Elastase-2 Knockout Mice Display Anxiogenic- and Antidepressant-Like Phenotype: Putative Role for BDNF Metabolism in Prefrontal Cortex.

Several pieces of evidence indicate that elastase-2 (ELA2; chymotrypsin-like ELA2) is an alternative pathway to the generation of angiotensin II (ANGII). Elastase-2 knockout mice (ELA2KO) exhibit alterations in the arterial blood pressure and heart rate. However, there is no data on the behavioral consequences of ELA2 deletion. In this study, we addressed this question, submitting ELA2KO and wild-type (WT) mice to several models sensitive to anxiety- and depression-like, memory, and repetitive behaviors. Our data indicates a higher incidence of barbering behavior in ELA2KO compared to WT, as well as an anxiogenic phenotype, evaluated in the elevated plus maze (EPM). While a decrease in locomotor activity was observed in ELA2KO in EPM, this feature was not the main source of variation in the other parameters analyzed. The marble-burying test (MBT) indicated increase in repetitive behavior, observed by a higher number of buried marbles. The actimeter test indicated a decrease in total activity and confirmed the increase in repetitive behavior. The spatial memory was tested by repeated exposure to the actimeter in a 24-h interval. Both ELA2KO and WT exhibited decreased activity compared to the first exposure, without any distinction between the genotypes. However, when submitted to the cued fear conditioning, ELA2KO displayed lower levels of freezing behavior in the extinction session when compared to WT, but no difference was observed during the conditioning phase. Increased levels of BDNF were found in the prefrontal cortex but not in the hippocampus of ELA2KO mice compared to WT. Finally, in silico analysis indicates that ELA2 is putatively able to cleave BDNF, and incubation of the purified enzyme with BDNF led to the degradation of the latter. Our data suggested an anxiogenic- and antidepressant-like phenotype of ELA2KO, possibly associated with increased levels of BDNF in the prefrontal cortex.

Purification and characterization of the hemorrhagic factor II from the venom of the Bushmaster snake (Lachesis muta muta).

Hemorrhagic factor II (LHF-II) was isolated from Lachesis muta muta (Bushmaster snake) venom using column chromatographies on Sephadex G-100, CM-Sepharose CL-6B and two cycles on Sephadex G-50. This preparation was devoid of phospholipase A2 as well as of the enzymes active on arginine synthetic substrates (TAME and BAPNA) which are present in the crude venom. LHF-II was homogeneous by SDS-polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. Also, a single symmetrical boundary with a value of 2.59 S was obtained by ultracentrifugation. LHF-II contains 180 amino acid residues, has a molecular weight of 22,300, and an isoelectric point of 6.6. It contains one gatom zinc and two gatoms calcium per mol protein. The hemorrhagic factor possesses proteolytic activity toward various substrates such as, casein, dimethylcasein, hide powder azure, fibrinogen and fibrin. It hydrolyzes selectively the A alpha-chain of fibrinogen, leaving the B beta- and gamma-chains unaffected. LHF-II is activated by Ca2+ and inhibited by Zn2+. The hemorrhagic as well as the proteinase activity is inhibited by cysteine and by metal chelators such as EDTA, EGTA and 1,10-phenanthroline. Inhibitors of serine proteinases such as phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI) have no effect on the hemorrhagic factor.

The complete amino acid sequence of the haemorrhagic factor LHFII, a metalloproteinase isolated from the venom of the bushmaster snake (Lachesis muta muta).

The complete amino acid sequence the haemorrhagic agent LHFII, a Zn and Ca containing metalloproteinase isolated from the venom of the Bushmaster snake Lachesis muta muta was determined by automated and DABITC/PITC microsequencing of the intact protein, fragments derived by cleavage with cyanogen bromide, and peptides resulting from enzymatic digestions with trypsin and the protease from S. aureus V8. The protein is composed of 200 residues and exhibits considerable sequence homology with the haemorrhagic toxins from a number of other snake venoms, and some metalloproteinases in the region of the putative Zn-binding sites.

The complete amino acid sequence of a thrombin-like enzyme/gyroxin analogue from venom of the bushmaster snake (Lachesis muta muta).

