A metal-free blue chromophore derived from plant pigments.

Blue natural pigments are rare, especially among plants. However, flowering species that evolved to attract Hymenoptera pollinators are colored by blue anthocyanin-metal complexes. Plants lacking anthocyanins are pigmented by betalains but are unable to produce blue hues. By extending the π-system of betalains, we designed a photostable and metal-free blue dye named BeetBlue that did not show toxicity to human hepatic and retinal pigment epithelial cells and does not affect zebrafish embryonal development. This chiral dye can be conveniently synthesized from betalamic acid obtained from hydrolyzed red beetroot juice or by enzymatic oxidation of l-dopa. BeetBlue is blue in the solid form and in solution of acidified polar molecular solvents, including water. Its capacity to dye natural matrices makes BeetBlue the prototype of a new class of low-cost bioinspired chromophores suitable for a myriad of applications requiring a blue hue.

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: biological effects benchmarking relatively to jararhagin, a P-III SVMP.

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding.

Enzymatic and immunochemical characterization of Bothrops insularis venom and its neutralization by polyspecific Bothrops antivenom.

Herein we compared the biological activities of Bothrops insularis and Bothrops jararaca venoms as well as their neutralization by polyspecific Bothrops antivenom (PBA). On account of that, we investigated their antigenic cross-reactivity and the neutralization of lethal, myotoxic and defibrinating activities by polyspecific and species-specific antivenoms. Silver-stained SDS-PAGE gels evidenced many common bands particularly above 47 kDa between B. jararaca and B. insularis venoms. However, some protein bands between 46 and 28 kDa were observed exclusively in B. jararaca venom. Both venoms presented gelatinolytic, caseinolytic, fibrinogenolytic and phospholipase A(2) activities. No hyaluronidase activity was detected in both venoms by zymography. Polyspecific and species-specific antivenoms showed similar titers to B. jararaca and B. insularis venoms by ELISA, and recognized similar components by immunoblotting. The PBA was effective in neutralizing the lethal, myotoxic and defibrinating activities of both venoms as well as to abrogate microcirculatory disturbances induced by B. insularis venom. No statistically significant difference was observed for minimal hemorrhagic doses between both venoms. Antigenic cross-reactivity was evident between both venoms. Since toxic and enzymatic activities were similar, we speculate that B. insularis venoms can induce a local damage in humans comparable to that observed in other Bothrops venoms. Besides, the PBA was effective in neutralizing the toxic activities of B. insularis venom.

Transcriptome analysis of expressed sequence tags from the venom glands of the fish Thalassophryne nattereri.

Thalassophryne nattereri (niquim) is a venomous fish found on the northern and northeastern coasts of Brazil. Every year, hundreds of humans are affected by the poison, which causes excruciating local pain, edema, and necrosis, and can lead to permanent disabilities. In experimental models, T. nattereri venom induces edema and nociception, which are correlated to human symptoms and dependent on venom kininogenase activity; myotoxicity; impairment of blood flow; platelet lysis and cytotoxicity on endothelial cells. These effects were observed with minute amounts of venom. To characterize the primary structure of T. nattereri venom toxins, a list of transcripts within the venom gland was made using the expressed sequence tag (EST) strategy. Here we report the analysis of 775 ESTs that were obtained from a directional cDNA library of T. nattereri venom gland. Of these ESTs, 527 (68%) were related to sequences previously described. These were categorized into 10 groups according to their biological functions. Sequences involved in gene and protein expression accounted for 14.3% of the ESTs, reflecting the important role of protein synthesis in this gland. Other groups included proteins engaged in the assembly of disulfide bonds (0.5%), chaperones involved in the folding of nascent proteins (1.4%), and sequences related to clusterin (1.5%), as well as transcripts related to calcium binding proteins (1.0%). We detected a large cluster (1.3%) related to cocaine- and amphetamine-regulated transcript (CART), a peptide involved in the regulation of food intake. Surprisingly, several retrotransposon-like sequences (1.0%) were found in the library. It may be that their presence accounts for some of the variation in venom toxins. The toxin category (18.8%) included natterins (18%), which are a new group of kininogenases recently described by our group, and a group of C-type lectins (0.8%). In addition, a considerable number of sequences (32%) was not related to sequences in the databases, which indicates that a great number of new toxins and proteins are still to be discovered from this fish venom gland.

