Publications by authors named "Léa Rodrigues-Simioni"

62 Publications

and plant extracts against the local effects of and snake venoms.

J Venom Res 2020 20;10:32-37. Epub 2020 Jul 20.

Laboratory of Pharmacology, Department of Physiological Sciences, Pontifical University Catholic of São Paulo (PUC/SP). Rua Joubert Wey, 290, Vila Boa Vista, 18030-070, Sorocaba, SP, Brazil.

produces similar local effects to snakebite, which can induce misidentification and bothropic antivenom administration. Antivenom therapy is effective, but has its limitations regarding local damage. Since plants are used in folk medicine to treat snakebite victims, we evaluated the protective properties of and extracts against and venoms. Preparations pretreated with both extracts inhibited > 90% the venom-induced neuromuscular blockade, and 52% to 81% the venom-induced blockade. inhibited the myonecrosis promoted by both venoms; however, prevented only the myofilaments hypercontraction. Regarding haemorrhagic activity, was more effective against venom, while was more effective against venom. On the other hand, for oedema-forming activity the results were the opposite. Considering that both extracts prevented (to different levels) the main manifestations of both snakebites (local symptoms), we endorse further studies involving these plants as coadjuvant in snakebite therapeutics.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7512476PMC
July 2020

Inhibition of Kv2.1 Potassium Channels by MiDCA1, A Pre-Synaptically Active PLA-Type Toxin from Coral Snake Venom.

Toxins (Basel) 2019 06 12;11(6). Epub 2019 Jun 12.

Interdisciplinary Centre for Research in Biotechnology (CIPBiotec), Federal University of Pampa (UNIPAMPA), Campus São Gabriel, São Gabriel 97300-000, RS, Brazil.

MiDCA1, a phospholipase A (PLA) neurotoxin isolated from coral snake venom, inhibited a major component of voltage-activated potassium (Kv) currents (41 ± 3% inhibition with 1 μM toxin) in mouse cultured dorsal root ganglion (DRG) neurons. In addition, the selective Kv2.1 channel blocker guangxitoxin (GxTx-1E) and MiDCA1 competitively inhibited the outward potassium current in DRG neurons. MiDCA1 (1 µM) reversibly inhibited the Kv2.1 current by 55 ± 8.9% in a oocyte heterologous system. The toxin showed selectivity for Kv2.1 channels over all the other Kv channels tested in this study. We propose that Kv2.1 channel blockade by MiDCA1 underlies the toxin's action on acetylcholine release at mammalian neuromuscular junctions.
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http://dx.doi.org/10.3390/toxins11060335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628393PMC
June 2019

Presynaptic Activity of an Isolated Fraction from Rhinella schneideri Poison.

Adv Pharm Bull 2018 Aug 29;8(3):517-522. Epub 2018 Aug 29.

School of Medicine Faculdade Israelita de Ciências da Saúde Albert Einstein, São Paulo, SP, Brazil.

Rhinella schneideri is a toad found in many regions of the South America. The poison of the glands has cardiotoxic effect in animals and neuromuscular effects in mice and avian preparation. The purpose of this work was to identify the toxin responsible for the neuromuscular effect in avian and mice neuromuscular preparation. The methanolic extract from R. schneideri poison was fractioned by reversed phase HPLC. The purity and molecular mass were determined by LC/MS mass spectrometry. Chick biventer cervicis and mouse phrenic-nerve diaphragm were used as neuromuscular preparations to identify the toxin. The purification resulted in 32 fractions, which 4 of them were active in neuromuscular preparation. The toxin of fraction 20 were chosen for better reproducibility of the whole extract activity and its molecular mass was 730.6 Da. The toxin produced facilitation of the muscle contraction followed by a complete neuromuscular blockade in chick biventer cervicis preparation in 90 min without interfering with the exogenous response to ACh and KCl. The quantal content was increased from 128 ± 13 (control) to 216 ± 44 (after 5 min and sustained until 60 min) in the presence of the toxin. In conclusion, our results demonstrated that the neuromuscular action of the poison of Rhinella schneideri is a multitoxin effect. More, the present work first isolated a 730.6 Da toxin that better represent the whole poison neuromuscular effect, to which is attributed a presynaptic action in avian and mouse neuromuscular preparation.
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http://dx.doi.org/10.15171/apb.2018.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156484PMC
August 2018

A survey on some biochemical and pharmacological activities of venom from two Colombian colubrid snakes, Erythrolamprus bizona (Double-banded coral snake mimic) and Pseudoboa neuwiedii (Neuwied's false boa).

Toxicon 2017 Jun 9;131:29-36. Epub 2017 Mar 9.

Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 255, Cidade Universitária Zeferino Vaz, 13083-862 Campinas, SP, Brazil. Electronic address:

Colombian colubrid snake venoms have been poorly studied. They represent a great resource of biological, ecological, toxinological and pharmacological research. We assessed some enzymatic properties and neuromuscular effects of Erythrolamprus bizona and Pseudoboa neuwiedii venoms from Colombia. Proteolytic, amidolytic and phospholipase A (PLA) activities were analyzed using colorimetric assays and the neuromuscular activity was analyzed in chick biventer cervicis (BC) preparations. The venom of both species showed very low PLA and amidolytic activities; however, both exhibited high proteolytic activity, which in E. bizona venom surpassed that of P. neuwiedii venom. E. bizona and P. neuwiedii venoms provoked partial neuromuscular blockade, which was more prominent in P. neuwiedii venom. E. bizona venom (30 μg/ml) induced a significant potentiation of the contracture response to exogenous ACh (110 μM), which was not accompanied by twitch height alteration, whereas the highest venom concentration (100 μg/ml) inhibited contracture responses to both ACh and KCl (40 mM). In contrast, P. neuwiedii venom (30 and 100 μg/ml) caused significant reduction in the contracture responses to exogenous ACh and KCl. The morphological analyses showed high myotoxic effects in the muscle fibers of BC incubated with either venoms; however, they are more prominent in the P. neuwiedii venom. Our results suggest that the myotoxicity of the venom of the two Colombian species can be ascribed to their high proteolytic activity. An interesting data was the potentiation of the ACh-induced contracture, but not the twitch height, caused by E. bizona venom, at a concentration that is harmless to muscle fibers integrity. This phenomenon remains to be further elucidated, and suggest that a possible involvement of post-synaptic receptors cannot be discarded. This work is a contribution to expand the knowledge on colubrid venoms; it allows envisaging that the two venoms offer the potential to go further in the identification of their components and biological targets.
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http://dx.doi.org/10.1016/j.toxicon.2017.02.030DOI Listing
June 2017

