration. Sessions were performed both at 30% of the MVC and 50% of the MVC, using a unilateral IHG protocol.\ud Both intensities were compared with a control session without exercise. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) at rest (R), during peak exercise (PE), and after 5, 10, 15, 30, 45, and 60 minutes of post-exercise recovery were evaluated. Results: No significant changes were observed after isometric exercise corresponding to 30% MVC for either SBP (R: 121 ± 10; PE: 127 ± 14; 5 min: 125 ± 13; 10 min: 123 ± 12; 15 min: 122 ± 11; 30 min: 124 ± 11; 45 min: 124 ± 10; 60 min: 121 ± 10 mmHg) or DBP (R: 74 ± 9; PE: 76 ± 6; 5 min: 74 ± 5; 10 min: 72 ± 8; 15 min: 72 ± 5; 30 min: 72 ± 8; 45 min: 73 ± 6; 60 min: 75 ± 7 mmHg). Similarly, the 50% MVC did not promote post-isometric exercise hypotension for either SBP (R: 120 ± 7; PE: 125 ± 11; 5 min: 120 ± 9; 10 min: 122 ± 9; 15 min: 121 ± 11; 30 min: 121 ± 9; 45 min: 121 ± 9; 60 min: 120 ± 7 mmHg) or DBP (R: 72 ± 8; PE: 78 ± 7; 5 min: 72 ± 7; 10 min: 72 ± 8; 15 min: 71 ± 7; 30 min: 72 ± 8; 45 min: 75 ± 10; 60 min: 75 ± 7 mmHg). Conclusion: Our data reveal that cardiovascular overload or post-exercise hypotension did not occur in elderly women with controlled hypertension when they undertook an IHG session. Thus this type of resistance exercise, with mild to moderate intensity, with short time of contraction appears to be safe for this populationFAPESP 2011/03528-0CAPE

High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidylinositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. the present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. the cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. the cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. the recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. the enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per titer of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.Universidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Nefrol, BR-04023062 São Paulo, BrazilUniv Mogi Cruzes, São Paulo, BrazilUniv Fed Minas Gerais, Dept Fisiol & Biofis, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Nefrol, BR-04023062 São Paulo, BrazilWeb of Scienc

Exercise during pregnancy protects adult mouse offspring from diet-induced obesity

Physical exercise induces positive alterations in gene expression involved in the metabolism of obesity. Maternal exercise provokes adaptations soon after birth in the offspring. Here, we investigated whether adult mouse offspring of swim-trained mothers is protected against the development of the deleterious effects of high fat diet (HFD). Our study comprises two parts. First, female C57BL/6 mice were divided into one sedentary and one swim-trained group (before and during pregnancy, n = 18). In the second part, adult offspring (n = 12) of trained and sedentary mothers was challenged to HFD for 16 weeks. Notably, most of the analysis was done in male offspring. Our results demonstrate that maternal exercise has several beneficial effects on the mouse offspring and protects them from the deleterious effects of HFD in the adult. Specifically, swimming during pregnancy leads to lower birth weight in offspring through 2 months of age. When subjected to HFD for 4 month in the adulthood, our study presents novel data on the male offspring’s metabolism of trained mothers. The offspring gained less weight, which was accompanied by less body fat, and they used more calories during daytime compared with offspring of sedentary mothers. Furthermore, we observed increased adiponectin expression in skeletal muscle, which was accompanied by decreased leptin levels and increased insulin sensitivity. Decreased interleukin-6 expression and increased peptide PYY levels were observed in sera of adult offspring of mothers that swam during pregnancy. Our results point to the conclusion that maternal exercise is beneficial to protect the offspring from developing obesity, which could be important for succeeding generations as well12FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2013/04757-

Lymphatic fluctuation in the parenchymal remodeling stage of acute interstitial pneumonia, organizing pneumonia, nonspecific interstitial pneumonia and idiopathic pulmonary fibrosis