Importance of jararhagin disintegrin-like and cysteine-rich domains in the early events of local inflammatory response.

Jararhagin is a multi-domain SVMP from Bothrops jararaca venom comprising catalytic, disintegrin-like and cysteine-rich domains, which cause a local reaction manifested by hemorrhage, edema, cytokine release and inflammatory cell recruitment. In this study, the importance of disintegrin-like/cysteine-rich domains of jararhagin was addressed by analyzing the effects of jararhagin-C, which lacks the catalytic domain, in induction of leukocyte rolling and release of pro-inflammatory cytokines. Jararhagin-C was isolated from B. jararaca venom conserving the same ability of complete jararhagin molecule in inhibiting collagen-induced platelet-aggregation. Treatment of trans-illuminated cremaster muscle in vivo with jararhagin-C increased number of rolling leukocytes (approximately 250%) in post-capillary venules in all periods analyzed, without interfering with microvasculature haemodynamic, like vessel diameter, the erythrocyte speed or the blood flow rate. The release of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 was significantly enhanced in the local of jararhagin-C injection, showing the maximum levels in periods between 2 and 4 h after treatment. Besides the action of jararhagin-C, the presence of the inactivated catalytic domain in o-phenanthrolin-treated jararhagin was related to a higher increase in the number of rolling leukocytes. Moreover, the levels of IL-6 and IL-1beta induced by catalytically active jararhagin were higher than those induced by jararhagin-C. In conclusion, our findings suggest that the disintegrin-like/cysteine-rich domains of jararhagin are sufficient to locally activate the early events of an acute inflammatory response as leukocyte rolling and pro-inflammatory cytokine release and this action may add to the effect of catalysis, which enhances the primary cell activation.

Natterins, a new class of proteins with kininogenase activity characterized from Thalassophryne nattereri fish venom.

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.

Immunosuppresive role of principal toxin (crotoxin) of Crotalus durissus terrificus venom.

The composition of the crotalic venom and the immunochemistry and/or pathophysiological characterization and main components were well studied. However, few studies have been carried out to investigate the effect of toxins of this venom on the development of the immune response. The objective of this work was to find out if venom or crotoxin of Crotalus durissus terrificus was able to modulate the immune response through its ability to change the mediators involved in the immune response by an unrelated antigen. We observed in the murine model, that venom as well as crotoxin have inhibitory effect on splenic cells proliferation induced by Con-A. Moreover, CB did not inhibit the proliferative response, suggesting that the integrity of crotoxin complex is necessary for the development of this phenomenon. Moreover, we showed that the effect on cellular proliferation was unrelated to cytotoxicity activity. We also observed that venom or crotoxin inhibited cytokine release induced in HSA immunised mice, mainly IL-2, IL-4 and IL-10, however, crotoxin did not inhibit the release of IFN-gamma. The involvement of T or B cells in the suppressive effect of venom was evaluated through the transference of purified splenic cells from venom-mice to normal mice that also produced low IgG1 anti-HSA levels, indicating the participation of these cells in this process. Mechanism of action of the crotalic venom on development of immune response to an unrelated antigen is much more complex, therefore it must not only involve the interaction of distinct cellular populations, but activation or inhibition of signalling proteins, need to be further investigated.

Thalassophryne nattereri fish venom: biological and biochemical characterization and serum neutralization of its toxic activities.