Erratum to "Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic antivenom" [Comp. Biochem. Physiol. C 191 (2017) 86-100].

Comp Biochem Physiol C Toxicol Pharmacol 2017 06 6;196:53. Epub 2017 Mar 6.

Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, 13083-887 Campinas, SP, Brazil.

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http://dx.doi.org/10.1016/j.cbpc.2017.03.001DOI Listing
June 2017

Influence of phospholipasic inhibition on neuromuscular activity of Bothrops fonsecai snake venom.

Toxicon 2017 May 27;130:35-43. Epub 2017 Feb 27.

Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, Cidade Universitária Zeferino Vaz, 13083-887 Campinas, SP, Brazil.

Bothrops fonsecai (B. fonsecai), a pitviper endemic to southeastern Brazil, has a venom mainly composed by snake venom phospholipases (PLA) and metalloproteases, compounds that could interfere with neuromuscular junction in vitro. In this work, we investigated the role of PLA in the myotoxicity and neuromuscular blockade caused by B. fonsecai venom using different procedures frequently associated with PLA activity inhibition: 24 °C bath temperature, Ca - Sr replacement and chemical modification with p-bromophenacyl bromide (p-BPB). Mice extensor digitorum longus preparations (EDL) were incubated with usual or modified Tyrode solution (prepared with Ca or Sr respectively) at 24 °C or 37 °C (as controls) and in addition of B. fonsecai venom (100 μg/mL) alone or after its incubation with buffer (24 h, 23 °C) on the absence (alkylation control) and presence of p-BPB; all muscle were processed for histological analysis. The PLA, proteolytic and amidolytic activities under the same conditions (24 °C or 37 °C, Ca - Sr replacement, absence or presence p-BPB) were also assessed. The B. fonsecai venom caused total neuromuscular blockade after 100 min of incubation, in Ca Tyrode solution at 37 °C (usual conditions); on Sr Tyrode solution (37 °C) the twitch height were 31.7 ± 7.4% of basal, and at 24 °C (Ca Tyrode solution) were 53.6 ± 7.0% of basal. The alkylation of PLA with p-BPB promoted a great blockade decrease at 100 min of incubation (88.7 ± 5.7% of basal), but it was also observed on alkylation control preparations (66.2 ± 6.6%). The venom produced 50% of blockade at 40.5 ± 5.9 min, in Ca Tyrode solution at 37 °C. The protocols delayed the time for 50% blockade: 105.7 ± 7.1 min (at 24 °C, in Ca Tyrode solution) and 71.1 ± 9.0 min (at 37 °C, in Sr Tyrode solution). Regarding p-BPB incubation and alkylation control preparations, 50% of blockade was not reached during the 120 min of venom incubation. Regarding to enzymatic activities, the 24 °C protocol reduced not only PLA (to 62.3%) but also proteolytic (52.3%) and amidolytic (73.4%) activities, as well as observed on p-BPB alkylation protocol which markedly inhibited all enzymes (<10%). The alkylation control promoted the same proteolytic and amidolytic inhibition but no reduction of PLA activity; Ca - Sr replacement reduced only the PLA activity (to 15.3%). We observed a strict relation between the inhibition of PLA activity and the myotoxicity. On the other hand, this relation was not observed with neuromuscular blockade, suggesting that blockade and muscle damage may not be strictly related. It suggests that the neuromuscular blockade may be induced by non-catalytic PLA or other venom components, such as metalloproteinases.
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http://dx.doi.org/10.1016/j.toxicon.2017.02.027DOI Listing
May 2017

Ultrastructural aspects of mouse nerve-muscle preparation exposed to Bothrops jararacussu and Bothrops bilineatus venoms and their toxins BthTX-I and Bbil-TX: Unknown myotoxic effects.

Microsc Res Tech 2016 Nov 18;79(11):1082-1089. Epub 2016 Aug 18.

Multidisciplinary Research Laboratory, São Francisco University (USF), Av. São Francisco de Assis, 218, Zip Code 12916-900, Bragança Paulista, SP, Brazil.

Bites by Bothrops snakes normally induce local pain, haemorrhage, oedema and myonecrosis. Mammalian isolated nerve-muscle preparations exposed to Bothrops venoms and their phospholipase A toxins (PLA ) can exhibit a neurotoxic pattern as increase in frequency of miniature end-plate potentials (MEPPs) as well as in amplitude of end-plate potentials (EPPs); neuromuscular facilitation followed by complete and irreversible blockade without morphological evidence for muscle damage. In this work, we analysed the ultrastructural damage induced by Bothrops jararacussu and Bothrops bilineatus venoms and their PLA toxins (BthTX-I and Bbil-TX) in mouse isolated nerve-phrenic diaphragm preparations (PND). Under transmission electron microscopy (TEM), PND preparations previously exposed to B. jararacussu and B. bilineatus venoms and BthTX-I and Bbil-TX toxins showed hypercontracted and loosed myofilaments; unorganized sarcomeres; clusters of edematous sarcoplasmic reticulum and mitochondria; abnormal chromatin distribution or apoptotic-like nuclei. The principal affected organelles, mitochondria and sarcoplasmic reticulum, were those related to calcium buffering and, resulting in sarcomeres and myofilaments hypercontraction. Schwann cells were also damaged showing edematous axons and mitochondria as well as myelin sheath alteration. These ultrastructural changes caused by both of Bothrops venoms and toxins indicate that the neuromuscular blockade induced by them in vitro can also be associated with nerve and muscle degeneration.
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http://dx.doi.org/10.1002/jemt.22748DOI Listing
November 2016

Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic venom.

