Carlos R. Diniz

Biological activities of venoms from South American snakes

Standard assay procedures for the characterization of snake venoms, developed by the World Health Organization (WHO) Collaborating Centre for the Control of Antivenoms (CCCA), were used to analyse 33 snake venoms including eight International Reference Venoms for the assessment of lethal, defibrinogenating, procoagulant, haemorrhagic and necrotizing properties. The International Reference Venoms were assayed as part of an International Collaborative Programme for the evaluation of Venoms and Antivenoms; the results showed a close relationship to those obtained by the CCCA. Twenty-five venoms from 13 different species of medically important snakes from South America were assayed as standardized by the WHO-CCCA. Additionally, evaluation of lethal activity by the i.p. and intra cerebroventricular routes, proteolytic effects and venom-induced edema were also determined. Venom yields from captive snakes are also presented. Among the venoms examined, from the subfamily Crotalinae, the members of the genera Bothrops and Lachesis had strong haemorrhagic, proteolytic and edema-inducing activities, whereas all Crotalus durissus species had none. The Elapinae, Micrurus frontalis showed no procoagulant, defibrinogenating, haemorrhagic, necrotizing or proteolytic activities. The results reflect differences among individual samples of the same species and of different geographical regions. The results suggest that there is little or no relationship between the properties of the different venoms and that the determination of one effect cannot predict the value of the others. Therefore, the characterization of the different activities of snake venoms is necessary if toxicity is to be properly evaluated and neutralized.

Purification and amino acid sequences of six Tx3 type neurotoxins from the venom of the Brazilian ‘armed’ spider Phoneutria Nigriventer (keys.)

Six neurotoxic peptides (Tx3-1 to Tx3-6) were purified from the venom of the spider Phoneutria nigriventer by a combination of gel filtration, reverse phase FPLC on PEP-RPC and PRO-RPC columns, reverse phase HPLC on Vydac C18, and ion exchange HPLC on cationic and anionic columns. These toxins caused different neurological symptoms in mice after intracerebroventricular injection. At dose levels of 5 micrograms/mouse, Tx3-3 and Tx3-4 caused rapid general flaccid paralysis followed by death in 10-30 min; Tx3-2 induced immediate clockwise gyration and flaccid paralysis after 6 hr; Tx3-1, Tx3-5 and Tx3-6 produced paralysis only in the posterior limbs and gradual decreases in movement and aggression during 24 hr. The mol. wt of these cystine-rich peptides were found to be in the range of 3500-8500 by mass spectroscopy and SDS-PAGE. The complete amino acid sequences of the neurotoxins Tx3-1 (40 residues), Tx3-2 (34 residues) and Tx3-6 (55 residues), and the N-terminal sequences of Tx3-3 (34 res.), Tx3-4 (40 res.) and Tx3-5 (36 res.) were established by direct automated Edman degradation, and manual DABITC/PITC microsequence analyses of peptides obtained from digests with various proteases. The structures of these Tx3 neurotoxins from Phoneutria nigriventer exhibited sequence similarities to one another and to the neurotoxins from the venoms of the spiders Hololena curta and Agelenopsis aperta, which were most evident in the location of the Cys residues.

The purification and amino acid sequences of four Tx2 neurotoxins from the venom of the Brazilian ‘armed’ spider Phoneutria nigriventer (Keys)

Four neurotoxic polypeptides (Tx2.1, Txt2‐5, Tx2‐6 and Tx2‐9) were purified from the venom of the South American ‘armed’ spider Phoneutria nigriventer (Keys) by gel filtration and reverse phase FPLC and HPLC. These cysteine‐rich polypeptides exhibited different levels of neurotoxicity in mice after intracerebroventricular injection. Tx2‐1, Tx2‐5 and Tx2‐6 caused spastic paralysis and death, but the less toxic Tx2‐9 produced only tail erection and scratching. The molecular weights of the polypeptides as determined by desorption mass spectroscoopy were 5838.8 for Tx2‐1, 5116.6 (Tx2‐5), 5291.3 (Tx2‐6) and 3742.1 (Tx2‐9). The complete amino acid sequences of the neurotoxins were determined by automated Edman degradation and by manual DABITC‐PITC microseqeunce analysis of peptides obtained after digestions with various proteases. The amino acid sequences of Tx2‐1 (53 residues), Tx2‐5 (48 residues) and Tx2‐6 (48 residues) were homologous, but had only limited similarities to the less toxic Tx2‐9 (32 residues). All four polypeptides had varying sequence identities with other neurotoxins from different spider species and biologically active peptides from scorpions, a sea snail and seeds of Mirabilis jalapa.

