Evaluation of the immunomodulatory effect of Bougainvillea xbuttiana (Var. Orange) on the cytokine production induced by Botrhops jararaca venom in macrophages / Evaluación del efecto inmunomodulador de Bougainvillea xbuttiana (var. naranja) sobre la producción de citoquinas de macrófagos inducida pelo veneno de Bothrops jararaca

To study the effect of 50% ethanol extract of Bougainvillea xbuttiana on the enzymatic activity, cell via bility and cytokine production provoked by the venom of Bothrops jararaca in macro - phages. Three assays were used to study the effects of B. xbuttiana extract on the damage pro - duced by B. jararaca : Enzymatic activity was detected by measuring the proteoly tic and phos - pholipase A2; macrophages cytotoxicity was determined by the MTT method; levels of cytokine were evaluated using ELISA and a biological assay. After treatment with 300 µg/mL B. xbuttiana extract for 30 min, the proteolytic and phospholipase A2 activities of the venom were reduced to 95 and 61%, respectively. In macrophages cultures treated with B. xbuttiana extract combined with venom, the production of TNF - α, IL - 6 and IFN - γ was reduced, whereas IL - 10 was potenti - ated. Our results support the potential effect of the B. xbuttiana extract as a complementary therapy against the toxicity caused by the venom of B . jararaca snakes

Estudiar el efecto del extracto etanólico al 50% de Bougainvillea xbuttiana sobre la actividad enzimática viabilidad celular y producci ón de citoquinas provocada por el veneno de Bothrops jararaca en macrófagos Se utilizaron tres ensayos para estudiar los efectos del extracto de B. xbuttiana sobre el daño producido por B. jararaca : Se detectó actividad enzimática mediante la medición del proteolítico y fosfolipasa A2; la citotoxicidad de los macrófagos se determinó por el método MTT; Los niveles de citoquinas se evaluaron utilizando ELISA y un ensayo biológico. Después del tratamiento con 300 µg/mL de extracto de B. xbuttiana durante 30 mi n, las actividades proteolíticas y de fosfolipasa A2 del veneno se redujeron a 95 y 61%, respectivamente. En cultivos de macrófagos tratados con extracto de B. xbuttiana combinado con veneno, la producción de TNF - α, IL - 6 e IFN - γ se redujeron, mientras que IL - 10 se potenció. Nuestros resultados apoyan el efecto potencial del extracto de B. xbuttiana como terapia complementaria frente a la toxicidad provocada por el veneno de B. jararaca .

Fruit Bagasse Phytochemicals from Malpighia Emarginata Rich in Enzymatic Inhibitor with Modulatory Action on Hemostatic Processes.

Agro-industrial wastes are promising sources of phytochemicals for the development of products to be used in health promotion and maintenance. In this study, extracts from acerola bagasse (AB) were characterized by HPLC, and evaluated according to its modulatory action on phospholipases A2 and proteases involved in processes such as inflammation and blood clotting. Snake venoms were used as biological tools once they have high functional and structural homology between their enzymes and human enzymes. Two types of extracts were prepared from AB: aqueous and methanolic. These extracts, evaluated at different proportions (venom:extract, w:w), significantly inhibited the phospholipase activity induced by the venoms of Bothrops moojeni, Bothrops atrox (11% to 31%), and Crotalus durissus terrificus (C. d. t.) (11% to 19%). The hemolytic activity induced by the venoms of B. moojeni and C. d. t. was better inhibited by the methanolic extract (inhibition between 23% and 48%). Thrombolysis induced by the venoms of B. moojeni and C. d. t. was inhibited by both extracts, with inhibition ranging from 13% to 63% for the aqueous extract, and from 12% to 92% for the methanolic one. Both extracts increased the time of coagulation induced by the venoms of B. moojeni and Lachesis muta muta in 26 and up to 68 s. These inhibitory actions were related to the following phenolic compounds present in the extract of AB: gallic acid, catechin, epigallocatechin gallate, epicatechin, syringic acid, p-coumaric acid, and quercetin. Additional studies are needed to confirm their potential use for nutraceutical purposes. PRACTICAL APPLICATION: Agro-industrial wastes are promising sources of phytochemicals for the development of products that can be used by pharmaceutical, cosmetics, and food industries. Studies report the use of the acerola bagasse extract in health improvement. However, its toxic-pharmacological characterization is still scarce. In this study, the extracts of acerola bagasse presented phenolic compounds that can modulate the activity of enzymes such as phospholipases A2 and proteases that act on the coagulant/anticoagulant and thrombotic/thrombolytic activities and the break of phospholipids, decreasing the inflammation and platelet aggregation. Although the in vivo effects of the extracts are not fully understood, this study shed light upon the possibilities of their usage.