Comp Biochem Physiol C Toxicol Pharmacol 2017 Jan 30;191:86-100. Epub 2016 Aug 30.

Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, 13083-887, Campinas, SP, Brazil.

In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA, proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2μg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30μg/site) and significantly inhibited by both ratios. Venom (10-300μg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai.
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http://dx.doi.org/10.1016/j.cbpc.2016.08.008DOI Listing
January 2017

Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins from Bothrops marajoensis (Marajó Lancehead) Snake Venom.

Biochem Res Int 2016 18;2016:2053459. Epub 2016 Aug 18.

Multidisciplinary Research Laboratory, São Francisco University (USF), Avenida São Francisco de Assis 218, Jardim São José, 12916-350 Bragança Paulista, SP, Brazil; Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 255, Cidade Universitária Zeferino Vaz, 13083-365 Campinas, SP, Brazil.

Neuromuscular preparations exposed to B. marajoensis venom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposed in vitro to BmjeTX-I and BmjeTX-II toxins from B. marajoensis venom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.
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http://dx.doi.org/10.1155/2016/2053459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007310PMC
September 2016

Functional and structural studies of a Phospholipase A-like protein complexed to zinc ions: Insights on its myotoxicity and inhibition mechanism.

Biochim Biophys Acta Gen Subj 2017 Jan 13;1861(1 Pt A):3199-3209. Epub 2016 Aug 13.

Departamento de Física e Biofísica, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Rua Prof. Dr. Antonio Celso Wagner Zanin, s/n, 18618-689 Botucatu, SP, Brazil. Electronic address:

Background: One of the main challenges in snakebite envenomation treatment is the development of stable, versatile and efficient anti-venom therapies. Local myotoxicity in accidents involving snakes from the Bothrops genus is still a consequence of serum therapy inefficient neutralization that may lead to permanent sequelae in their victims. One of the classes of toxins that participate in muscle necrosis is the PLA-like proteins. The aim of this work was to investigate the role of zinc ions in the inhibition of PLA-like proteins and to advance the current knowledge of their action mechanism.

Methods: Myographic and electrophysiological techniques were used to evaluate the inhibitory effect of zinc ions, isothermal titration calorimetry assays were used to measure the affinity between zinc ions and the toxin and X-ray crystallography was used to reveal details of this interaction.

Results: We demonstrated that zinc ions can effectively inhibit the toxin by the interaction with two different sites, which are related to two different mechanism of inhibition: preventing membrane disruption and impairing the toxin state transition. Furthermore, structural study presented here included an additional step in the current myotoxic mechanism improving the comprehension of the allosteric transition that PLA-like proteins undergo to exert their function.

Conclusions: Our findings show that zinc ions are inhibitors of PLA-like proteins and suggest two different mechanisms of inhibition for these ions.

General Significance: Zinc is a new candidate that can assist in anti-venom treatments and can promote the design of new and even more accurate structure-based inhibitors for PLA-like proteins.
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http://dx.doi.org/10.1016/j.bbagen.2016.08.003DOI Listing
January 2017

Biological activities of Leptodeira annulata (banded cat-eyed snake) venom on vertebrate neuromuscular preparations.

Toxicon 2016 Sep 4;119:345-51. Epub 2016 Jul 4.

Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 255, Cidade Universitária Zeferino Vaz, 13083-862, Campinas, SP, Brazil. Electronic address:

The physiological properties of colubrid snake venoms are largely unknown and less frequently investigated. In this study, we assessed the enzymatic properties and biological activities of Leptodeira annulata (banded cat-eyed snake) venom, an opistoglyphous snake from Colombia. The proteolytic, phospholipase A2 and amidolytic activities are assessed using colorimetric assays and the biological activities were analyzed in avian and mammalian neuromuscular preparations. L. annulata venom caused neuromuscular blockade in chick biventer cervicis (BC) preparations (40± 15% and 50± 3% of twitch reduction for 30 and 100 μg/ml, respectively; p < 0.05) following 120 incubation; 10 μg/ml of venom did not induce blockade. There was a mild reduction in contracture response to exogenous acetylcholine (110 μM) in BC preparations exposed to 10 and 30 μg of venom/ml (∼4% and ∼32% of reduction, respectively, p > 0.05, n = 4) compared to basal values whereas the highest concentration (100 μg/ml) abolished it after 120 min. The venom caused a significant reduction in contracture response elicited by KCl (∼58 and ∼90 of reduction for 30 and 100 μg/ml, respectively, p < 0.05, n = 4). In mouse phrenic nerve-diaphragm (PND) preparations, L. annulata venom induced a progressive muscle membrane depolarization [from -85.9 ± 1.6 mV (t0) to -72.2 ± 2.9 mV (t120), p < 0.05, n = 4); the postsynaptic receptors remained functional as shown by carbachol-induced depolarization. The morphological analyses showed a concentration-dependent number of pathological states in muscle fibers from both BC and PND preparations pre-exposed to venom. The venom showed high proteolytic activity and low phospholipase A2 activity; there was no evidence for serine protease activity. These results indicate that the neuromuscular effect induced by L. annulata venom resulted from damaged muscle fibers that lead to the blockade of twitches response. The findings suggest that the myotoxicity might be related to the presence of metalloproteases in this venom.
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http://dx.doi.org/10.1016/j.toxicon.2016.07.004DOI Listing
September 2016

Neutralization of the neuromuscular inhibition of venom and taipoxin from the taipan (Oxyuranus scutellatus) by F(ab')2 and whole IgG antivenoms.