Because the superficial lymphatics in the lungs are distributed in the subpleural, interlobular and peribroncovascular interstitium, lymphatic impairment may occur in the lungs of patients with idiopathic interstitial pneumonias (IIPs) and increase their severity. We investigated the distribution of lymphatics in different remodeling stages of IIPs by immunohistochemistry using the D2-40 antibody. Pulmonary tissue was obtained from 69 patients with acute interstitial pneumonia/diffuse alveolar damage (AIP/DAD, N = 24), cryptogenic organizing pneumonia/organizing pneumonia (COP/OP, N = 6), nonspecific interstitial pneumonia (NSIP/NSIP, N = 20), and idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP, N = 19). D2-40+ lymphatic in the lesions was quantitatively determined and associated with remodeling stage score. We observed an increase in the D2-40+ percent from DAD (6.66 +/- 1.11) to UIP (23.45 +/- 5.24, P = 0.008) with the advanced process of remodeling stage of the lesions. Kaplan-Meier survival curves showed a better survival for patients with higher lymphatic D2-40+ expression than 9.3%. Lymphatic impairment occurs in the lungs of IIPs and its severity increases according to remodeling stage. The results suggest that disruption of the superficial lymphatics may impair alveolar clearance, delay organ repair and cause severe disease progress mainly in patients with AIP/DAD. Therefore, lymphatic distribution may serve as a surrogate marker for the identification of patients at greatest risk for death due to IIPs.CNPqCNPqFAPESP [07/52785-0, 08/53022-3, 08/57130-5]FAPESPLaboratorio Diagnostika, Hospital das Clinicas and Faculdade de Medicina, Universidade de Sao PauloLaboratorio Diagnostika, Hospital das Clinicas and Faculdade de Medicina, Universidade de Sao Paul

Balance between the two kinin receptors in the progression of experimental focal and segmental glomerulosclerosis in mice

Focal and segmental glomerulosclerosis (FSGS) is one of the most important renal diseases related to end-stage renal failure. Bradykinin has been implicated in the pathogenesis of renal inflammation, whereas the role of its receptor 2 (B2RBK; also known as BDKRB2) in FSGS has not been studied. FSGS was induced in wild-type and B2RBK-knockout mice by a single intravenous injection of Adriamycin (ADM). in order to further modulate the kinin receptors, the animals were also treated with the B2RBK antagonist HOE-140 and the B1RBK antagonist DALBK. Here, we show that the blockage of B2RBK with HOE-140 protects mice from the development of FSGS, including podocyte foot process effacement and the re-establishment of slit-diaphragm-related proteins. However, B2RBK-knockout mice were not protected from FSGS. These opposite results were due to B1RBK expression. B1RBK was upregulated after the injection of ADM and this upregulation was exacerbated in B2RBK-knockout animals. Furthermore, treatment with HOE-140 downregulated the B1RBK receptor. the blockage of B1RBK in B2RBK-knockout animals promoted FSGS regression, with a less-inflammatory phenotype. These results indicate a deleterious role of both kinin receptors in an FSGS model and suggest a possible cross-talk between them in the progression of disease.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Clin & Expt Immunol Lab, Div Nephrol, BR-04023900 São Paulo, BrazilUniv São Paulo, Inst Biomed Sci 4, Dept Immunol, Lab Transplantat Immunobiol, BR-05508000 São Paulo, BrazilUniversidade Federal de São Paulo, Translat Med Div, Clin & Expt Immunol Lab, BR-04039002 São Paulo, BrazilInst Butantan, Lab Cellular Biol, BR-05503900 São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Biophys, BR-04023062 São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilINSERM, Unite Mixte Rech 699, F-75870 Paris, FranceAlbert Einstein Hosp, Inst Israelita Ensino & Pesquisa Albert Einst, Renal Transplantat Unit, BR-05521000 São Paulo, BrazilUniversidade Federal de São Paulo, Clin & Expt Immunol Lab, Div Nephrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Translat Med Div, Clin & Expt Immunol Lab, BR-04039002 São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Biophys, BR-04023062 São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilFAPESP: 2012/05605-5FAPESP: 07/07139-3FAPESP: 12/02270-2CNPq: 140739/2008-4Web of Scienc

