Triacontyl p-coumarate: an inhibitor of snake venom metalloproteinases.

Snake venom metalloproteinases (SVMPs) participate in a number of important biological, physiological and pathophysiological processes and are primarily responsible for the local tissue damage characteristic of viperid snake envenomations. The use of medicinal plant extracts as antidotes against animal venoms is an old practice, especially against snake envenomations. Such plants are sources of many pharmacologically active compounds and have been shown to antagonize the effects of some venoms and toxins. The present study explores the activity of triacontyl p-coumarate (PCT), an active compound isolated from root bark of Bombacopsis glabra vegetal extract (Bg), against harmful effects of Bothropoides pauloensis snake venom and isolated toxins (SVMPs or phospholipase A(2)). Before inhibition assays, Bg or PCT was incubated with venom or toxins at ratios of 1:1 and 1:5 (w/w; venom or isolated toxins/PCT) for 30 min at 37°C. Treatment conditions were also assayed to simulate snakebite with PCT inoculated at either the same venom or toxin site. PCT neutralized fibrinogenolytic activity and plasmatic fibrinogen depletion induced by B. pauloensis venom or isolated toxin. PCT also efficiently inhibited the hemorrhagic (3MDH - minimum hemorrhagic dose injected i.d into mice) and myotoxic activities induced by Jararhagin, a metalloproteinase from B. jararaca at 1:5 ratio (toxin: inhibitor, w/w) when it was previously incubated with PCT and injected into mice or when PCT was administered after toxin injection. Docking simulations using data on a metalloproteinase (Neuwiedase) structure suggest that the binding between the protein and the inhibitor occurs mainly in the active site region causing blockade of the enzymatic reaction by displacement of catalytic water. Steric hindrance may also play a role in the mechanism since the PCT hydrophobic tail was found to interact with the loop associated with substrate anchorage. Thus, PCT may provide a alternative to complement ophidian envenomation treatments.

Action of neuwiedase, a metalloproteinase isolated from bothrops neuwiedi venom, on skeletal muscle: an ultrastructural and immunocytochemistry study

The damaging effects of neuwiedase, a non-hemorrhagic snake venom metalloproteinase from P-I class, on gastrocnemius muscle are studied herein. Following neuwiedase injection, ultrastructural alterations were detected early showing disarrangement of skeletal muscle fibers (characterized by discontinuity of Z lines), mitochondrial swelling, and disruption of plasma membrane and basal lamina. Degradation of skeletal muscle and the appearance of an amorphous substance, primarily composed of cellular debris, were noted after 24 hours. The presence of neuwiedase at the injection site (detected by immunocytochemistry) revealed highly specific labeling of myofibril components of damaged myocytes. In addition, proteolysis of muscle proteins assayed through myofibrils extracted from gastrocnemius muscle indicated that neuwiedase provoked degradation of myofibrils, especially myosin. These results suggest that skeletal muscle damage, induced by neuwiedase, is probably due to its proteolytic action on myofibrils, which are responsible for the maintenance of the cellular architecture.

Peptide mimicking antigenic and immunogenic epitope of neuwiedase from Bothrops neuwiedi snake venom.

Peptides derived from a phage display library may mimic essential features of epitopes (mimotopes), including their immunogenicity. A recombinant peptide library of 12 amino acids displayed on the phage capsid was used to obtain peptides that mimic epitopes of antigens that are reactive to specific polyclonal antibodies anti-neuwiedase (NEU), a toxin from Bothrops neuwiedi snake venom. These polyclonal antibodies are protective against NEU activity and were used as target for the peptide library biopannings, resulting in the selection of 80 peptides. Antibody-binding epitopes were obtained by sequence alignment with the primary and tertiary structures of the NEU protein. Antigenicity and specificity of the mimotopes mixture were confirmed by dot blot, immuno dot blot, plaque reduction and Western blot assays. Their immunogenicity was demonstrated by immunization of BALB/c mice and ELISA tests. The NEU toxin is an important antigen that has many common structural regions to several toxic venom metalloproteinases, in which two epitope regions have been detected. The two mapped epitopes were found in primary sequences of several snake venom toxins, thus demonstrating the potential application of these NEU mimotopes as possible antigen components that are toxicity free.