Toxicol Lett 2016 Jan 24;241:175-83. Epub 2015 Nov 24.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

The neuromuscular junction activity of Oxyuranus scutellatus venom and its presynaptic neurotoxin, taipoxin, and their neutralization by two antivenoms were examined in mouse phrenic nerve-diaphragm preparations. The action of taipoxin was also studied at 21°C. The efficacy of the antivenoms was also assessed in an in vivo mouse model. Both antivenoms were effective in neutralizing the neuromuscular blocking activity in preincubation-type experiments. In experiments involving independent addition of venom and antivenoms, neutralization depended on the time interval between venom addition and antivenom application. When taipoxin was incubated for 5, 10 or 20min at 21°C, and antivenom added and temperature increased to 37°C, neutralization was achieved only when the toxin was incubated for 5 or 10min. The neutralization by the two antivenoms in an in vivo model showed that both whole IgG and F(ab')2 antivenoms were effective in neutralizing lethality. Our findings highlight the very rapid action of taipan venom at the nerve terminal, and the poor capacity of antivenoms to revert neurotoxicity as the time interval between venom or taipoxin application and antivenom addition increased. Additionally the disparity between molecular masses of the active substances of the two antivenoms did not result in differences in neutralization.
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http://dx.doi.org/10.1016/j.toxlet.2015.11.020DOI Listing
January 2016

Bp-13 PLA2: Purification and Neuromuscular Activity of a New Asp49 Toxin Isolated from Bothrops pauloensis Snake Venom.

Biochem Res Int 2015 19;2015:826059. Epub 2015 Feb 19.

Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), 13083-881 Campinas, SP, Brazil.

A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°-45°C. Full Bp-13 PLA2 activity required Ca(2+); its PLA2 activity was inhibited by Mg(2+), Mn(2+), Sr(2+), and Cd(2+) in the presence and absence of 1 mM Ca(2+). In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca(2+) by Sr(2+) and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.
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http://dx.doi.org/10.1155/2015/826059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350628PMC
March 2015

The neuromuscular activity of Bothriopsis bilineata smaragdina (forest viper) venom and its toxin Bbil-TX (Asp49 phospholipase A2) on isolated mouse nerve-muscle preparations.

Toxicon 2015 Mar 5;96:24-37. Epub 2015 Jan 5.

Centre for Venom and Toxin Drug Discovery, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Cathedral Street, 161, G4 0RE, Glasgow, UK. Electronic address:

The presynaptic action of Bothriopsis bilineata smaragdina (forest viper) venom and Bbil-TX, an Asp49 PLA2 from this venom, was examined in detail in mouse phrenic nerve-muscle (PND) preparations in vitro and in a neuroblastoma cell line (SK-N-SH) in order to gain a better insight into the mechanism of action of the venom and associated Asp49 PLA2. In low Ca(2+) solution, venom (3μg/ml) caused a quadriphasic response in PND twitch height whilst at 10μg/ml the venom additionally induced an abrupt and marked initial contracture followed by neuromuscular facilitation, rhythmic oscillations of nerve-evoked twitches, alterations in baseline and progressive blockade. The venom slowed the relaxation phase of muscle twitches. In low Ca(2+), Bbil-TX [210nM (3μg/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10μg/ml) and Bbil-TX (210nM) caused minor changes in the compound action potential (CAP) amplitude recorded from sciatic nerve preparations, with no significant effect on rise time and latency; tetrodotoxin (3.1nM) blocked the CAP at the end of the experiments. In mouse triangularis sterni nerve-muscle (TSn-m) preparations, venom (10μg/ml) and Bbil-TX (210nM) significantly reduced the perineural waveform associated with the outward K(+) current while the amplitude of the inward Na(+) current was not significantly affected. Bbil-TX (210nM) caused a progressive increase in the quantal content of TSn-m preparations maintained in low Ca(2+) solution. Venom (3μg/ml) and toxin (210nM) increased the calcium fluorescence in SK-N-SH neuroblastoma cells loaded with Fluo3 AM and maintained in low or normal Ca(2+) solution. In normal Ca(2+), the increase in fluorescence amplitude was accompanied by irregular and frequent calcium transients. In TSn-m preparations loaded with Fluo4 AM, venom (10μg/ml) caused an immediate increase in intracellular Ca(2+) followed by oscillations in fluorescence and muscle contracture; Bbil-TX did not change the calcium fluorescence in TSn-m preparations. Immunohistochemical analysis of toxin-treated PND preparations revealed labeling of junctional ACh receptors but a loss of the presynaptic proteins synaptophysin and SNAP25. Together, these data confirm the presynaptic action of Bbil-TX and show that it involves modulation of K(+) channel activity and presynaptic protein expression.
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http://dx.doi.org/10.1016/j.toxicon.2015.01.001DOI Listing
March 2015

Neuromuscular activity of Bothrops fonsecai snake venom in vertebrate preparations.

J Venom Res 2014 18;5:6-15. Epub 2014 Jun 18.

Serpentário do Centro de Estudos da Natureza e Instituto de Pesquisa & Desenvolvimento (IP&D), Universidade do Vale do Paraíba (UNIVAP), Avenida Shishimi Hifumi, 2911, Urbanova, 12240-000, São José dos Campos, SP, Brazil.

The neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160μg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160μg/ml, respectively) and attenuated the contractures to 110μM ACh (78-100% inhibition) and 40mM KCl (45-90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200μg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200μg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K(+) were abolished by preincubating venom with commercial antivenom. These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4095944PMC
July 2014

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad.

J Venom Anim Toxins Incl Trop Dis 2014 4;20:30. Epub 2014 Jul 4.

Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, Campinas, SP 13083-970, Brasil.

Background: Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity.

Methods: The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording - in response to indirect stimulation - and for electrophysiological measurements.

Results: Venom extract (50 μg/mL) increased the muscle twitch tension in PND preparations but did not significantly alter the resting membrane potential values. Electrophysiological evaluations showed that the extract (50 μg/mL) significantly augmented the frequency of miniature end-plate potential (from 38 ± 3.5 to 88 ± 15 after 60 minutes; n = 5; p < 0.05) and quantal content (from 128 ± 13 to 272 ± 34 after five minutes; n = 5; p < 0.05). Pretreatment with ouabain (1 μg/mL) for five minutes prevented the increase in quantal content (117 ± 18 and 154 ± 33 after five and 60 minutes, respectively).

Conclusion: These results indicate that the methanolic extract of R. schneideri venom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.
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http://dx.doi.org/10.1186/1678-9199-20-30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094905PMC
July 2014

In vitro antiophidian mechanisms of Hypericum brasiliense choisy standardized extract: quercetin-dependent neuroprotection.

Biomed Res Int 2013 31;2013:943520. Epub 2013 Dec 31.

Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), P.O. Box 6111, 13083-970 Campinas, SP, Brazil.

The neuroprotection induced by Hypericum brasiliense Choisy extract (HBE) and its main active polyphenol compound quercetin, against Crotalus durissus terrificus (Cdt) venom and crotoxin and crotamine, was enquired at both central and peripheral mammal nervous system. Cdt venom (10 μg/mL) or crotoxin (1 μg/mL) incubated at mouse phrenic nerve-diaphragm preparation (PND) induced an irreversible and complete neuromuscular blockade, respectively. Crotamine (1 μg/mL) only induced an increase of muscle strength at PND preparations. At mouse brain slices, Cdt venom (1, 5, and 10 μg/mL) decreased cell viability. HBE (100 μg/mL) inhibited significantly the facilitatory action of crotamine (1 μg/mL) and was partially active against the neuromuscular blockade of crotoxin (1 μg/mL) (data not shown). Quercetin (10 μg/mL) mimicked the neuromuscular protection of HBE (100 μg/mL), by inhibiting almost completely the neurotoxic effect induced by crotoxin (1 μg/mL) and crotamine (1 μg/mL). HBE (100 μg/mL) and quercetin (10 μg/mL) also increased cell viability in mice brain slices. Quercetin (10 μg/mL) was more effective than HBE (100 μg/mL) in counteracting the cell lysis induced by Cdt venom (1 and 10 μg/mL, resp.). These results and a further phytochemical and toxicological investigations could open new perspectives towards therapeutic use of Hypericum brasiliense standardized extract and quercetin, especially to counteract the neurotoxic effect induced by snake neurotoxic venoms.
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http://dx.doi.org/10.1155/2013/943520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3892476PMC
August 2014

Neuromuscular activity of Micrurus laticollaris (Squamata: Elapidae) venom in vitro.

Toxins (Basel) 2014 Jan 17;6(1):359-70. Epub 2014 Jan 17.

Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos, CP. 62210, Mexico.

In this work, we have examined the neuromuscular activity of Micrurus laticollaris (Mexican coral snake) venom (MLV) in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations, the MLV induced an irreversible concentration- and time-dependent (1-30 µg/mL) neuromuscular blockade, with 50% blockade occurring between 8 and 30 min. Muscle contractures evoked by exogenous acetylcholine were completely abolished by MLV, whereas those of KCl were also significantly altered (86% ± 11%, 53% ± 11%, 89% ± 5% and 89% ± 7% for one, three, 10 and 30 µg of venom/mL, respectively; n = 4; p < 0.05). In mouse phrenic nerve-diaphragm preparations, MLV (1-10 µg/mL) promoted a slight increase in the amplitude of twitch-tension (3 µg/mL), followed by neuromuscular blockade (n = 4); the highest concentration caused complete inhibition of the twitches (time for 50% blockade = 26 ± 3 min), without exhibiting a previous neuromuscular facilitation. The venom (3 µg/mL) induced a biphasic modulation in the frequency of miniature end-plate potentials (MEPPs)/min, causing a significant increase after 15 min, followed by a decrease after 60 min (from 17 ± 1.4 (basal) to 28 ± 2.5 (t15) and 12 ± 2 (t60)). The membrane resting potential of mouse diaphragm preparations pre-exposed or not to d-tubocurarine (5 µg/mL) was also significantly less negative with MLV (10 µg/mL). Together, these results indicate that M. laticollaris venom induces neuromuscular blockade by a combination of pre- and post-synaptic activities.
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http://dx.doi.org/10.3390/toxins6010359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920266PMC
January 2014

VdTX-1, a reversible nicotinic receptor antagonist isolated from venom of the spider Vitalius dubius (Theraphosidae).

Toxicon 2013 Aug 10;70:135-41. Epub 2013 May 10.

Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas-UNICAMP, Campinas, SP, Brazil.