Isolation of neurotoxic peptides from the venom of the ‘armed’ spider Phoneutria nigriventer

Three neurotoxic fractions, lethal to mice, were isolated from the venom of the spider Phoneutria nigriventer, by gel filtration and reverse phase chromatography (Phoneutria toxins 1, 2 and 3). These toxins have mol. wts in the range 6000-9000, and have different amino acid compositions and N-terminal amino acid sequences. The toxins also differ in the lethality and signs they cause in mice after intracerebro-ventricular injection. The median LD50 being respectively for the whole venom, toxins 1, 2 and 3, 47 +/- 5 micrograms, 45 +/- 4 micrograms, 1.7 +/- 0.7 micrograms and 137 +/- 10 micrograms/kg mouse. Toxins 1 and 2 induce excitatory symptoms in mice and toxin 3 a flaccid paralysis with an ED50 of 40 +/- 5 micrograms/kg mouse as measured also by intracerebro-ventricular injection. The presence in the venom of a non-neurotoxic, smooth muscle active peptide is also described.

EFFECT OF SCORPION VENOM, TITYUSTOXIN, ON THE RELEASE OF ACETYLCHOLINE FROM INCUBATED SLICES OF RAT BRAIN

Abstract— Purified tityustoxin (TsTX) from the venom of the scorpion, Tityus serrulatus, when incubated in vitro with slices of rat cerebral cortex, increased the amount of free ace‐tylcholine (ACh) in the incubation medium and, simultaneously, reduced the amount of bound ACh in the slices. The effect was optimal at pH 7.4 and was dependent upon time of incubation, an energy source and the concentration of toxin. Tityustoxin increased the synthesis of ACh, but this effect seemed to be related to an increase in the release of ACh. The effect of the TsTX was independent of the concentration of K+ ion but was dependent on the presence of Na+ and Ca2+ in the incubation medium. Hexamethonium and hemicholinium reduced the effect of tityustoxin, but cocaine had no effect on the release of ACh stimulated by the TsTX. Tetrodotoxin blocked completely the stimulation caused by the tityustoxin. We suggest that probably both tityustoxin and tetrodotoxin exert different and antagonistic effects competing in the Na+ channels.

Action of metalloproteinases mutalysin I and II on several components of the hemostatic and fibrinolytic systems.

The zinc endopeptidases mutalysin I (100 kDa) and mutalysin II (22.5 kDa) have been previously isolated from bushmaster (Lachesis muta muta) snake venom. Hemorrhagic activity was observed with as little as 0.5 microg (2000 units/mg) and 17.8 microg (56.2 units/mg) for mutalysin I and II, respectively. Additionally, the proteases hydrolyse the Aalpha>Bbeta chain of fibrinogen without clot formation. The specific fibrinogenolytic activity was estimated as 5. 25 and 16.3 micromol fibrinogen/min/micromol protein for mutalysin I and II, respectively. In vitro, the enzymes act directly on fibrin and are not inhibited by serine proteinase inhibitors (SERPINS). Analysis by SDS-PAGE of fibrin hydrolysis by both enzymes showed that mutalysin II (0.22 microM) completely digested the alpha- and gamma-gamma chains and partially the beta-chain (in 120 min incubation). In contrast, mutalysin I (three fold higher concentration than mutalysin II) hydrolyzed selectively the alpha-chain of fibrin leaving the beta and gamma-gamma chains unaffected. Unlike with the plasminogen activator-based thrombolytic agents (e.g., streptokinase), mutalysins do not activate plasminogen. Neither enzyme had an effect on protein C activation. Mutalysin II does not inhibit platelet aggregation in human PRP induced by collagen or ADP. However, mutalysin I showed a selective inhibitory effect on collagen-induced aggregation of human PRP; it did not affect platelet aggregation with ADP as the agonist. The present investigation demonstrates that both native and EDTA-inactivated mutalysin I dose dependently blocked aggregation of human PRP elicited by 10 microg/mL of collagen with an IC(50) of 180 and 580 nM, respectively. These studies suggest that, in addition to the metalloprotease region of mutalysin I, the disintegrin-like domain also participates in the inhibitory effect. The proteolytic activity of mutalysin II against dimethylcasein and fibrin was completely abolished by alpha2-macroglobulin (alpha2-M). The stoichiometry of inhibition was 1.0 mol of enzyme per mol of alpha2-M. In contrast, the proteolytic effect of mutalysin I against the same substrates was not significantly inhibited by alpha2-M. Therefore, the data explain why mutalysin I contributes significantly not only to local but also to systemic bleeding associated with the observed pathological effects of the venom.