Purification and functional characterization of two fibrinogenolytic enzymes from Bothrops alternatus venom

Two fibrinogenolytic enzymes, Bothrops alternatus metalloprotease isoform (BaltMP)-I and II, were purified from Bothrops alternatus venom using Diethylaminoethyl (DEAE) Sephacel, Sephadex G-75 and Heparin-Agarose column chromatography. Purified BaltMP-I and II ran as single protein bands on analytical polyacrylamide gel electrophoresis and showed molecular weights of 29000 and 36000, respectively, under reducing conditions in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). BaltMP-II, but not BaltMP-I, displayed blood-clotting activity in bovine plasma, which was about 10-fold higher than that of the crude venom. Both enzymes were proteolytically active against bovine fibrinogen as substrate. When fibrinogen and each enzyme were incubated at 37°C, at a ratio of 1:100 (w/w), BaltMP-II cleaved preferentially the Aalpha -chain and more slowly the Bbeta -chain. The action of BaltMP-I was similar, but lower. None of the proteases degraded the gamma-chain of fibrinogen. The fibrinogenolytic activity of the enzymes was inhibited by 1,10-phenanthroline, suggesting they are metalloproteases. Since both enzymes were found to cause defibrinogenation when intraperitoneally (i.p.) administered to mice, they can be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.(AU)

Neutralizing properties of Musa paradisiaca L. (Musaceae) juice on phospholipase A2, myotoxic, hemorrhagic and lethal activities of crotalidae venoms.

The use of plants as medicine has been referred to since ancient peoples, perhaps as early as Neanderthal man. Plants are a source of many biologically active products and nowadays they are of great interest to the pharmaceutical industry. The study of how people of different culture use plants in particular ways has led to the discovery of important new medicines. In this work, we verify the possible activity of Musa paradisiaca L. (Musaceae) against the toxicity of snake venoms. Musa paradisiaca, an important source of food in the world, has also been reported to be popularly used as an anti-venom. Interaction of Musa paradisiaca extract (MsE) with snake venom proteins has been examined in this study. Phospholipase A2 (PLA2), myotoxic and hemorrhagic activities, including lethality in mice, induced by crotalidae venoms were significantly inhibited when different amounts of MsE were mixed with these venoms before assays. On the other hand, mice that received MsE and venoms without previous mixture or by separated routes were not protected against venom toxicity. Partial chemical characterization of MsE showed the presence of polyphenols and tannins and they are known to non-specifically inactivate proteins. We suggest that these compounds can be responsible for the in vitro inhibition of the toxic effects of snake venoms. In conclusion, according to our results, using mice as experimental model, MsE does not show protection against the toxic effects of snake venoms in vivo, but if was very effective when the experiments were done in vitro.

Neutralization of some hematological and hemostatic alterations induced by neuwiedase, a metalloproteinase isolated from Bothrops neuwiedi pauloensis snake venom, by the aqueous extract from Casearia mariquitensis (Flacourtiaceae).

The aqueous extract from the leaves of Casearia mariquitensis (C. m.), a plant found in Brazilian open pastures, was assayed for its ability to inhibit some hematological and hemostatic effects induced by neuwiedase, a 22 kDa class P-I metalloproteinase from the venom of the South American pit viper Bothrops neuwiedi pauloensis. The aqueous extract from C. m. was able to neutralize the hematological alterations induced by the crude venom (C.V.) upon erythrocytes when the venom was incubated at a ratio of 1:10 (w/w, venom/extract), but it did not neutralize the platelet decreasing ability of C.V. The plasma fibrinogen concentration decreased approximately 36% and 83% when 0.6 LD(50) of the C.V. or neuwiedase, respectively, were injected by i.p. route in mice, and the aqueous extract from C. m. was able to inhibit this effect. The Bbeta fibrinogen chain was protected against degradation caused by crude venom and neuwiedase when the venom or toxin were incubated with C. m. extract. We also observed that this extract exerted a very slight effect on the clotting time, prolonging it only to a little extent. The pulmonary hemorrhage induced by neuwiedase when injected intravenously with 0.6 LD(50) was completely inhibited when this toxin was incubated with the extract at a ratio of 1:10 (w/w, toxin/extract). It is concluded that C. m. displays components able to inhibit some hematological and systemic alterations induced by C.V.