Theraphosid spider venoms can block neurotransmission in vertebrate nerve-muscle preparations in vitro, but few of the components involved have been characterized. In this work, we describe the neuromuscular activity of venom from the Brazilian theraphosid Vitalius dubius and report the purification and pharmacological characterization of VdTX-1, a 728 Da toxin that blocks nicotinic receptors. Neuromuscular activity was assayed in chick biventer cervicis preparations and muscle responses to exogenous ACh and KCl were determined before and after incubation with venom or toxin. Changes in membrane resting potential were studied in mouse diaphragm muscle. The toxin was purified by a combination of filtration through Amicon® filters, cation exchange HPLC and RP-HPLC; toxin purity and mass were confirmed by mass spectrometry. Venom caused progressive neuromuscular blockade and muscle contracture; the blockade but not the contracture was reversible by washing. Venom attenuated contractures to exogenous ACh and KCl. Filtration yielded low (LM, <5 kDa) and high (HM, >5 kDa) fractions, with the latter reproducing the contracture seen in venom but with a slight and progressive twitch blockade. The LM fraction caused reversible blockade and attenuated contractures to ACh, but had no effect on contractures to KCl. VdTX-1 (728 Da) purified from the LM fraction was photosensitive and reduced the E(max) to ACh in biventer cervicis muscle without affecting the EC₅₀; VdTX-1 also abolished carbachol-induced depolarizations. V. dubius venom contains at least two components that affect vertebrate neurotransmission. One component, VdTX-1, blocks nicotinic receptors non-competitively to produce reversible blockade without muscle contracture.
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http://dx.doi.org/10.1016/j.toxicon.2013.04.020DOI Listing
August 2013

Pharmacological study of a new Asp49 phospholipase A(2) (Bbil-TX) isolated from Bothriopsis bilineata smargadina (forest viper) venom in vertebrate neuromuscular preparations.

Toxicon 2013 Jul 26;69:191-9. Epub 2013 Mar 26.

Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, Cidade Universitária Zeferino Vaz, 13083-887 Campinas, SP, Brazil.

The neuromuscular activity of Bbil-TX, a PLA2 with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5-10 μg/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87 ± 7, 41 ± 7 and 19 ± 2 min (mean ± SEM; n = 4-6) for 1, 5 and 10 μg/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10 μg/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10 μg/ml) BC preparations. However, Bbil-TX (10 μg/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116 ± 17 IU/ml (basal) to 710 ± 91 IU/ml after 45 min). Bbil-TX (5 μg/ml)-induced blockade was markedly inhibited at 22-24 °C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3-30 μg/ml, n = 4-6) caused partial time- and concentration-dependent blockade in PND preparations (52 ± 2% at the highest concentration). Bbil-TX (30 μg/ml) also markedly reduced the MEPPs frequency [from 26 ± 2.5 (basal) to 10 ± 1 after 60 min; n = 5; p < 0.05] and the quantal content [from 94 ± 14 (basal) to 24 ± 3 after 60 min; n = 5; p < 0.05] of PND preparations, but caused only minor depolarization of the membrane resting potential [from -80 ± 1 mV (basal) to -66 ± 2 mV after 120 min; n = 5; p < 0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA2 that contributes to the neuromuscular blockade caused by B. b. smargadina venom.
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http://dx.doi.org/10.1016/j.toxicon.2013.03.006DOI Listing
July 2013

Biochemical, pharmacological, and structural characterization of new basic PLA2 Bbil-TX from Bothriopsis bilineata snake venom.

Biomed Res Int 2013 30;2013:612649. Epub 2012 Dec 30.

Department of Biochemistry, Institute of Biology (IB), Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil.

Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25-37°C. Maximum PLA2 activity required Ca(2+) and in the presence of Cd(2+), Zn(2+), Mn(2+), and Mg(2+) it was reduced in the presence or absence of Ca(2+). Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinflammatory effect, the phospholipid hydrolysis may be relevant for these phenomena.
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http://dx.doi.org/10.1155/2013/612649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591176PMC
November 2013

Beneficial effect of crotamine in the treatment of myasthenic rats.

Muscle Nerve 2013 Apr 4;47(4):591-3. Epub 2013 Mar 4.

Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paolo, Brazil.

Introduction: Crotamine is a basic, low-molecular-weight peptide that, at low concentrations, improves neurotransmission in isolated neuromuscular preparations by modulating sodium channels. In this study, we compared the effects of crotamine and neostigmine on neuromuscular transmission in myasthenic rats.

Methods: We used a conventional electromyographic technique in in-situ neuromuscular preparations and a 4-week treadmill program.

Results: During the in-situ electromyographic recording, neostigmine (17 μg/kg) caused short-term facilitation, whereas crotamine induced progressive and sustained twitch-tension enhancement during 140 min of recording (50 ± 5%, P < 0.05). On the treadmill evaluation, rats showed significant improvement in exercise tolerance, characterized by a decrease in the number of fatigue episodes after 2 weeks of a single-dose treatment with crotamine.

Conclusions: These results indicate that crotamine is more efficient than neostigmine for enhancing muscular performance in myasthenic rats, possibly by improving the safety factor of neuromuscular transmission.
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http://dx.doi.org/10.1002/mus.23714DOI Listing
April 2013

Suramin affects metalloproteinase-9 activity and increases β-dystroglycan levels in the diaphragm of the dystrophin-deficient mdx mouse.

Muscle Nerve 2012 Nov;46(5):810-3

Departamento de Anatomia, Biologia Celular, Fisiologia e Biofísica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São Paulo 13083-970, Brazil.

Introduction: In Duchenne muscular dystrophy and in the mdx mouse, muscle fiber degeneration and subsequent fibrosis lead to cardiorespiratory failure. Previously, we demonstrated that the anti-fibrotic agent suramin was effective in decreasing fibrosis in mdx muscles. In this study, we were interested to see whether suramin could affect metalloproteinases (MMP) and improve the functional activity of the mdx diaphragm muscle.

