Antioxidant dietary deficiency induces caspase activation in chick skeletal muscle cells

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambdaexc = 320 nm and lambdaem = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BioquímicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BiofísicaUniversidade de São Paulo Faculdade de Zootecnia e Engenharia de Alimentos Departamento de CiênciasUNIFESP, EPM, Depto. de BioquímicaUNIFESP, EPM, Depto. de BiofísicaSciEL

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 µM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 µM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 µM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BioquímicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BiofísicaUNIFESP, EPM, Depto. de BioquímicaUNIFESP, EPM, Depto. de BiofísicaSciEL

The component of Carica papaya seed toxic to A-aegypti and the identification of tegupain, the enzyme that generates it

As Aedes aegypti transmits the etiologic agents of both yellow and dengue fever; vector control is considered essential to minimise their incidence. the aim of this work was to identify the component of Carica papaya seed toxic to A. aegypti, and the identification of tegupain, the enzyme that generates it. Aqueous extracts (1%, w/v) of the seed tegument and cotyledon of C. papaya are not larvicidal isolately. However, a mixture of 17 mu g mL(-1) tegument extract and 27 mu g mL(-1) cotyledon extract caused 100% larval mortality in a bioassay. the mixture was no longer larvicidal after the tegument extract was pre-treated at 100 degrees C for 10 min. the enzyme tegupain efficiently hydrolysed the substrate Z-Phe-Arg-pNan (K-m 58.8 mu M, K-cat 28020 s(-1), K-cat/K-m 5 x 10(8) M-1 s(-1)), and its activity increased with 2 mM dithiothreitol (DTT), at 37 degrees C, pH 5.0. the chelating agent EDTA did not modify the enzyme activity. Inhibition of tegupain by cystatin (K-iapp 2.43 nM), E64 (3.64 nM, 83% inhibition), and the propeptide N-terminal sequence indicate that the toxic activity is due to a novel cysteine proteinase-like enzyme, rendered active upon the hydrolysis of a cotyledon component of C. papaya seeds. (c) 2013 Elsevier B.V. All rights reserved.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)Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Estadual Norte Fluminense, Biotechnol Lab, Campos Dos Goytacazes, RJ, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilFAPESP: 2009/17058-6FAPESP: 2009/53766-5Web of Scienc

Bradykinin Release Avoids High Molecular Weight Kininogen Endocytosis

Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate H-kininogen endocytosis. in the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. in CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. the H-kininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. the anti-pain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. the present data also demonstrates that serine or cysteine proteases in lipid raft domains/caveolae on the CHO cell can hydrolyze H-kininogen, thus releasing kinins.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Universidade Federal de São Paulo-FAP/UNIFESPUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Bioquim, São Paulo, SP, BrazilUniv Anhanguera São Paulo UNIAN SP, Programa Biomat, São Paulo, SP, BrazilUniv Anhanguera São Paulo UNIAN SP, Programa Biotecnol, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Biofis, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Bioquim, São Paulo, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Escola Paulista Med, Dept Biofis, São Paulo, SP, BrazilCNPq: CNPq 472403/2007-9FAPESP: FAPESP 13/05822-9FAPESP: FAPESP 2012/50219-6Web of Scienc

Action of plant proteinase inhibitors on enzymes of physiopathological importance

Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundo de Auxilio aos Docentes e Alunos/Fundacao de Apoio a UNIFESP (FADA/FAP)Univ Fed Sao Paulo, Dept Bioquim, Escola Paulista Med, BR-04044020 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Bioquim, Escola Paulista Med, BR-04044020 Sao Paulo, BrazilWeb of Scienc

PURIFICATION AND PARTIAL CHARACTERIZATION OF A THIOL PROTEINASE-INHIBITOR FROM ENTEROLOBIUM-CONTORTISILIQUUM BEANS

ESCOLA PAULISTA MED,DEPT BIOQUIM,CAIXA POSTAL 20372,BR-04034 SAO PAULO,BRAZILUNIV FED MINAS GERAIS,CTR UNIV CORUMBA,CORUMBA,BRAZILESCOLA PAULISTA MED,DEPT BIOQUIM,CAIXA POSTAL 20372,BR-04034 SAO PAULO,BRAZILWeb of Scienc

PURIFICATION AND PRELIMINARY CHARACTERIZATION OF TORRESEA-CEARENSIS TRYPSIN-INHIBITOR

ESCOLA PAULISTA MED SCH,DEPT BIOQUIM,CAIXA POSTAL 20372,BR-04023 SAO PAULO,SP,BRAZILUNIV FED MATO GROSSO DO SUL,BR-79300 CORUMBA,BRAZILESCOLA PAULISTA MED SCH,DEPT BIOQUIM,CAIXA POSTAL 20372,BR-04023 SAO PAULO,SP,BRAZILWeb of Scienc