Identification of bradykinin: related peptides from Phyllomedusa nordestina skin secretion using electrospray ionization tandem mass spectrometry after a single-step liquid chromatography

Amphibian skin secretions are a source of potential new drugs with medical and biotechnological applications. Rich in peptides produced by holocrine-type serous glands in the integument, these secretions play different roles, either in the regulation of physiological skin functions or in the defense against predators or microorganisms. The aim of the present work was to identify novel peptides with bradykinin-like structure and/or activity present in the skin of Phyllomedusa nordestina. In order to achieve this goal, the crude skin secretion of this frog was pre-fractionated by solid phase extraction and separated by reversed-phase chromatography. The fractions were screened for low-molecular-mass peptides and sequenced by mass spectrometry. It was possible to identify three novel bradykinin-related peptides, namely: KPLWRL-NH2 (Pnor 3), RPLSWLPK (Pnor 5) and VPPKGVSM (Pnor 7) presenting vascular activities as assessed by intravital microscopy. Pnor 3 and Pnor 7 were able to induce vasodilation. On the other hand, Pnor 5 was a potent vasoconstrictor. These effects were reproduced by their synthetic analogues.

Antivenom action on renal effects induced by Thalassophryne nattereri venom

Thalassophryne nattereri (niquim) is a venomous fish responsible for numerous accidents involving fishermen in northern and northeastern Brazil. The aim of the present investigation was to evaluate the action of antivenom on renal effects caused by Thalassophryne nattereri venom. Isolated kidneys of Wistar rats were perfused with a previously dialyzed Krebs-Henseleit solution containing 6 g% bovine serum albumin. The antivenom action was studied through perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). The niquim venom (1 miug/mL), the antivenom alone (1 miug/mL) or the venom incubated with antivenom were added to the system 30 minutes after the beginning of each perfusion. Previous works have shown venom induced-alterations of renal function parameters. In the isolated rat Kidney, T. nattereri venom (1 miug/mL) increased the perfusion pressure and renal vascular resistance at 60, 90 and 120 minutes. UF and GFR also increased at 60, 90 and 120 minutes when compared with the control group; however, no effects were observed on the percent of sodium (% TNa more control equal 81.1 more or less 0.86; % TNa more 60 equal 78.04 more or less 1.18; % TNa more 90 equal -5.16 more or less 3.34; %TNa more 120 equal 79.49 more or less 0.87) and potassium (%TKcontrol equal 72.29 more or less 1.12; %TK more 60 equal 75.41 more or less 0.65; % TK more 90 equal 71.23 more or less 2.55; % TK more 120 equal 76.62 more or less 1.04) tubular transporto. The administration of the antivenom (1 miug/mL) incubated with venom (1 miug/mL) reduced the changes in PP, RVR, UF and GFR provoked by Thalassophryne nattereri venom. The group perfused with venom alone showed a moderate deposit of a proteinaceous material in the tubules and urinary space.(...)

A C-type lectin from Bothrops jararacussu venom can adhere to extracellular matrix proteins and induce the rolling of leukocytes

Purification of a lectin from Bothrops jararacussu venom (BjcuL) was carried out using agarose-D-galactose affinity gel. MALDI-TOF gave a major signal at m/z 32028, suggesting the presence of a dimmer composed of two identical subunits. Divalent cations were required for the lectin activity, as complete absence of such ions reduced hemagglutination. BjcuL was more effective at neutral pH and showed total loss of activity at pH values below 4.0 and above 9.0. Its agglutinating activity remained stable at 25°C until 60min, but increased when at 35°C for at least 15min. Adhesion assays to extracellular matrix (ECM) glycoproteins showed that the biotinylated lectin (0.039-5.0µg/100µl) was capable of binding to fibronectin and vitronectin in a dose-dependent manner. The binding was partially inhibited in the presence of D-galactose. BjcuL (1.25-10µg/30µl) potential was investigated for leukocyte rolling and adhesion to endothelial cells in living microvessels using intravital microscopy, which showed that it induced a dose-dependent increase in rolling and adherence of leukocytes, acting directly on endothelial cells of postcapillary venules. The specific association between lectins and their ligands, either on the cell surface or on the ECM, is related to a variety of biological processes. The complementary characterization of BjcuL, shown here, is useful to further understand the venom effects and as a background for future investigation for therapeutic strategies.

A C-type lectin from Bothrops jararacussu venom can adhere to extracellular matrix proteins and induce the rolling of leukocytes

Purification of a lectin from Bothrops jararacussu venom (BjcuL) was carried out using agarose-D-galactose affinity gel. MALDI-TOF gave a major signal at m/z 32028, suggesting the presence of a dimmer composed of two identical subunits. Divalent cations were required for the lectin activity, as complete absence of such ions reduced hemagglutination. BjcuL was more effective at neutral pH and showed total loss of activity at pH values below 4.0 and above 9.0. Its agglutinating activity remained stable at 25°C until 60min, but increased when at 35°C for at least 15min. Adhesion assays to extracellular matrix (ECM) glycoproteins showed that the biotinylated lectin (0.039-5.0µg/100µl) was capable of binding to fibronectin and vitronectin in a dose-dependent manner. The binding was partially inhibited in the presence of D-galactose. BjcuL (1.25-10µg/30µl) potential was investigated for leukocyte rolling and adhesion to endothelial cells in living microvessels using intravital microscopy, which showed that it induced a dose-dependent increase in rolling and adherence of leukocytes, acting directly on endothelial cells of postcapillary venules. The specific association between lectins and their ligands, either on the cell surface or on the ECM, is related to a variety of biological processes. The complementary characterization of BjcuL, shown here, is useful to further understand the venom effects and as a background for future investigation for therapeutic strategies.