Methods: Zymography was performed to evaluate MMP-2 and MMP-9 activity. Western blotting was used to analyze the levels of beta-dystroglycan. Muscle function was assessed in hemidiaphragm in vitro preparations.

Results: We found that suramin affects metalloproteinase-9 activity and increases beta-dystroglycan. Furthermore, suramin also protects against diaphragm muscle fatigue over time.

Conclusions: These results show the potential benefits of suramin in maintaining the structure of the dystrophin-glycoprotein complex.
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http://dx.doi.org/10.1002/mus.23468DOI Listing
November 2012

Guanidine affects differentially the twitch response of diaphragm, extensor digitorum longus and soleus nerve-muscle preparations of mice.

Molecules 2012 Jun 15;17(6):7503-22. Epub 2012 Jun 15.

Department of Histology and Embryology, Institute of Biology, University of Campinas-UNICAMP, P.O. Box 6109, 13083-970 Campinas, SP, Brazil.

Guanidine has been used with some success to treat myasthenia gravis and myasthenic syndrome because it increases acetylcholine release at nerve terminals through K⁺, Na⁺ and Ca²⁺ channels-involving mechanisms. Currently, guanidine derivatives have been proposed for treatment of several diseases. Studies aimed at providing new insights to the drug are relevant. Experimentally, guanidine (10 mM) induces on mouse phrenic nerve-diaphragm (PND) preparations neurotransmission facilitation followed by blockade and a greatest secondary facilitation after its removal from bath. Herein, we hypothesized that this peculiar triphasic response may differ in muscles with distinct twitch/metabolic characteristics. Morphological alterations and contractile response of PND, extensor digitorum longus (EDL) and soleus (SOL) preparations incubated with guanidine (10 mM) for 15, 30, 60 min were analyzed. Guanidine concentrations of 5 mM (for PND and EDL) and 1 mM (for EDL) were also tested. Guanidine triphasic effect was only observed on PND regardless the concentration. The morphological alterations in muscle tissue varied along time but did not impede the PND post-wash facilitation. Higher doses (20-25 mM) did not increase EDL or SOL neurotransmission. The data suggest a complex mechanism likely dependent on the metabolic/contractile muscle phenotype; muscle fiber types and density/type of ion channels, sarcoplasmic reticulum and mitochondria organization may have profound impact on the levels and isoform expression pattern of Ca²⁺ regulatory membrane proteins so reflecting regulation of calcium handling and contractile response in different types of muscle.
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http://dx.doi.org/10.3390/molecules17067503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6268877PMC
June 2012

Protection by Mikania laevigata (guaco) extract against the toxicity of Philodryas olfersii snake venom.

Toxicon 2012 Sep 4;60(4):614-22. Epub 2012 Jun 4.

Departamento de Ciências Fisiológicas, Faculdade das Ciências Médicas e da Saúde, Pontifícia Universidade Católica de São Paulo (PUC/SP), Praça José Ermírio de Moraes 290, Sorocaba 18030-095, SP, Brazil.

Philodryas olfersii is responsible for most colubrid snakebites in Brazil. In this work, we examined the ability of an ethanolic extract from Mikania laevigata (guaco) leaves to protect against the in vitro neuromuscular activity of P. olfersii venom in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) preparations. M. laevigata extract caused moderate twitch-tension facilitation at low concentrations (107.4 ± 6.2% with 20 μl/ml and 118.9 ± 9.3% with 40 μl/ml in PND, and 120.7 ± 7.7% with 40 μl/ml and 114.5 ± 4.4% with 50 μl/ml in BC after 120 min; n = 4-6, mean ± SEM). In PND, the ethanol alone (40 μl/ml, n = 4) did not change the twitch-tension when compared with control. However, in BC, the ethanol produced a higher facilitation when compared to control. At higher concentrations (>50 μl/ml) the extract caused total and reversible blockade in both preparations. Venom (50 μg/ml) caused partial blockade in PND (58.5 ± 12%, n = 4) and almost total blockade in BC (93.5 ± 2.2%, n = 4). Pretreatment of the preparations with extract (40 μl/ml) for 30 min before incubation with venom (50 μg/ml) completely protected PND from neuromuscular blockade and delayed the blockade in BC. The extract alone caused only mild morphological alterations (12.5 ± 0.5% and 10.9 ± 2.3% fiber damage in PND and BC, respectively, compared to 2.3 ± 0.3% and 3 ± 0 in controls; n = 3), with no increase in expression of the inflammatory cytokines TNFα and IFNγ. The ethanol alone also caused slight muscle damage: 4.3 ± 2.4% in PND and 6.7 ± 3.3% in BC (both n = 3) and little or no TNFα and IFNγ expression in both preparations as observed in control. Venom (50 μg/ml) caused 53.5 ± 8.5% and 55.8 ± 4.3% fiber damage in PND and BC, respectively; (n = 3, p < 0.05 vs. controls) and enhanced expression of TNFα and IFNγ. Pretreatment of the preparations with extract protected against venom-induced muscle damage by 80.3 and 60.4 in PND and BC, respectively, and prevented TNFα and IFNγ expression. These results indicate that the M. laevigata extract protected nerve-muscle preparations against the myotoxic, neurotoxic and inflammatory effects of P. olfersii venom.
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http://dx.doi.org/10.1016/j.toxicon.2012.05.014DOI Listing
September 2012

Mipartoxin-I, a novel three-finger toxin, is the major neurotoxic component in the venom of the redtail coral snake Micrurus mipartitus (Elapidae).

Toxicon 2012 Oct 4;60(5):851-63. Epub 2012 Jun 4.

Programa de Ofidismo y Escorpionismo, Universidad de Antioquia, Medellín, Colombia.