Preclinical evaluation of the neutralizing ability of a monospecific antivenom for the treatment of envenomings by Bothrops lanceolatus in Martinique.

Bothrops lanceolatus is an endemic viperid species in the Lesser Caribbean island of Martinique. Envenomings by this species are characterized by local and systemic effects, among which the development of thrombosis in various organs is the most severe complication. An experimental toxicological characterization of this venom was performed using in vivo mouse tests and various in vitro assays. The venom induced lethal, local and systemic hemorrhagic, edema-forming, myotoxic, thrombocytopenic, proteinase and phospholipase A2 activities. The preclinical efficacy of a batch of monospecific Bothrofav® antivenom currently in use in Martinique was assessed. The antivenom was highly effective in the neutralization of all activities tested, in agreement with its described clinical efficacy. This batch of antivenom showed a higher preclinical efficacy as compared to a previous batch used in the past.

Evaluation of Rhamnetin as an Inhibitor of the Pharmacological Effect of Secretory Phospholipase A2.

Rhamnetin (Rhm), 3-O-methylquercetin (3MQ), and Rhamnazin (Rhz) are methylated derivatives of quercetin commonly found in fruits and vegetables that possess antioxidant and anti-inflammatory properties. Phospholipase A2 (PLA2) displays several important roles during acute inflammation; therefore, this study aimed at investigating new compounds able to inhibit this enzyme, besides evaluating creatine kinase (CK) levels and citotoxicity. Methylated quercetins were compared with quercetin (Q) and were incubated with secretory PLA2 (sPLA2) from Bothrops jararacussu to determine their inhibitory activity. Cytotoxic studies were performed by using the J774 cell lineage incubated with quercertins. In vivo tests were performed with Swiss female mice to evaluate decreasing paw edema potential and compounds' CK levels. Structural modifications on sPLA2 were made with circular dichroism (CD). Despite Q and Rhz showing greater enzymatic inhibitory potential, high CK was observed. Rhm exhibited sPLA2 inhibitory potential, no toxicity and, remarkably, it decreased CK levels. The presence of 3OH on the C-ring of Rhm may contribute to both its anti-inflammatory and enzymatic inhibition of sPLA2, and the methylation of ring A may provide the increase in cell viability and low CK level induced by sPLA2. These results showed that Rhm can be a candidate as a natural compound for the development of new anti-inflammatory drugs.

Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme.

Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous expression of a serine protease from Crotalus durissus collilineatus, named collinein-1, in Pichia pastoris, as well as the enzymatic comparative characterization of the toxin in native and recombinant forms. The complementary DNA (cDNA) encoding collinein-1 was amplified from cDNA library of C. d. collilineatus venom gland and cloned into the pPICZαA vector. The recombinant plasmid was used to transform cells of KM71H P. pastoris. Heterologous expression was induced by methanol and yielded 56 mg of recombinant collinein-1 (rCollinein-1) per liter of culture. The native collinein-1 was purified from C. d. collilineatus venom, and its identity was confirmed by amino acid sequencing. The native and recombinant enzymes showed similar effects upon bovine fibrinogen by releasing preferentially fibrinopeptide A. Although both enzymes have induced plasma coagulation, native Colinein-1 has shown higher coagulant activity. The serine proteases were able to hydrolyze the chromogenic substrates S-2222, S-2238, and S2302. Both enzymes showed high stability on different pH and temperature, and their esterase activities were inhibited in the presence of Zn2+ and Cu2+. The serine proteases showed similar k cat/K m values in enzyme kinetics assays, suggesting no significant differences in efficiency of these proteins to hydrolyze the substrate. These results demonstrated that rCollinein-1 was expressed with functional integrity on the evaluated parameters. The success in producing a functionally active recombinant SVSP may generate perspectives to their future therapeutic applications.

A comparison of the ability of Bellucia dichotoma Cogn. (Melastomataceae) extract to inhibit the local effects of Bothrops atrox venom when pre-incubated and when used according to traditional methods.

Bellucia dichotoma Cogn. (Melastomataceae) is one of various plant species used in folk medicine in the west of the state of Pará, Brazil, to treat snake bites. Many studies have been carried out to evaluate the effectiveness of anti-snake bite plants, but few of these use the same preparation methods and doses as those traditionally used by the local populations. This study therefore compared inhibition of the main local effects of B. atrox venom (BaV) by aqueous extract of B. dichotoma (AEBd) administered according to traditional methods and pre-incubated with BaV). The concentrations of phenolic compounds (tannins and flavonoids) in AEBd were determined by colorimetric assays. The effectiveness of AEBd in inhibiting the hemorrhagic and edematogenic activities of BaV was evaluated in mice in four different experimental in vivo protocols: (1) pre-incubation (venom:extract, w/w); (2) pre-treatment (p.o.); (3) post-treatment (p.o.); and (4) AEBd (p.o.) in combination with Bothrops antivenom (BA) (i.v.). To assess in vitro inhibition of BaV phospholipase A2 activity, the pre-incubation method or incorporation of AEBd or BA in agarose gels were used. The effect of AEBd on BaV was determined by SDS-PAGE, zymography and Western blot. Colorimetric assays revealed higher concentrations of (condensed and hydrolyzable) tannins than flavonoids in AEBd. Hemorrhagic activity was completely inhibited using the pre-incubation protocol. However, with pre-treatment there was no significant inhibition for the concentrations tested, and with the post-treatment only the 725 mg/kg dose of AEBd was able to inhibit 40.5% (p = 0.001) of the hemorrhagic activity of BaV. Phospholipase A2 activity was only inhibited when AEBd was pre-incubated with BaV. BaV-induced edema was completely inhibited with pre-incubation (p < 0.05) and significantly reduced (p < 0.05) with pre- and post-treatment (p.o.) for the concentrations tested. The reduction in local edema was even greater when AEBd was administered in combination with BA. The SDS-PAGE profiles showed that several of the BaV protein (SDS-PAGE) and enzyme (zymography) bands were not detected when the venom was pre-incubated, and Western blot revealed that this was not caused by the AEBd enzymes observed in the zymogram. The "pseudo inhibition" observed after pre-incubation in this study may be due to the presence of tannins in the extract, which could act as chelating agents, removing metalloproteins and Ca²âº ions and thus inhibiting hemorrhagin and PLA2 activity. However, when administered according to traditional methods, B. dichotoma extract was effective in blocking BaV-induced edematogenic activity and had an additional effect on inhibition of this activity by BA.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom.

BACKGROUND: Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A2 are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A2 drugs. METHODS: HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated. RESULTS: HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 ± 0.28 µg/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA2 inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid. CONCLUSION: HP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents.

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes Aα followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Protective effect of schizolobium parahyba flavonoids against snake venoms and isolated toxins.