Effects of a toxic fraction, PhTx2, from the spider Phoneutria nigriventer on the sodium current.

SummaryThe toxic fraction PhTx2 of the spider Phoneutria nigriventer was studied with a modified loose patch clamp technique on frog skeletal muscle. At saturating concentration (8 μg/ml) potassium currents were unaffected whereas there was a 7-fold increase in the time constant of sodium current inactivation (at −13 mV test potential). The time course of tail current deactivation was at least 3-fold slower than the control. The steady state (100 ms) inactivation and the conductance activation were shifted toward more negative potentials by 12.2 and 7.0 mV, respectively. The reversal of the sodium current was shifted 7.6 mV to more negative potential. We conclude that PhTx2 prolongs the inactivation and deactivation processes of sodium ion channels. These effects may account for the toxicity of PhTx2.

Amino acid sequence of toxin VII, A β-toxin from the venom of the scorpton Tityus serrulatus

The sequence of the 61 amino acids of toxin VII, a β-toxin from the venom of the South American scorpion Tityus serrulatus, has been determined by automatic sequencing of the reduced and S-[14C] car☐ymethylated protein and of tryptic peptides obtained before or after citraconylation of this protein. This toxin, the most active β-toxin from this venom, is the first Tityus toxin to be fully sequenced. The results clearly show that toxin VII belongs to the structural group of scorpion toxins originating from Central and North America.

In vivo protection against scorpion toxins by liposomal immunization

The possibility of raising a humoral immune response able to induce protection from the lethal effects of scorpion toxins was evaluated in the mouse model. A toxic fraction from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes which yielded a conveniently detoxified immunogen. After three injections of this immunogen, all but three of a group of 18 mice developed an IgG response which was shown to be both specific and of good affinity for the toxic antigen. In vitro neutralization assays indicated that pre-incubation of a lethal dose of the toxic fraction with immune sera strongly diminished its toxicity. In vivo protection assays showed that mice with the highest levels of circulating anti-toxin antibodies could resist the challenge by double the normal LD50 of the toxic fraction, which killed all control non-immune mice. The protection was, however, found to be limited both in its duration and its effectiveness against higher amounts of toxin.

Phoneutria nigriventer toxin Tx3-1 blocks A-type K+ currents controlling Ca2+ oscillation frequency in GH3 cells.

Abstract: GH3 cells present spontaneous Ca2+ action potentials and oscillations of intracellular Ca2+, which can be modified by altering the activity of K+ or Ca2+ channels. We took advantage of this spontaneous activity to screen for effects of a purified toxin (Tx3‐1) from the venom of Phoneutria nigriventer on ion channels. We report that Tx3‐1 increases the frequency of Ca2+ oscillations, as do two blockers of potassium channels, 4‐aminopyridine and charybdotoxin. Whole‐cell patch clamp experiments show that Tx3‐1 reversibly inhibits the A‐type K+ current (IA) but does not block other K+ currents (delayed‐rectifying, inward‐rectifying, and large‐conductance Ca2+‐sensitive) or Ca2+ channels (T and L type) in these cells. In addition, we describe the sequence of a full cDNA clone of Tx3‐1, which shows that Tx3‐1 has no homology to other known blockers of K+ channels and gives insights into the processing of this neurotoxin. We conclude that Tx3‐1 is a selective inhibitor of IA, which can be used to probe the role of this channel in the control of cellular function. Based on the effect of Tx3‐1, we suggest that IA is an important determinant of the frequency of Ca2+ oscillations in unstimulated GH3 cells.