The major venom component of Micrurus mipartitus, a coral snake distributed from Nicaragua to northern South America, was characterized biochemically and functionally. This protein, named mipartoxin-I, is a novel member of the three-finger toxin superfamily, presenting the characteristic cysteine signature and amino acid sequence length of the short-chain, type-I, α-neurotoxins. Nevertheless, it varies considerably from related toxins, with a sequence identity not higher than 70% in a multiple alignment of 67 proteins within this family. Its observed molecular mass (7030.0) matches the value predicted by its amino acid sequence, indicating lack of post-translational modifications. Mipartoxin-I showed a potent lethal effect in mice (intraperitoneal median lethal dose: 0.06 μg/g body weight), and caused a clear neuromuscular blockade on both avian and mouse nerve-muscle preparations, presenting a post-synaptic action through the cholinergic nicotinic receptor. Since mipartoxin-I is the most abundant (28%) protein in M. mipartitus venom, it should play a major role in its toxicity, and therefore represents an important target for developing a therapeutic antivenom, which is very scarce or even unavailable in the regions where this snake inhabits. The structural information here provided might help in the preparation of a synthetic or recombinant immunogen to overcome the limited venom availability.
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http://dx.doi.org/10.1016/j.toxicon.2012.05.023DOI Listing
October 2012

Neuromuscular activity of Bothrops alcatraz snake venom in chick biventer cervicis preparations.

Toxicon 2012 Feb 1;59(2):294-9. Epub 2011 Dec 1.

Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), P.O. Box 6111, 13083-970 Campinas, SP, Brazil.

Venom (10-100 μg/ml) from Bothrops alcatraz, a pitviper from the Alcatrazes Archipelago off the coast of southeastern Brazil, caused progressive, irreversible neuromuscular blockade in chick isolated biventer cervicis preparations. The venom also inhibited contractures to exogenous ACh (110 μM) and KCl (20 mM), caused myofiber damage and increased creatine kinase release. Commercial bothropic antivenom raised against mainland Bothrops species neutralized the neuromuscular activity, depending on the venom concentration.
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http://dx.doi.org/10.1016/j.toxicon.2011.11.011DOI Listing
February 2012

Biological characterization of Bothrops marajoensis snake venom.

J Venom Res 2011 19;2:37-41. Epub 2011 Oct 19.

This study describes the effects of Bothrops marajoensis venom (Marajó lancehead) on isolated neuromuscular preparations of chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND). At low concentrations (1µg/ml for CBC and 5µg/ml for PND), the venom exhibited a neuromuscular blocking without any damaging effect on the muscle integrity. At higher concentration (20μg/ml for PND), together with the neuromuscular blockade, there was a moderate myonecrosis. The results show differences between mammalian and avian preparations in response to venom concentration; the avian preparation was more sensitive to venom neurotoxic effect than the mammalian preparation. The possible presynaptic mechanism underlying the neuromuscular blocking effect was reinforced by the observed increase in MEPPs at the same time (at 15min) when the facilitation of twitch tension occurred. These results indicate that the B. marajoensis venom produced neuromuscular blockade, which appeared to be presynaptic at low concentrations with a postsynaptic component at high concentrations, leading to muscle oedema. These observations demand the fractionation of the crude venom and characterization of its active components for a better understanding of its biological dynamics.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3211074PMC
November 2011

Presynaptic effect of a methanolic extract of toad (Rhinella schneideri) poison in avian neuromuscular preparation.

J Venom Res 2011 22;2:32-6. Epub 2011 Sep 22.

The neurotoxicity of a methanolic extract of toad (Rhinella schneideri) poison was examined in chick biventer cervicis preparations. The methanolic extract (1, 3, 10 and 30µg/ml) caused concentration-dependent blockade at the three highest concentrations (time for 50% blockade, mean±SEM: 84±10, 51±3 and 12±0.8min for 3, 10 and 30µg/ml, respectively; n=6-8 each) that was preceded by significant, transient facilitation at 10μg/ml. Contractures to exogenous ACh (110μM) or KCl (20mM) were unaffected by the blockade. In curarized (d-Tc, 1μg/ml) preparations, the extract (10µg/ml) caused complete, irreversible blockade that persisted after extensive washing. The extract did not significantly alter the creatine kinase release or morphology of biventer cervicis muscle. These results indicate that the methanolic extract of R. schneideri poison acts primarily presynaptically to enhance neurotransmitter release in this avian preparation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3189579PMC
November 2011

The neuromuscular activity of Micrurus pyrrhocryptus venom and its neutralization by commercial and specific coral snake antivenoms.

J Venom Res 2011 24;2:24-31. Epub 2011 Jun 24.

The neuromuscular activity ofMicrurus pyrrochryptus venom was studied in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. The venom (0.5-50μg/ml) caused irreversible, time- and concentration-dependent blockade, with BC being more sensitive than PND (50% blockade with 10μg/ml in 22±;3min and 62±4min, respectively; mean±SEM, n=6; p<0.05). In BC preparations, venom (0.5μg/ml) progressively abolished ACh-induced contractures, whereas contractures to exogenous KCl and muscle twitches in curarized preparations were unaffected. The venom neither altered creatine kinase release (venom: 25.8±1.75IU/l vs control: 24.3±2.2IU/l, n=6, after 120min), nor it caused significant muscle damage (50μg of venom/ml vs control: 3.5±0.8% vs 1.1±0.7% for PND; 4.3±1.5% vs 1.2±0.5% for BC, n=5). The venom had low PLA(2) activity. Neurotoxicity was effectively neutralized by commercial Micrurus antivenom and specific antivenom. These findings indicate that M. pyrrhocryptus venom acts postsynaptically on nicotinic receptors, with no significant myotoxicity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3132105PMC
November 2011