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Fonseca K.C.,Federal University of Uberlandia | Morais N.C.G.,Federal University of Uberlandia | Queiroz M.R.,Federal University of Uberlandia | Silva M.C.,Federal University of Uberlandia | And 10 more authors.
Phytochemistry | Year: 2010

A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatographic steps using DEAE-Sephacel and gel-filtration with Sephadex G-75. Eumiliin is a monomeric protein with an apparent molecular mass of 30 kDa by SDS-PAGE under reducing conditions and gave one main peak at 29,814 KDa in MALDI-TOF/TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the Aα-chain of fibrinogen and, more slowly, the Bβ-chain. Its fibrinogenolytic activity is inhibited by β-mercaptoethanol and leupeptin. In contrast, EDTA and benzamidine did not affect the activity of Eumiliin. The caseinolytic activity of Eumiliin had a pH optimum of 8.0 and was stable in solution at up to 40 °C; activity was completely lost at ≥80 °C. Intraplantar injection of Eumiliin (1-25 μg/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1-5 h after enzyme injection. Intraplantar injection of Eumiliin (1-25 μg/paw) also caused an oedematogenic response that was maximal after 1 h. Morphological analyses indicated that Eumiliin induced an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 h after injection. © 2010 Elsevier Ltd. All rights reserved. Source


Nunes D.C.O.,Federal University of Uberlandia | Rodrigues R.S.,University of Sao Paulo | Lucena M.N.,University of Sao Paulo | Cologna C.T.,University of Sao Paulo | And 8 more authors.
Comparative Biochemistry and Physiology - C Toxicology and Pharmacology | Year: 2011

In the present study, an acidic PLA 2, designated Bl-PLA 2, was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA 2 was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000 Da and pI was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA 2s from snake venoms. Its specific activity was 159.9 U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA 2 induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-α, IL-1β and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA 2 induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism. © 2011 Elsevier Inc. Source


Naves de Souza D.L.,Federal University of Uberlandia | Naves de Souza D.L.,Instituto Nacional Of Ciencia E Tecnologia Em Nano Biofarmaceutica N Biofar | Gomes M.S.R.,Federal University of Uberlandia | Gomes M.S.R.,State University of Southwest Bahia | And 10 more authors.
Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology | Year: 2012

Snake Venom Metalloproteinases (SVMPs) are the most abundant components present in Viperidae venom. They are important in the induction of systemic alterations and local tissue damage after envenomation. In the present study, a metalloproteinase named BpMPI was isolated from Bothropoides pauloensis snake venom and its biochemical and enzymatic characteristics were determined. BpMPI was purified in two chromatography steps on ion exchange CM-Sepharose Fast flow and Sephacryl S-300. This protease was homogeneous on SDS-PAGE and showed a single chain polypeptide of 20. kDa under non reducing conditions. The partial amino acid sequence of the enzyme showed high similarity with other SVMPs enzymes from snake venoms. BpMPI showed proteolytic activity upon azocasein and bovine fibrinogen and was inhibited by EDTA, 1,10 phenanthroline and β-mercaptoethanol. Moreover, this enzyme showed stability at neutral and alkaline pH and it was inactivated at high temperatures. BpMPI was able to hydrolyze glandular and tissue kallikrein substrates, but was unable to act upon factor Xa and plasmin substrates. The enzyme did not induce local hemorrhage in the dorsal region of mice even at high doses. Taken together, our data showed that BpMP-I is in fact a fibrinogenolytic metalloproteinase and a non hemorrhagic enzyme. © 2011 Elsevier Inc. Source


Menaldo D.L.,University of Sao Paulo | Bernardes C.P.,University of Sao Paulo | Pereira J.C.,University of Sao Paulo | Silveira D.S.C.,University of Sao Paulo | And 9 more authors.
International Immunopharmacology | Year: 2013

The present study aimed to evaluate the effects of two serine proteases from Bothrops pirajai snake venom, named BpirSP27 and BpirSP41, on the complement system and the inflammatory response. The effects of these enzymes on the human complement system were assessed by kinetic hemolytic assays, evaluating the hemolysis promoted by the classical/lectin (CP/LP) and alternative (AP) pathways after incubation of normal human serum with the serine proteases. The results suggested that these enzymes were able to induce modulation of CP/LP and AP at different levels: BpirSP41 showed higher inhibitory effects on the hemolytic activity of CP/LP than BpirSP27, with inhibition values close to 40% and 20%, respectively, for the highest concentration assayed. Regarding AP, both enzymes showed percentages of inhibition of the hemolytic activity around 20% for the highest concentrations tested, indicating similar effects on this complement pathway. The proinflammatory effects of B. pirajai serine proteases were evaluated regarding their ability to induce paw edema, variations in the pain threshold and leukocyte recruitment at the site of injection. Both showed mild effects on these inflammatory processes, leading to low levels of increase of paw volumes and decrease in pain thresholds in rats up to 6 h after injection, and inducing neutrophil recruitment without significant increases in the total number of leukocytes in the inflammatory exudates after 6 and 24 h of administration into mice peritoneal cavity. These results suggest that serine proteases must present a minor role in the inflammation caused by B. pirajai snake venom. © 2013 Elsevier B.V. All rights reserved. Source


Bernardes C.P.,University of Sao Paulo | Menaldo D.L.,University of Sao Paulo | Mamede C.C.N.,Federal University of Uberlandia | Mamede C.C.N.,Instituto Nacional Of Ciencia E Tecnologia Em Nano Biofarmaceutica N Biofar | And 8 more authors.
Molecular Immunology | Year: 2015

In this study, we evaluated the edema and hyperalgesic response induced by BpirMP, a P-I class metalloproteinase isolated from Bothrops pirajai snake venom. The animals were injected with the metalloproteinase or sterile PBS (control group) and evaluated for 1, 2, 3, 4, 5, 6 and 24. h. The intraplantar injection of BpirMP (5-50. μg/paw) induced a dose- and time-dependent response. BpirMP (50. μg) induced paw edema in rats rapidly, with peak response two hours after injection of the toxin. Also, BpirMP injection caused a significant reduction in the nociceptive threshold of the animals tested, with peak response three hours after injection of the toxin. The inflammatory mediators involved in these responses were assayed by pretreatment of animals with synthesis inhibitors or receptor antagonists. Peak responses were significantly reduced by pretreatment of animals with pyrilamine, a histamine receptor antagonist, sodium cromoglycate, a mast cell degranulation inhibitor and valeryl salicylate and meloxicam, cyclooxygenase inhibitors. The analysis of the peritoneal cavity exudate revealed an acute inflammatory response with recruitment of leukocytes, increased levels of total proteins, nitric oxide and the cytokines IL-6, TNF-α and IL-10. In conclusion, our results demonstrated that BpirMP induces inflammation mediated by mast cell degranulation, histamine, prostaglandins and cytokine production. © 2015 Elsevier Ltd. Source

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