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This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and "de novo amino acid sequencing", exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitro-3-(octanoyloxy)-benzoic acid, determined its V(max) (7.56nmoles/min) and K(M) (2.76mM). Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca(2+). Intrinsic fluorescence measurements suggested that Ca(2+) induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg(2+), Mn(2+), Sn(2+) and Cd(2+) apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 degrees C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75mM and 0.4muM, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2.

Human envenoming by Lachesis muta muta venom, although infrequent, is rather severe, being characterized by pronounced local tissue damage and systemic dysfunctions. Studies on the pharmacological actions of L. m. muta venom are relatively scant and the direct actions of the crude venom and its purified phospholipase A(2) (PLA(2)) have not been addressed using in vitro models. In this work, we investigated the cytotoxicity of L. m. muta venom and its purified PLA(2) isoform LmTX-I in cultured Madin-Darby canine kidney (MDCK) and in a skeletal muscle (C2C12) cell lines. As revealed by neutral red dye uptake assay, the crude venom (10 or 100 microg/ml) induced a significant decrease in cell viability of MDCK cells. LmTX-I at the concentrations tested (70-270 microg/ml or 5-20 microM) displayed no cytotoxicity in both MDCK and C2C12 cell lines. Morphometric analysis of Feulgen nuclear reaction revealed a significant increase in chromatin condensation (pyknosis), apparent reduction in the number of mitotic nuclei and nuclear fragmentation of some MDCK cells after incubation with L. m. muta venom. Monolayer exposure to crude venom resulted in morphological changes as assessed by scanning electron microscopy. The staining with TRITC-labelled phalloidin showed a marked disarray of the actin stress fiber following L. m. muta venom exposure. In contrast, LmTX-I had no effect on nucleus and cell morphologies as well as on stress fiber organization. These results indicate that L. m. muta venom exerts toxic effects on cultured MDCK cells. The LmTX-I probably does not contribute per se to the direct venom cytotoxicity, these effects are mediated by metalloproteinases/disintegrins and other components of the venom.

A novel l-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 microM and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 microg/ml.

Tendons are parallel arrays of collagenous fibers which are specialized in resisting and transmitting tensile forces. In this work we examined the structure of the superficial digital flexor tendon (SDFT) and the deep digital flexor tendon (DDFT) of pigs, which are considered "wrap around" tendons and so receive compression and tension forces. In both tendons, fibrocartilaginous areas were observed in the regions subjected to compression plus frictional loading. Histological and ultrastructural analyses of the tensional region showed an extracellular matrix (ECM) rich in collagen bundles, that were all arranged in the same direction. Fibroblasts were seen closely associated with the collagen bundles. Chondrocyte-like cells and high levels of glycosaminoglycans (GAGs) were observed in the compressional regions. The collagen bundles in the compressional region were arranged in several directions and were associated with proteoglycans (PGs). The crimp pattern detected in the tensional region showed that the collagen fibrils were ordered aggregates which formed helical superstructures.

Bothrops jararacussu myotoxin I (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) were purified by ion-exchange chromatography and reverse phase HPLC. In this work we used the isolated perfused rat kidney method to evaluate the renal effects of B. jararacussu myotoxins I (Lys49 PLA2) and II (Asp49 PLA2) and their possible blockage by indomethacin. BthTX-I (5 microg/ml) and BthTX-II (5 microg/ml) increased perfusion pressure (PP; ct120=110.28+/-3.70 mmHg; BthTX I=171.28+/-6.30*mmHg; BthTX II=175.50+/-7.20*mmHg), renal vascular resistance (RVR; ct120=5.49+/-0.54 mmHg/ml.g(-1)min(-1); BthTX I=8.62+/-0.37*mmHg/ml g(-1)min(-1); BthTX II=8.9+/-0.36*mmHg/ml g(-1)min(-1)), urinary flow (UF; ct(120)=0.14+/-0.01ml g(-1)min(-1); BthTX I=0.32+/-0.05*ml g(-1)min(-1); BthTX II=0.37+/-0.01*ml g(-1)min(-1)) and glomerular filtration rate (GFR; ct120=0.72+/-0.10 ml g(-1)min(-1); BthTX I=0.85+/-0.13*ml g(-1)min(-1); BthTX II=1.22+/-0.28*ml g(-1)min(-1)). In contrast decreased the percent of sodium tubular transport (%TNa(+); ct(120)=79,76+/-0.56; BthTX I=62.23+/-4.12*; BthTX II=70.96+/-2.93*) and percent of potassium tubular transport (%TK(+);ct120=66.80+/-3.69; BthTX I=55.76+/-5.57*; BthTX II=50.86+/-6.16*). Indomethacin antagonized the vascular, glomerular and tubular effects promoted by BthTX I and it's partially blocked the effects of BthTX II. In this work also evaluated the antibacterial effects of BthTx-I and BthTx-II against Xanthomonas axonopodis. pv. passiflorae (Gram-negative bacteria) and we observed that both PLA2 showed antibacterial activity. Also we observed that proteins Also we observed that proteins chemically modified with 4-bromophenacyl bromide (rho-BPB) decrease significantly the antibacterial effect of both PLA2. In conclusion, BthTx I and BthTX II caused renal alteration and presented activity antimicrobial. The indomethacin was able to antagonize totally the renal effects induced by BthTx I and partially the effects promoted by BthTx II, suggesting involvement of inflammatory mediators in the renal effects caused by myotoxins. In the other hand, other effects could be independently of the enzymatic activity of the BthTX II and the C-terminal domain could be involved in both effects promoted for PLA2.

Reference is usually made to the parallel orientation towards the main line of exerted tension at the pubic joint in mice, for supporting forces applied to the joint. Despite the wealth of morphological information about the extracellular matrix in this joint, little is known regarding the involvement of the crimp of collagen fibers in the dramatic transformations occurring in this region during the last 3 days of pregnancy. Examination of the collagenous architecture suggests that the biomechanical properties are directly related to fibril diameters, composition of ground substance and changes in the bundle morphology, particularly in the crimp structure. The purpose of this study was to further describe the transformation of the collagen fibers of the pubic symphysis during late mouse pregnancy. We examined the architecture of collagen fibers in the symphysis and pubic ligament through the Picrosirius-polarization method and also through scanning electron microscopy to directly visualize and measure the crimping from pregnant and virgin mice. The crimp angle and the length of five consecutive crimps were measured according to Patterson-Kane et al. [Connect. Tissue Res. 36 (1997) 253]. It could be demonstrated that the angles progressively decreased and the crimp length increased, denoting that the fibers have untwisted during the relaxation process. Our findings suggest that a disruption of the helical arrangement of the collagen containing fibers may contribute to explaining the rapid remodeling that occurs at the end of pregnancy and that is responsible for an increase in pliancy and length of the pubic ligament in mice.