Four compounds (isoquercitrin, myricetin-3-O-glucoside, catechin and gallocatechin) were isolated from lyophilized aqueous extract of Schizolobium parahyba leaves by chromatography on Sephadex LH-20, followed by semipreparative HPLC using a C-18 column, and identified by 1H and 13C NMR. The compounds were then, tested against hemorrhagic and fibrinogenolytic activities of Bothrops crude venoms and isolated metalloproteinases. The inhibitors neutralized the biological and enzymatic activities of Bothrops venoms and toxins isolated from B. jararacussu and B. neuwiedi venoms. The results showed that gallocatechin and myricetin-3-O-glucoside are good inhibitors of hemorrhagic and fibrinogenolytic activities of metalloproteinases, respectively. Gallocatechin also inhibited the myotoxic activity of both B. alternatus venom and BnSP-6 (Lys49 PhospholipaseA2 from B. neuwiedi). Circular dichroism and docking simulation studies were performed in order to investigate the possible interaction between BnSP-6 and gallocatechin. This is the first time these compounds and their anti-ophidian properties are reported for S. parahyba species. Forthcoming studies involving X-ray co-crystallization, will be of great importance for the development of new therapeutic agents for the treatment of ophidian accidents and for the better understanding of the structure/function relationship of venom toxins.

Molecular cloning and characterization of cDNAs encoding metalloproteinases from snake venom glands.

Snake venom metalloproteinases (SVMPs) are a superfamily of zinc-dependent proteases and participate in a number of important biological, physiological and pathophysiological processes. In this work, we simultaneously amplified nine cDNAs encoding different classes of metalloproteinases from glands of four different snake species (Agkistrodon contortrix laticinctus, Crotalus atrox, Crotalus viridis viridis and Agkistrodon piscivorus leucostoma) by RT-PCR with a pair of primers. Among the encoded metalloproteinases, two enzymes (AclVMP-I and AplVMP-I), three enzymes (CaVMP-II, CvvVMP-II and AplVMP-II) and four enzymes (AclVMP-III, CaVMP-III, CvvVMP-III and AplVMP-III) with the characteristic motif (HEXXHXXGXXH) of metalloproteinase belong to type P-I, P-II and P-III enzymes, respectively. Disintegrin domains of CaVMP-II and CvvVMP-II from two Crotatus snakes contain RGD-motif whereas AplVMP-II from Agkistrodon snake has KGD-motif. Instead of R/KGD-motif within disintegrin domain of SVMP-II enzyme, CaVMP-III, CvvVMP-III and AplVMP-III enzymes contain SECD-motif, while AclVMP-III has DDCD-motif in their corresponding position of disintegrin-like domains. There are 12 Cys amino acids in cysterin-rich domains of each P-III enzyme. Moreover, a disintegrin precursor (AplDis) with RGD-motif also simultaneously amplified from the glands of A.p. leucostoma while amplifying AplVMP-II and AplVMP-III, which indicated that different types of SVMPs and related genes are present in a single species of snake and share a consensus sequence at the 3' and 5' untranslated regions. RT-PCR result also showed that P-III is highly expressed in Crotalus snakes than in Agkistrodon snakes. Aligning the deduced amino acid sequence of these enzymes with other SVMPs from GenBank database indicated that this is the first report on the isolation of cDNAs encoding P-II and P-III enzymes from C.v. viridis and A.p. leucostoma snakes. The availability of these SVMP sequences directly facilitated further studies of structure characterization and diversified function analysis.

Inhibition of snake venoms and phospholipases A(2) by extracts from native and genetically modified Eclipta alba: isolation of active coumestans.

We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing secondary metabolites with pharmacological properties against phospholipase A(2) and the myotoxic activities of snake venom. Extracts from in natura aerial parts and roots, both native and genetically modified (in vitro), were prepared and analysed by high-performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants collected from Botucatu and Ribeirão Preto were efficient in inhibiting snake venom phospholipase A(2) activity. Regarding in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A(2) isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX-I and II). The search for antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also as an attempt to complement serum therapy.