Neutralization of proteases from Bothrops snake venoms by the aqueous extract from Casearia sylvestris (Flacourtiaceae).

Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the hemorrhagic activity caused by Bothrops asper, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was also inhibited by the C. sylvestris extract in different levels. The alpha-fibrinogen chain was partially protected against degradation caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract partially increased the time of plasma coagulation caused by B. jararacussu, B. moojeni and B. neuwiedi venoms. C. sylvestris extract did not induce proteolysis in any substrate assayed.

Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A2.

The crude aqueous extract from the leaves of Casearia sylvestris, a plant found in Brazilian open pastures, was assayed for its ability to inhibit phospholipase A2 (PLA2) activity and some biological activities of bee and several snake venoms, and of a number of isolated PLA2s. The extract induced partial inhibition of the PLA2 activity of venoms containing class I, II and III PLA2s. When tested against the purified toxins, it showed the highest efficacy against class II PLA2s from viperid venoms, being relatively ineffective against the class I PLA2 pseudexin. In addition, C. sylvestris extract significantly inhibited the myotoxic activity of four Bothrops crude venoms and nine purified myotoxic PLA2s, including Lys-49 and Asp-49 variants. The extract was able to inhibit the anticoagulant activity of several isolated PLA2s, with the exception of pseudexin. Moreover, it partially reduced the edema-inducing activity of B. moojeni and B. jararacussu venoms, as well as of myotoxins MjTX-II and BthTX-I. The extract also prolonged the survival time of mice injected with lethal doses of several snake venoms and neutralized the lethal effect induced by several purified PLA2 myotoxins. It is concluded that C. sylvestris constitutes a rich source of PLA2 inhibitors.

The amino acid sequence of ribitol dehydrogenase-F, a mutant enzyme with improved xylitol dehydrogenase activity.

A mutant ribitol dehydrogenase (RDH-F) was purified from Klebsiella aerogenes strain F which evolved from the wild-type strain A under selective pressure to improve growth on xylitol, a poor substrate used as sole carbon source. The ratio of activities on xylitol (500 mM) and ribitol (50 mM) was 0.154 for RDH-F compared to 0.033 for the wild-type (RDH-A) enzyme. The complete amino acid sequence of RDH-F showed the mutations. Q60 for E60 and V215 for L215 in the single polypeptide chain of 249 amino acid residues. Structural modeling based on homologies with two other microbial dehydrogenases suggests that E60 --> Q60 is a neutral mutation, since it lies in a region far from the catalytic site and should not cause structural perturbations. In contrast, L215 --> V215 lies in variable region II and would shift a loop that interacts with the NADH cofactor. Another improved ribitol dehydrogenase, RDH-D, contains an A196 --> P196 mutation that would disrupt a surface alpha-helix in region II. Hence conformational changes in this region appear to be responsible for the improved xylitol specificity.

Purification and partial characterization of a new proteolytic enzyme from the venom of Bothrops moojeni (CAISSACA).

A basic serine protease which is active on casein and fibrinogen was purified from Bothrops moojeni venom using a single step chromatography on a CM-Sepharose fast flow column. The enzyme, MOO3, was not hemorrhagic and presented only a trace of blood-clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by MOO3. Using polyacrylamide gel electrophoresis at pH 4.3, MOO3 showed as a single protein band. Using sodium dodecyl sulfate-polyacrylamide electrophoresis, MOO3 behaved as a single-chain protein with an approximate mol. weight of 27,000, both in the presence and absence of beta-mercaptoethanol. Its pI was 7.8 by electrofocusing. The enzyme did not contain neutral carbohydrates and its N-terminal amino acid was alanine. The amino acid composition showed 249 residues/mole, a high content of hydrophilic amino acids and 14 half-cystine residues, which should account for 7 disulfide bonds. The protease cleaved the A-alpha chain faster than the B-beta of bovine fibrinogen and showed no effect on the delta-chain. Specific esterolytic activity of MOO3 on alpha-N-tosyl-l-arginine methyl ester was 29.64 mumol min-1 x mg-1. MOO3 represented 1.42% (w/w) of the initial desiccated venom. Its proteolytic activity was inhibited by beta-mercaptoethanol, leupeptin, phenylmethylsulphonyl fluoride and ethylenediamine tetraacetate.