The complete amino acid sequence of a thrombin-like enzyme with gyroxin activity isolated from the venom of the bushmaster snake Lachesis muta muta was determined by automated and DABITC/PITC microsequencing of the intact protein; fragments derived from it by separate cleavages with cyanogen bromide, iodosobenzoic acid and hydroxylamine; and peptides resulting from enzymatic digestions with trypsin, pepsin, chymotrypsin, and elastase. The protein, which is composed of 228 residues, contains four putative sites of N-linked glycosylation and exhibits significant sequence similarities with other serine proteases reported from snake venoms.

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 microsequence analysis of peptides obtained after digestions with various proteases. The amino acid sequences of Tx2-1 (53 residues), Tx2-5 (49 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.

The purification and amino acid sequence of the lethal neurotoxin Tx1 from the venom of the Brazilian 'armed' spider Phoneutria nigriventer.

A lethal neurotoxic polypeptide of Mr 8 kDa was purified from the venom of the South American 'armed' or wandering spider Phoneutria nigriventer by centrifugation, gel filtration on Superose 12, and reverse phase FPLC on columns of Pharmacia PepRPC and ProRPC. The purified neurotoxin Tx1 had an LD50 of 0.05 mg/kg in mice following intracerebroventricular injection. The complete amino acid sequence of the neurotoxin was determined by automated Edman degradation of the native and S-carboxymethylated protein in pulsed liquid and dual phase sequencers, and by the manual DABITC/PITC double coupling method applied to fragments obtained after digestions with the S. aureus V8 protease and trypsin. The neurotoxin Tx1 consists of a single chain of 77 amino acid residues, which contains a high proportion of cysteine. The primary structure showed no homology to other identified spider toxins.

Molecular cloning and nucleotide sequence analysis of a cDNA encoding the main beta-neurotoxin from the venom of the South American scorpion Tityus serrulatus.

A cDNA encoding the main Tityus serrulatus beta-neurotoxin was isolated from a venom gland cDNA library by using an oligonucleotide probe. The amino acid sequence deduced from the cDNA nucleotide sequence indicated that the toxin is the processed product of a precursor containing: (i) a signal peptide of 20 residues; (ii) the amino acid sequence of the mature toxin; and (iii) an extra Gly-Lys-Lys tail at the C-terminal end before the termination codon. Thus, in addition to the removal of the signal peptide by a signal peptidase, the generation of the mature toxin requires both a post-translational cleavage by a carboxypeptidase specific for basic residues and the action of an alpha-amidating enzyme. These results also show that the biosynthetic pathway for beta-toxins of 'New World' scorpion venoms is similar to that already described for alpha-toxins of 'Old World' scorpion venoms.

Biochemical, pharmacological and genomic characterisation of Ts IV, an alpha-toxin from the venom of the South American scorpion Tityus serrulatus.

The venom of the scorpion, Tityus serrulatus, was fractionated to investigate the chemical and pharmacological properties of its alpha-toxin content. Three alpha-toxins (Ts III, Ts IV and Ts V) were purified by conventional chromatography (gel filtration and ion-exchange chromatography), followed by immunoaffinity chromatography. Competition experiments using reference alpha- and beta-toxins suggested that these alpha-toxins were contaminated with around 0.01% of beta-toxin. The sequence of the first 30 amino acids of Ts IV was established. Using an oligonucleotide probe, a cDNA encoding its precursor was cloned from a venom gland cDNA library. The primary structure deduced from the cDNA nucleotide sequence provides possible explanations for the polymorphism of these three molecules.

The effect of crotoxin on the longitudinal muscle-myenteric plexus preparation of the guinea pig ileum.

The effects of crotoxin, the neurotoxin of the venom of the South American rattlesnake (Crotalus durissus terrificus), was studied by using the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. Crotoxin (0.02-4.0 microM) caused depression of the twitch response of the electrically stimulated preparation. This transitory depression depended on the concentration of crotoxin; since crotoxin diminished the output of acetylcholine, this depression may be due to the inhibition of the release of acetylcholine from the plexus. Crotoxin also induced an early contraction, followed by relaxation; as the contraction was inhibited by aspirin and indomethacin, it may have resulted from the release of prostaglandin. In addition, a late persistent contracture was observed after the early contraction. The contracture was resistant to blockage by muscarinic, histamine and serotonin antagonists, to hexamethonium, a non-depolarizing ganglionic blocking substance and to tetrodotoxin, a sodium channel blocker. The contracture was blocked by an elevated concentration of calcium (10 mM) and by verapamil, a calcium channel blocker.