Immunization with cDNA of a novel P-III type metalloproteinase from the rattlesnake Crotalus durissus durissus elicits antibodies which neutralize 69% of the hemorrhage induced by the whole venom.

Zinc-dependent metalloproteinases play a key role in the hemorrhage induced by viperid bite envenoming. In this work we report the cloning and sequencing of the cDNA from a novel P-III type metalloproteinase from Crotalus durissus durissus venom glands. The recombinant plasmid was used for DNA immunization in mice using accelerate DNA-coated microparticles with the Gene Gun system. The results showed that there is no significant difference in the efficiency of the immunization in mice when gold or tungsten microparticles were used. A pool of the sera from mice immunized with the metalloproteinase encoding DNA neutralized the hemorrhagic activity of C. d. durissus venom. The co-immunization with DNA encoding the metalloproteinase and a plasmid encoding the murine IL-2 increased the number of mice which show a specific antibody response towards C. d. durissus venom antigens. The neutralizing ability of the produced antibodies demonstrates that DNA immunization with tungsten microparticles may be used in strategies for antivenom production.

Anti-snake venom properties of Schizolobium parahyba (Caesalpinoideae) aqueous leaves extract.

Many medicinal plants have been recommended for the treatment of snakebites. The aqueous extracts prepared from the leaves of Schizolobium parahyba (a plant found in Mata Atlantica in Southeastern Brazil) were assayed for their ability to inhibit some enzymatic and biological activities induced by Bothrops pauloensis and Crotalus durissus terrificus venoms as well as by their isolated toxins neuwiedase (metalloproteinase), BnSP-7 (basic Lys49 PLA(2)) and CB (PLA(2) from crotoxin complex). Phospholipase A(2), coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by B. pauloensis and C. d. terrificus venoms, as well as by their isolated toxins were significantly inhibited when different amounts of S. parahyba were incubated previously with these venoms and toxins before assays. However, when S. parahyba was administered at the same route as the venoms or toxins injections, the tissue local damage, such as hemorrhage and myotoxicity was only partially inhibited. The study also evaluated the inhibitory effect of S. parahyba upon the spreading of venom proteins from the injected area into the systemic circulation. The neutralization of systemic alterations induced by i.m. injection of B. pauloensis venom was evaluated by measuring platelet and plasma fibrinogen levels which were significantly maintained when S. parahyba extract inoculation occurred at the same route after B. pauloensis venom injection. In conclusion, the observations confirmed that the aqueous extract of S. parahyba possesses potent snake venom neutralizing properties. It may be used as an alternative treatment to serum therapy and as a rich source of potential inhibitors of toxins involved in several physiopathological human and animal diseases.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13 percent of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper.

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 microg) and fibrinogen (minimum coagulant dose = 4.2 microg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 microg). In addition, when injected intravenously in mice at doses of 5 and 10 microg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the ;gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Triterpenoid saponins, new metalloprotease snake venom inhibitors isolated from Pentaclethra macroloba.

We report here the antiproteolytic and antihemorrhagic properties of triterpenoid saponin inhibitors, named macrolobin-A and B, from Pentaclethra macroloba, against Bothrops snake venoms. The inhibitors were able to neutralize the hemorrhagic, fibrin(ogen)olytic, and proteolytic activities of class P-I and P-III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. Clotting and fibrinogenolytic activities induced by snake venoms and isolated thrombin-like enzymes were partially inhibited. Furthermore, the potential use of these inhibitors to complement antivenom therapy as an alternative treatment and/or used as molecular models for development of new therapeutical agents in the treatment of snake bite envenomations needs to be evaluated in future studies.

Characterization and cDNA cloning of aminopeptidase A from the venom of Gloydius blomhoffi brevicaudus.

The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)(18)-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile.

Strong myotoxic activity of Trimeresurus malabaricus venom: role of metalloproteases.