Kinin B(1) receptor deficiency leads to leptin hypersensitivity and resistance to obesity

OBJECTIVE-Kinins mediate pathophysiological processes related to hypertension, pain, and inflammation through the activation of two G-protein-coupled receptors, named B(1) and B(2). Although these peptides have been related to glucose homeostasis, their effects on energy balance are still unknown.RESEARCH DESIGN and METHODS-Using genetic and pharmacological strategies to abrogate the kinin B(1) receptor in different animal models of obesity, here we present evidence of a novel role for kinins in the regulation of satiety and adiposity.RESULTS-Kinin B(1) receptor deficiency in mice (B(1)(-/-)) resulted in less fat content, hypoleptinemia, increased leptin sensitivity, and robust protection against high-fat diet-induced weight gain. Under high-fat diet, B(1)(-/-) also exhibited reduced food intake, improved lipid oxidation, and increased energy expenditure. Surprisingly, B(1) receptor deficiency was not able to decrease food intake and adiposity in obese mice lacking leptin (ob/ob-B(1)(-/-)). However, ob/ob-B(1)(-/-) mice were more responsive to the effects of exogenous leptin on body weight and food intake, suggesting that B(1) receptors may be dependent on leptin to display their metabolic roles. Finally, inhibition of weight gain and food intake by B(1) receptor ablation was pharmacologically confirmed by long-term administration of the kinin B(1) receptor antagonist SSR240612 to mice under high-fat diet.CONCLUSIONS-Our data suggest that kinin B(1) receptors participate in the regulation of the energy balance via a mechanism that could involve the modulation of leptin sensitivity.Universidade Federal de São Paulo, Dept Biophys, BR-04023062 São Paulo, BrazilUniv Mogi das Cruzes, Mogi Das Cruzes, BrazilUniversidade Federal de São Paulo, Dept Physiol, BR-04023062 São Paulo, BrazilSanofi Aventis, Montpellier, FranceUniversidade Federal de São Paulo, Dept Med, BR-04023062 São Paulo, BrazilInst Natl Sante & Rech Med, Dept Renal & Cardiac Remodeling, U858 I2MR, Toulouse, FranceUniv Toulouse 3, Inst Med Mol Rangueil, F-31062 Toulouse, FranceInst Natl Rech Agron AgroParisTech, UMR914 Nutr Physiol & Ingest Behav, Paris, FranceMax Delbruck Ctr Mol Med, Berlin, GermanyUniversidade Federal de São Paulo, Dept Biophys, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Physiol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, BR-04023062 São Paulo, BrazilWeb of Scienc

Carbamazepine inhibits angiotensin I-converting enzyme, linking it to the pathogenesis of temporal lobe epilepsy

We find that a common mutation that increases angiotensin I-converting enzyme activity occurs with higher frequency in male patients suffering from refractory temporal lobe epilepsy. However, in their brains, the activity of the enzyme is downregulated. As an explanation, we surprisingly find that carbamazepine, commonly used to treat epilepsy, is an inhibitor of the enzyme, thus providing a direct link between epilepsy and the renin-angiotensin and kallikrein-kinin systems. Translational Psychiatry (2012) 2, e93; doi:10.1038/tp.2012.21; published online 13 March 2012INNTConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Dept Biophys, BR-04023032 São Paulo, BrazilUniversidade Federal de São Paulo, BR-04023032 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pathol, BR-04023032 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Neurol & Neurosurg, BR-04023032 São Paulo, BrazilUniv São Paulo, Sch Arts Sci & Humanities, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Sci & Technol, BR-04023032 São Paulo, BrazilNove de Julho Univ UNINOVE, Dept Rehabil Sci, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04023032 São Paulo, BrazilUniversidade Federal de São Paulo, BR-04023032 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pathol, BR-04023032 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Neurol & Neurosurg, BR-04023032 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Sci & Technol, BR-04023032 São Paulo, BrazilWeb of Scienc