Envenomation by Thalassophryne nattereri fishes are an important medical problem in northeast of Brazil, causing in human victims considerable pain and edema followed by necrosis. Venom obtained from fresh captured specimens of this fish was tested in vitro or in animal models for a better characterization of its toxic activities. Intradermal injection of the venom in the foot pad of mice induced local edema and hemorrhage followed a few hours later by necrosis. Subcutaneous injection of the venom induced systemic effects consisting in jerking motions, paralysis of hind limbs, erection of hair, rotational movements and violent convulsions followed by death. Dead animals showed hyperemia of the small intestine and lungs. The venom showed distinct edematous, necrotizing and hemolytic activities, a low level of hemorrhagic, myotoxic and proteolytic activities and no detectable phospholipase A2 activity. SDS-PAGE analysis of the crude venom showed at least 17 components with the major band located around Mw = 19,000. Almost all proteins stained by amido black were also revealed by Western blotting with antibodies to T. nattereri venom. Fractionation of the venom by either gel filtration or cation exchange chromatography resulted in a few distinct peaks but in both situations the biological activities were located in only one of the peaks which corresponded to basic proteins with approximately Mw = 47,000. Heating of the venom at 56 degrees C for 60 min completely destroyed its biological activities. All venom toxic activities except edema were completely neutralized after in vitro incubation with anti-T. nattereri serum.

Neutralization of Thalassophryne nattereri (niquim) fish venom by an experimental antivenom.

T. nattereri (niquim) is a venomous fish involved in many human accidents in Brazil. The clinical picture includes mild local erythema, severe edema, intense pain and rapid progression to necrosis. The present therapy with anti-inflammatory and analgesic drugs is ineffective and, therefore, we decided to assess serum therapy as an alternative treatment using an experimental antivenom. The antivenom used was raised in rabbits showing an ELISA antibody titer of 1:8,192,000 and its ability to neutralize lethality, necrosis, nociception and edema was evaluated both by pre-incubating the venom with antivenom before injection into mice or by independent injections of venom and antivenom. Lethality was completely neutralized by pre-incubation (ED(50)=141.5 microl/mg) while necrosis and nociception were neutralized by pre-incubation or the independent injection of antivenom. Edema was only partially prevented even when large amounts of antivenom were used. These data suggest that antivenom may be a promising treatment for patients stung by T. nattereri and suggest the viability of producing a horse antivenom for use in clinical trials.

A comparative study of biological activities of crotoxin and CB fraction of venoms from Crotalus durissus terrificus, Crotalus durissus cascavella and Crotalus durissus collilineatus.

In Brazil, the Crotalus durissus terrificus subspecie is the most studied, particularly concerning its crotoxin. Crotoxin is the major toxic component of the South American rattlesnake Crotalus durissus venom. It is composed of two different subunits, CA called crotapotin and CB weakly toxic phospholipase A2 with high enzymatic activity. In this paper, we decided to make a study of the main toxic characteristics of crotoxin (CTX) and CB fraction from the other subspecies, Crotalus durissus cascavella and of Crotalus durissus collilineatus, in comparison with those of C. d. terrificus. Ours results have shown that the venoms presented similar chromatographic profiles and the purified fractions were free of contaminants. Regarding the toxic activities, the DL50 of the crotoxins showed no significant differences between the subspecies. The smaller toxicity of CB indicated that the toxicity of the crotoxin complex depends on the interaction between CA and CB. CTX and fraction CB of the three species of Crotalus showed negligible proteolytic activity. C. d. terrificus CTX presented higher PLA2 activity when compared with the others two subspecies. The oedema induced by CB developed later than the CTX and reached its peak 3 h after the injection. The myotoxic activity was determined by assaying serum CK levels. Mice injected with CTX of C. d. terrificus presented greater myotoxic activity compared to the others. The myotoxic activity of CB from the three subspecies was lower than the activity of the crotoxin, reinforcing the idea that the fraction CA increases the toxicity of CB.

Effects of Thalassophryne nattereri fish venom in isolated perfused rat kidney.

Thalassophryne nattereri, popularly known as Niquim, is a venomous fish responsible for many accidents in fishermen in the Northeast of Brazil. The effects of T. nattereri venom on renal physiology has not been tested. Isolated kidneys from Wistar rats of 240-280 g weight were perfused with Krebs-Henseleit solution containing 6g% of previously dialyzed bovine serum albumin. The effects of Niquim venom were studied on the perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF), glomerular filtration rate (GFR), percent of sodium tubular transport (%TNa(+)), percent of potassium tubular transport (%TK(+)) and percent of chloride tubular transport (%TCl(-)). The venom of T. nattereri (0.3, 1.0, and 3.0 microg/ml) was always added to the system 30 minutes after the beginning of each experiment (n=6). All experiments were preceded by 30 minutes internal control period and an external control group, where kidneys were perfused with only Krebs-Henseleit solution. All three doses tested promoted increases in PP and RVR. The first two doses also increased GFR and UF. The higher dose promoted decreases in GFR, UF, %TNa(+), %TK(+), %TCl(-). In the treated groups we observed hyalin casts inside all tubules and proteinaceous material in the urinary space. We conclude that the effects resulted from niquim venom agents that promoted a direct effect in kidney cells causing the release of vasoactive factors.