Inhibition of neuronal high-voltage activated calcium channels by the omega-phoneutria nigriventer Tx3-3 peptide toxin.

We have investigated the effect of omega-PnTx3-3 (referred to in previous papers simply as Tx3-3), a peptide toxin from the venom of the spider Phoneutria nigriventer, on neuronal high-voltage activated (HVA) Ca(2+) channels, using whole-cell patch-clamp. omega-PnTx3-3 (120 nM) blocked 74+/-8% of the total HVA Ca(2+) currents of cerebellar granule neurones, without affecting the low-voltage activated (LVA) current. P/Q/R-type currents in cerebellar granule neurones, isolated using 4 microM nicardipine and 100 nM omega-conotoxin GVIA, were markedly (79+/-6%) inhibited by 60 nM omega-PnTx3-3. R-type currents, isolated either by additional application of 0.5-1 microM of omega-agatoxin IVA or by pre-incubation with 5 microM omega-conotoxin MVIIC were inhibited almost totally by 120 nM of omega-PnTx3-3. omega-PnTx3-3 reversibly altered the kinetics of the P/Q/R current, increasing the degree of inactivation that occurred during a 50 ms pulse from 20% to 40%. N-type currents, recorded from neuroblastoma N18 cells, were partially (34+/-2%) inhibited by 320 nM omega-PnTx3-3. L-type currents, recorded from GH3 cells, were partially (45+/-12%) inhibited by 80 nM omega-PnTx3-3. We conclude that omega-PnTx3-3 inhibits all known HVA Ca(2+) channels, and most effectively the P/Q- and R-type currents.

A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis.

1. The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. 2. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. 3. Using omega-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by omega-agatoxin IVA, an antagonist of P/Q calcium channels. Omega-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. 4. It is concluded that Tx3-3 potently inhibits omega-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

Phoneutria nigriventer toxins block tityustoxin-induced calcium influx in synaptosomes.

Neurotoxins can help the understanding of mechanisms involved in neurotransmission. We here report that two neurotoxin isoforms, Tx3-3 and Tx3-4 obtained from the venom of the spider Phoneutria nigriventer inhibited the 45Ca2+ influx in rat cortical synaptosomes induced by the scorpion venom tityustoxin. The IC50 for Tx3-3 and Tx3-4 were 0.32 and 7.9 nM, respectively. The neurotoxins Tx3-3 and Tx3-4 are very effective in inhibiting 45Ca2+ influx and they should be useful in studies involving Ca(2+)-dependent processes.

Inhibition of glutamate uptake by Tx3-4 is dependent on the redox state of cysteine residues.

Glutamate transporters are essential for the homeostasis of glutamate and normal function of glutamatergic synapses. Their function was shown to be regulated by redox agents and dimerizations that involves redox changes of cysteine residues. Peptide neurotoxins are also known to be rich in cysteine residues that contribute to their activity and stability. Among them is the toxin Tx3-4, from the spider Phoneutria nigriventer, which is able to inhibit glutamate uptake in rat hippocampal synaptosomes. Based on results obtained with manipulation of the redox state of cysteine residues in synaptosomes and in Tx3-4, we suggest that the effect of this toxin on glutamate uptake is due to interactions that involve cysteines both in the toxin and in the transporters.

Efficacy of antivenom therapy for neutralizing circulating venom antigens in patients stung by Tityus serrulatus scorpions.

