Navegando por Palavras-chave "metalloproteinase"
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- ItemSomente MetadadadosThe effect of post-translational modifications on the hemorrhagic activity of snake venom metalloproteinases(Elsevier B.V., 2004-05-01) Garcia, L. T.; Silva, LTPE; Ramos, OHP; Carmona, A. K.; Bersanetti, P. A.; Selistre-de-Araujo, H. S.; Universidade Federal de São Carlos (UFSCar); Universidade Federal de São Paulo (UNIFESP)Metalloproteinases (MPs) are Zn+-dependent endoproteolytic enzymes, abundant in crotalid and viperid snake venoms. Most snake venom metalloproteinases (svMPs) are active on extracellular matrix components and this effect is thought to result in bleeding as a consequence of the basement membrane disruption in capillaries. Jararhagin and ACLH are hemorrhagic svMPs from Bothrops jararaca and Agkistrodon contortrix laticinctus venom, respectively. Both enzymes demonstrate proteolytic activity on fibrinogen and fibronectin and jararhagin inhibits collagen-induced platelet aggregation in vitro. This work describes the expression, purification and successful refolding of the recombinant ACLH zymogen (rPRO-ACLH) as well as the catalytic domain of jararhagin (rCDJARA). the heterologous proteins were produced in E. coli, an in vivo expression system that does not make post-translational modifications. the recombinant refolded proteins did not show any hemorrhagic activity in mice skin, as well as the native deglycosylated jararhagin and ACLH. However, they preserved their proteolytic activity on fibrinogen and fibronectin. It seems that the hemorrhagic properties of these hemorrhagins are dependent on post-translational modifications, whereas their proteolytic activity is not dependent on such modifications. (C) 2004 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosIsolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom(Elsevier B.V., 2006-02-01) Bello, C. A.; Hermogenes, ALN; Magalhaes, A.; Veiga, S. S.; Gremski, L. H.; Richardson, M.; Sanchez, E. F.; Ezequiel Dias Fdn; Univ Fed Parana; Universidade Federal de São Paulo (UNIFESP)In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed-jararaca). the proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography oil CM Sepharose. the enzyme called leucrolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. the amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. the protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. the proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity = 21.6 units/mg and 17.5 units/Pg, respectively; Crude venom = 8.0 units/mg and 9.5 units/mu g). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a. cleaved the Ala(14)-Leu(15) and Tyr(16)-Leu(17) bonds in oxidized insulin B chain. the pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the Purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. in vitro studies revealed that leuc-a dissolves clots made either from Purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 mu g) Subcutaneously into mice. (c) 2005 Elsevier SAS. All rights reserved.
- ItemAcesso aberto (Open Access)Matriz Metaloproteinase 2: um importante marcador genético para colesteatomas(ABORL-CCF Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial, 2007-02-01) Morales, Douglas Salmazo Rocha; Penido, Norma de Oliveira [UNIFESP]; Silva, Ismael Dale Cotrim Guerreiro da [UNIFESP]; Stávale, João Norberto [UNIFESP]; Guilherme, Arnaldo [UNIFESP]; Fukuda, Yotaka [UNIFESP]; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP); Hospital do Servidor Público Estadual de São PauloAIM: This study is to determine the MMP2 s presence in cholesteatomas and whether complicating cholesteatomas show a higher immunohistochemical expression of matrix metalloproteinase 2. Cholesteatoma produces enzymesthat cause bone erosion like Matrixmetalloproteinase 2 (MMP2). MATERIAL AND METHODS: We analyzed the expression of MMP2 in invasive (causing complications) compared to latent cholesteatomas (not causing complications). A crosssectional study with nineteen slides and paraffin blocks of cholesteatomas derived from mastoidectomies were located and processed, including 8 invasive and 11 latent cholesteatomas. Immunohistochemical thecnique was empregated to MMP2. RESULTS: The results are expressed as 0, + (to low), ++ and +++(high) according to the quantity and color of the immunohistochemical staining of MMP2. Higher expression of MMP2 was observed in 7 (87.5%) of the 8 invasive cholesteatomas. With respect to latent cholesteatomas, higher expression of MMP2 was observed in 27.3% (3 cases), with Fisher s exact test indicating a significant difference (p=0.015). CONCLUSIONS: Cholesteatoamas express MMP2 and Invasive cholesteatomas had high MMP2 compared to latent cholesteatomas.