A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: biochemical characterization, crystallization, myotoxic and edematogenic activity.

Bothtrops moojeni snake venom was fractionated on a CM-Sepharose column which was previously equilibrated with 0.05 M ammonium bicarbonate buffer at pH 8.0 and subsequently eluted with an ammonium bicarbonate concentration gradient from 0.05 to 0.5 M at constant pH (8.0) and temperature (25 degrees C). The fraction which eluted last (M-VI) showed, after direct lyophilization, a single band by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, indicating an approximate Mr of 14000 and 27000, in the presence and absence of dithiothreitol, respectively. Its amino acid composition revealed a high level of hydrophobic and basic amino acids as well as 14 half-cystine residues. Its isoelectric point and extinction coefficient (E(1.0 mg/ml) (1.0 cm) at 278 nm and pH 7.0) were 8.2 and 1.170, respectively. M-VI was devoid of phospholipase A2 (PLA2) activity on egg yolk, as well as of hemorrhagic, anticoagulant and coagulant activities, but could induce drastic necrosis on skeletal muscle fibres as well as rapid and transient edema on the rat paw. Its N-terminal sequence: SLFELGKMILQETGKNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYK... revealed high homology with other Lys 49 PLA2-like myotoxins from other bothropic venoms. Orthorhombic crystals of M-VI, which diffracted to a maximal resolution of 1.6 A, were obtained and indicated the presence of a dimer in the asymmetrical unit.

Geographic variations in the composition of myotoxins from Bothrops neuwiedi snake venoms: biochemical characterization and biological activity.

(1) Venom pools from Bothrops neuwiedi (Bn) and from two subspecies, namely Bothrops neuwiedi pauloensis (Bnp) and Bothrops neuwiedi urutu (Bnu), collected in the States of São Paulo (SP) and Minas Gerais (MG), Brazil, were electrophoretically examined. Basic toxins with different isoelectric points were identified in the venom collected in São Paulo (BnSP). These toxins were absent in the corresponding pools from Minas Gerais (BnMG, BnpMG and BnuMG). (2) BnSP, but not BnMG, BnpMG or BnuMG, showed two myotoxins (pI approximately equal to 8.6 and 8.8, respectively) which were isolated by ion-exchange chromatography on CM-Sepharose. (3) From BnMG, three myotoxic isoforms (pI approximately equal to 8.2 and M(r) = 13,600) were isolated by chromatography on CM-Sepharose followed by reversed-phase high-performance liquid chromatography. (4) The chemical and biological characterization of these toxins showed a high similarity with the Lys-49 myotoxins from other bothropic venoms. (5) Doses up to 5 LD50 (i.p.) of p-bromophenacyl bromide alkylated BnSP-7 caused a total loss of lethality in 18-22-g mice, thus indicating that the LD50 was increased by greater than 5-fold. At this dose myotoxicity was also not detectable, but the edematogenic activity on the rat paw apparently did not change.

Inhibition of proteases, myotoxins and phospholipases A2 from Bothrops venoms by the heteromeric protein complex of Didelphis albiventris opossum serum.

The antibothropic complex (ABC) from opossum (species Didelphis albiventris) serum was purified by chromatography on DEAE-Sephacel. It showed an acidic character and two polypeptide chains of ca. 45 kDa and 48 kDa, respectively. Lyophilized opossum serum or the ABC (100 micrograms), as well as ethylenediamine tetraacetate (0.25 mumoles) were able to completely neutralise the hemorrhagic effect of 50 micrograms of the desiccated venoms of Bothrops moojeni, Bothrops pirajai and Bothrops jararacussu. The myotoxic (100 micrograms venom in mice) and edematogenic (90 micrograms venom in rats) activities of Bothrops moojeni and Bothrops jararacussu venoms, as well as of the major myonecrotic protein (myotoxin-I) isolated from Bothrops moojeni venom, were also totally inhibited by the ABC (200 micrograms and 270 micrograms, respectively). The lyophilized opossum serum (30 micrograms) and the ABC (30 micrograms) reduced to 50% the phospholipase A2 activity of Bothrops moojeni venom (10 micrograms). The clotting activity of Bothrops alternatus and Bothrops moojeni (20 micrograms) on bovine plasma was also significantly inhibited by the ABC (60 micrograms).