Enzyme-linked immunosorbent assays for detection of Tityus serrulatus venom antigen and of horse anti-T. serrulatus venom antibodies were carried out before antivenom treatment and at 1, 6, 12, and 24 hr after antivenom therapy in 18 patients with systemic manifestations following T. serrulatus scorpion sting. Increased levels of circulating venom antigens were detected in the patients before antivenom treatment, but were no longer detected 1 hr after specific antivenom therapy. High titers of antivenom persisted for at least 24 hr after treatment with antivenom. The evolution of clinical and laboratory manifestations of envenoming showed that vomiting and local pain decreased within 1 hr and hyperglycemia was no longer detected 12 hr after antivenom therapy. The cardiorespiratory manifestations disappeared 6-24 hr after the administration of antivenom and all patients recovered completely. This study demonstrates the efficacy of antivenom therapy in neutralizing circulating venom antigens and supports the prompt administration of a potent antivenom to patients with systemic manifestations of envenoming.

PhTx4, a new class of toxins from Phoneutria nigriventer spider venom, inhibits the glutamate uptake in rat brain synaptosomes.

We report the characterization of a new class of glutamate uptake inhibitors isolated from Phoneutria nigriventer venom. Glutamate transport activity was assayed in rat cerebrocortical synaptosomes by using [(3)H]-L-glutamate. PhTx4 inhibited glutamate uptake in a dose dependent manner. The IC(50) value obtained was 2.35+/-0.9 microg/ml which is in the observed range reported for glutamate uptake blockers. Tx4-7, one of PhTx4 toxins, showed the strongest inhibitory activity (50.3+/-0.69%, n=3).

Acetylcholine release from rat brain cortical slices evoked by the fraction P4 of the venom of the spider Phoneutria nigriventer has Ca2+ and temperature independent components.

Fractionation of the venom of the spider Phoneutria nigriventer revealed that it was a mixture of several neurotoxic peptides. The peptides so far characterized either inhibited or induced neurotransmitter release. These effects were mediated by Ca2+ channels or increasing Na+ permeability through voltage sensitive Na(+)-channels, respectively. The pooled toxic components (fraction P4) showed stimulatory effects on acetylcholine release from brain cortical slices. In addition, a component of the observed effects resembling that of alpha-latrotoxin was identified, which was characterized by the ability to provoke release of acetylcholine (ACh) at low temperature and in a manner independent of extracellular Ca2+ and of voltage sensitive Na(+)-channels.

The effect of PhTx3 on the release of 3H-acetylcholine induced by tityustoxin and potassium in brain cortical slices and myenteric plexus.

The venom of the Brazilian spider Phoneutria nigriventer possesses several neurotoxic polypeptidic fractions. Previous work has established that one of the toxic components, PhTx3, inhibited Ca(2+)-dependent glutamate release and the increase in cytosolic free Ca2+ in response to membrane depolarization. In the present work, we investigated the effect of PhTx3 on the release of acetylcholine (ACh) from brain and peripheral neurons. PhTx3 decreased the release of [3H]-ACh induced by tityustoxin and KCl in brain cortical slices and myenteric plexus. The inhibitory effect of myenteric plexus had the same magnitude as that obtained in the absence of extracellular Ca2+. However, in brain PhTx3 was less efficient at decreasing the evoked release of ACh. These experiments suggest that the target of PhTx3 is coupled to the process of release of ACh in brain and autonomic nervous system.

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.

Snake bite and antivenom complications in Belo Horizonte, Brazil.

Factors associated with clinical complications of snake bite and antivenom therapy were studied in 310 hospital patients admitted with snake bite over 6 years to a tertiary referral hospital in Belo Horizonte, southeast Brazil. Overall, 17.4% had early clinical complications including tissue loss associated with abscess and necrosis, acute renal failure, shock, acute lung oedema and intracranial haemorrhage. 3% had permanent sequelae, caused by muscle contractures and amputations, chronic renal failure, or death. Early complications were associated with the following: age under 9 years (P = 0.04), residence in a rural area (P = 0.04), and a delay of more than 8 h in seeking clinical care (P < 0.01). Antivenom was administered to 98.1% of patients; 13.8% presented with anaphylaxis and 11.8% with pyrexia. Individuals from a rural area had a higher occurrence of anaphylactic reactions (P = 0.03). Neither anaphylaxis nor pyrexia was linked with antivenom type and dosage. This study suggested that antivenom might be associated with a reduced risk of serious injuries related to snake bite, especially when administered within the first 8 h. Complications appeared to be a far greater risk than adverse reactions to the antivenom.

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

The 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 micrograms/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.