- ItemSomente MetadadadosRelaxation of the mouse pubic symphysis during late pregnancy is not accompanied by the influx of granulocytes(Wiley-Blackwell, 2008-03-01) Rosa, Renata Giardini; Badin Tarsitano, Christiane Aparecida; Hyslop, Stephen; Yamada, Aureo Tatsumi; Toledo, Olga Maria S. [UNIFESP]; Joazeiro, Paulo Pinto; Universidade Estadual de Campinas (UNICAMP); Universidade Federal de São Paulo (UNIFESP)In some animals, such as mice and guinea pigs, a hormonally controlled mechanism increases the flexibility of the pubic symphysis and enhances the cervical remodeling necessary for safe delivery. Cervical ripening during pregnancy is associated with a paradoxical influx of leukocytes. However, the changes in cell metabolism during relaxation of the mouse pubic symphysis for delivery have not been extensively studied. in this work, we used light microscopy and transmission and scanning electron microcopy, as well as immunohistochemistry and Western blotting for MMP-8, to investigate the involvement of granulocytes or resident stromal cells in the relaxation of the virgin pubic symphysis during late pregnancy (days 18 and 19, before delivery) in vivo and in explanted joints. MMP-8 was studied because this collagenase is a hallmark for cervical ripening associated with the influx of granulocytes during late pregnancy. Extensive dissolution and disorganization of the extracellular matrix was seen around fibroblastic-like cells in late pregnancy. in contrast to the cervix (positive control), morphological and immunohistochemical analyses revealed that there was no characteristic cellular inflammatory response in the interpubic tissue. Staining for MMP-8 was observed in chondroid and fibroblastic-like cells of virgin and relaxed interpubic ligament, respectively. However, no granulocytes were seen during the extensive remodeling of the pubic joint in late pregnancy. These results indicate that constitutive stromal cells may have an important role in tissue relaxation during remodeling of the pubic symphysis in pregnancy.
- ItemSomente MetadadadosStructural and functional characterization of a P-III metalloproteinase, leucurolysin-B, from Bothrops leucurus venom(Elsevier B.V., 2007-12-15) Sanchez, Eladio F.; Gabriel, Lucilene M.; Gontijo, Silela; Gremski, Luiza H.; Veiga, Silvio S.; Evangelista, Karla S.; Eble, Johannes A.; Richardson, Michael; Ezequiel Dias Fdn; Univ Fed Parana; Univ Munster; Hosp Santa Casa; Universidade Federal de São Paulo (UNIFESP)Leucurolysin-B (leuc-B) is an hemorrhagic metalloproteinase found in the venom of Bothrops leucurus (white-tailed-jararaca) snake. By means of liquid chromatography consisting of gel filtration on Sephracryl S-200, S-300 and ion-exchange on DEAE Sepharose, leuc-B was purified to homogeneity. the proteinase has an apparent molecular mass of 55 kDa as revealed by the reduced SDS-PAGE, and represents approximately 1.2% of the total protein in B. leucurus venom. the partial amino acid sequence of leuc-B was determined by automated Edman sequencing of peptides derived from digests of the S-reduced and alkylated protein with trypsin. Leuc-B exhibits the characteristic motif of metalloproteinases, HEXXHXXGXXH and a methonine-containing turn of similar conformation (Metturn), which forms a hydrophobic basis for the zinc ions and the three histidine residues involved as ligands. Leuc-B has been characterized as a P-III metalloproteinase and possesses a multidomain structure including a metalloproteinase, a disintegrin-like (ECD sequence instead of the typical RGD motif) and a cysteine-rich C-terminal domain. Leuc-B contains three potential sites of N-glycosylation. the enzyme only cleaves the Ala(14)-Leu(15) peptide bond of the oxidized insulin B-chain and preferentially hydrolyzes the A alpha-chain of fibrinogen and the a-chain of fibrin. Its proteolytic activity was completely inhibited by metal chelating agents but not by other typical proteinase inhibitors. in addition, its enzymatic activity was stimulated by the divalent cations Ca2+ and Mg2+ but inhibited by Zn2+ and CU2+. the catalytic activity of leuc-B on extracellular matrix proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites (MHD = 30 ng in rabbit), with alterations in platelet function. in summary, here we report the isolation and the structure-function relationship of a P-III snake venom metalloproteinase. ((c)) 2007 Elsevier Inc. All rights reserved.