Trimeresurus malabaricus is an endemic snake found in the Southern region of Western Ghats section of India along with the more widely distributed species like Naja naja and Daboia russelii. T. malabaricus venom is not lethal when injected (i.p.) up to 20 mg/kg body weight in mice, but causes extensive local tissue degeneration. N. naja and D. russelii are highly toxic (i.p.) with minimum local tissue damage in experimental mice. In this study a comparative analysis of local tissue damage of T. malabaricus venom is made with N. naja and D. russelii snake venoms of the Southern regions of Western Ghats. T. malabaricus venom exhibits caseinolytic activity 16 and 24 times more than N. naja and D. russelii venom. Inhibition studies with specific protease inhibitors reveal that the major proteases belong to metalloproteases. T. malabaricus venom hydrolyses gelatin and induces strong hemorrhagic activity in mice. Both N. naja and D. russelii fail to hydrolyze gelatin even at very high concentration and did not induce any hemorrhagic activity. With D. russelii venom small hemorrhagic spot was observed at the site of injection. The hemorrhagic activity of T. malabaricus venom is completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor. The i.m. injection of T. malabaricus venom causes extensive degradation of muscle tissue within 24 h. The light microscopic observation of muscle tissue showed congestion of blood vessels and hemorrhage at the early stage followed by extensive necrosis of muscle fibers. The elevated levels of serum CK and LDH activity further supported the muscle degeneration. Such pathological symptoms were not seen with N. naja and D. russelii snake venom. The hemorrhagic and the muscle necrosis was completely neutralized by metalloprotease inhibitors and not by serine protease inhibitor strongly suggests that the major toxin component in the T. malabaricus venom is metalloprotease and its activity can be easily neutralized using chelating agents and its use in the first aid as chelation therapy is beneficial.

Inhibitory effects of Piper umbellatum and Piper peltatum extracts towards myotoxic phospholipases A2 from Bothrops snake venoms: isolation of 4-nerolidylcatechol as active principle.

Phospholipases A(2) (PLA(2)) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA(2) from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA(2)s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of approximately 1mM. In addition, 4-nerolidylcatechol inhibited representatives of groups I and III of PLA(2)s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA(2) activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA(2)s studied.

Primary structure of brevilysin L4, an enzymatically active fragment of a disintegrin precursor from Gloydius halys brevicaudus venom.

Brevilysin L4 (L4) is a non-hemorrhagic P-I class metalloprotease (MP) isolated from Gloydius halys brevicaudus venom. Its complete amino acid sequence has been determined. L4 is a single-chain polypeptide and highly homologous to those of other snake venom MPs. A zinc-binding motif, HExxHxxGxxH, is located at residues 142-152. A characteristic feature of L4 is the presence of a spacer sequence (LRTDTVS) at the C-terminal that links metalloprotease and disintegrin domains and is usually removed by post-translational proteolysis, suggesting that L4 is expressed together with a spacer region and a disintegrin domain at the C-terminal. The nucleotide sequence of a cDNA clone encoding L4 has revealed that L4 is a disintegrin precursor and produced as a P-II class MP. The disintegrin coded after L4 sequence was brevicaudin 1, a disintegrin previously isolated from the same venom. P-II class MPs have been suspected to undergo autoproteolysis to release disintegrins. Although being P-I class MP, L4 itself autocatalytically degrades with a half-life of 30min at pH 8.5 and 37 degrees C in the absence of Ca(2+). Sequence analysis of several fragment peptides produced during the autolysis of L4 indicated that more than 40 peptide bonds were split, and the cleavages of Ser(60)-Asn(61), Thr(99)-Ala(100), and Phe(103)-Asp(104) bonds may trigger the autoproteolysis. Addition of Ca(2+) completely suppressed the cleavage of these particular bonds, resulting in a marked prevention of autoproteolysis. Thus, L4 provides a good model for the investigation of autolysis of some MPs.