Alterations of the microcirculatory network during the clearance of epimastigote forms of Trypanosoma cruzi: an intravital microscopic study.

The distribution of epimastigote forms of Trypanosoma cruzi in the microcirculatory network and the vessel alterations were observed using an intravital microscopy technique. Immediately after intravenous inoculation of 2 x 10(6) epimastigote suspension into normal mice, parasites were seen as circulating clumps, and their retention at some sites of the endothelium of venules and capillaries was observed. Injection of 2 x 10(7) and 2 x 10(8) parasite suspensions induced, respectively, intermittent or total stasis of venules and capillaries, probably via obstruction by clumping. The mobility of epimastigotes in the clumps indicates that parasites were alive in the lumen of vessels. The retention of clumps in the capillaries, although intense, could only be observed when labeled parasites were inoculated. These results suggest that the rapid clearance of epimastigote forms of T. cruzi from the blood circulation of mice may be due to the retention of parasites to the endothelium of venules and capillaries that, in turn, may facilitate phagocytosis. This may be a mechanism by which mice are able to eliminate epimastigote forms from the circulation. These findings are consistent with our previous observations showing that epimastigotes are not lysed by complement activation but are phagocytosed and destroyed by a distinct population of blood cells.

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B2and M1 muscarinic acetylcholine receptors.

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (

Role of crotoxin, a phospholipase A2 isolated from Crotalus durissus terrificus snake venom, on inflammatory and immune reactions.

BACKGROUND: Crotoxin (CTX) is a potent neurotoxin from Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions. AIM: The aim of this paper was to investigate the mechanisms involved in these impaired responses. MATERIALS AND METHODS: Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)-6, IL-10, interferon (IFN)-gamma and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL-4, IL-6, IL-10 or IFN-gamma secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment. RESULTS: Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSA-treated animals. Increased levels of IL-6, IL-10 and corticosterone were also detected in CTX-injected animals. IFN-gamma levels were not modified after treatments. In contrast, spleen cells obtained from CTX-treated animals and stimulated with concanavalin A secreted less IL-10 and IL-4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration. CONCLUSIONS: Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV. CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.

Skeletal muscle necrosis and regeneration after injection of Thalassophryne nattereri (niquim) fish venom in mice.

Stings by Thalassophryne nattereri are responsible for envenomation of fishermen in north-eastern Brazil. Its venom induces prominent local tissue damage, characterized by pain, oedema and necrosis. The pathogenesis of acute muscle damage induced by T. nattereri venom was studied in mice. Intramuscular injection induced myonecrosis within the first hours. Some muscle cells presented a hypercontracted morphology, but most necrotic fibres were not hypercontracted, being instead characterized by a disorganization of myofibrils, with Z line loss, mitochondrial swelling and sarcolemmal disruption. In addition, thrombosis was observed histologically in venules and veins, together with vascular congestion and stasis, evidenced by intravital microscopy. Venom induced a rapid increment in serum creatine kinase (CK) levels, concomitant with a reduction in gastrocnemius muscle CK activity, whereas no increments in muscle lactic acid were detected. A rapid cytolytic effect was induced by the venom on C2C12 murine myoblasts in culture. The inflammatory reaction in affected muscle was characterized by oedema and scarce cellular infiltrate of polymorphonuclear leucocytes and macrophages, with a consequent delay in the removal of necrotic material. Skeletal muscle regeneration was partially impaired, as evidenced by the presence of regenerating fibres of variable size and by the increase of fibrotic tissue in endomysium and perimysium. It is suggested that T. nattereri venom affects muscle fibres by a direct cytotoxic effect, and that the vascular alterations described preclude a successful regenerative process.

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.