Antagonism of the myotoxic effects of Bothrops jararacussu venom and bothropstoxin by polyanions.

The effects of heparin and other polyanions on the myotoxicity of Bothrops jararacussu venom and purified bothropstoxin (BthTX) were investigated. The release rate of creatine kinase (CK) from isolated extensor digitorum longus muscle and the plasma CK activity of mice were used to quantify the results. The myotoxic effects of B. jararacussu venom or BthTX were inhibited by preincubation of these agents with one of the following: a heterogeneous heparin preparation (designated 'heparin'), low mol. wt heparin (H-4500) or dextran sulfates (DS-8000 and DS-500,000). Non-sulfated dextran (D-40,000) and two chondroitin sulfates were ineffective. The antimyotoxic effects of the polyanions are ascribed to their forming inactive acid-base complexes with the basic myotoxins of Bothrops venoms. Gel-filtration experiments in Sephadex provided direct evidence for complex formation between heparin and BthTX. Intravenous (i.v.) administration of H-4500 or DS-8000 opposed the increase in plasma CK activity induced by a subsequent i.m. injection of venom or BthTX. In contrast, pretreatment with i.v. heparin or DS-500,000 enhanced the venom-induced increase in plasma CK activity. This effect was not observed (1) when the animals were treated with a polyvalent antivenom, which inhibits the coagulation and local stasis induced by Bothrops venoms, and (2) when BthTX, which has no thrombotic or hemorrhagic properties, was the myotoxic agent. The potentiation of the venom-induced increase in plasma CK activity by heparin and DS-500,000 is ascribed to improved washout of the CK released from damaged fibers, because of the anticoagulant properties of the drugs.

Effects induced by bothropstoxin, a component from Bothrops jararacussu snake venom, on mouse and chick muscle preparations.

Bothropstoxin, a 13,700 mol. wt myotoxic phospholipase homologue isolated from the venom of Bothrops jararacussu and devoid of PLA2, proteolytic or hemolytic activities, inhibited muscle twitch tension, evoked either directly or indirectly through stimulation of the motor nerve in the mouse phrenic-diaphragm preparations. The compound action potential of the muscle was also abolished with a similar time course. In addition, the toxin (0.7 mM) evoked membrane depolarization which was inhibited in the presence of 10 mM Ca2+. In chick biventer cervicis muscle, the toxin (2 mM) induced a contracture that reached its maximum amplitude in 44.8 +/- 15.6 min (n = 6) and was not blocked by either d-tubocurarine or tetrodotoxin. The time to maximum amplitude was reduced to 5.5 +/- 1.0 min (n = 4) in nominally Ca(2+)-free Krebs solution and was completely abolished in Ca(2+)-free Krebs solution containing 1 mM EGTA.

Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake Bothrops jararacussu.

A myotoxic phospholipase A2, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as "delta lesions" were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae.

Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin.

A myotoxin, bothropstoxin (BthTX), showing no detectable phospholipase A2 activity, was purified to homogeneity from the venom of the Brazilian snake Bothrops jararacussu by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on SP-Sephadex C-25. Four phospholipases (Sm-SP1 to Sm-SPIV) were also isolated, the latter showing, similarly to BthTX (Sm-SPv) myonecrotic activity. Approximate mol. wts, as determined by SDS-PAGE, and pI of Sm-SPI to Sm-SPIV are: 22,400-4.2; 15,500-4.8; 13,800-6.9; and 13,200-7.7, respectively. BthTX is a single chain protein, approximate mol. wt 13,000, with 16 half-cystine residues, pI = 8.2 and LD50 = 7.5 mg/kg (i.p.) and 4.8 mg/kg (i.v.) for 20 g mice. The ten first N-terminal amino acid residues show a significant homology to other toxins with phospholipase structure. BthTX is specifically myotoxic, contrary to crude B. jararacussu venom which, although also myotoxic, affects intramuscular arteries and veins leading to thrombosis. BthTX and Sm-SPIV also differ from toxins isolated from the venom of other Brazilian snakes which are strongly hemorrhagic.