Pharmacological and partial biochemical characterization of Bmaj-9 isolated from Bothrops marajoensis snake venom

Bmaj-9, a basic PLA2 (13679.33 Da), was isolated from Bothrops marajoensis snake venom through only one chromatographic step in reversed phase HPLC on ¼-Bondapak C-18 column. The amino acid composition showed that Bmaj-9 had a high content of Lys, His, and Arg, typical of a basic PLA2. The sequence of Bmaj-9 contains 124 amino acid residues with a pI value of 8.55, such as DLWQWGQMIL KETGKLPFSY YTAYGCYCGW GGRGGKPKAD TDRCCFVHDC, revealing a high homology with Asp49 PLA2 from other snake venoms. It also exhibited a pronounced phospholipase A2 activity when compared with crude venom. In chick biventer cervicis preparations, the time for 50% and 100% neuromuscular paralysis was respectively (in minutes): 110 ± 10 (1 µg/mL); 40 ± 6 and 90 ± 2 (5 µg/mL); 30 ± 3 and 70 ± 5 (10 µg/mL); 42 ± 1 and 60 ± 2 (20 µg/mL), with no effect on the contractures elicited by either exogenous ACh (110 µM) or KCl (20 mM). Bmaj-9 (10 µg/mL) neither interfered with the muscular response to direct electrical stimulation in curarized preparations nor significantly altered the release of CK at 0, 15, 30 and 60 minutes incubations (27.4 ± 5, 74.2 ± 8, 161.0 ± 21 and 353.0 ± 47, respectively). The histological analysis showed that, even causing blockade at the maximum dosage (5 µg/mL), the toxin does not induce significant morphological alterations such as necrosis or infiltration of inflammatory cells. These results identified Bmaj-9 as a new member of the basic Asp49 PLA2 family able to interact with the motor nerve terminal membrane, thereby inducing a presynaptic neuromuscular blockade181627

The Pharmacological Effect Of Bothrops Neuwiedii Pauloensis (jararaca-pintada) Snake Venom On Avian Neuromuscular Transmission.

The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 micro g/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 micro g/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 micro M acetylcholine alone and cumulative concentrations of 1 micro M to 10 mM were unaffected. At venom concentrations higher than 50 micro g/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24 degrees C, the venom (50 g/ml) produced only partial neuromuscular blockade (30.7 +/- 8.0%, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action.36617-2

Effects of manganese (Mn2+) on neurotoxic and myotoxic activities induced by Crotalus durissus terrificus and Bothrops jararacussu venoms in chick biventer cervicis preparations

In the present study, manganese (Mn2+), a neuromuscular blocker with pre and postsynaptic actions, was used to verify the neurotoxicity and myotoxicity induced by Crotalus durissus terrificus (Cdt) and Bothrops jararacussu (Bjssu) venoms in biventer cervicis preparations (BCp). Preparations pretreated with 0.66 and 1.6mM Mn2+ did not affect Cdt venom-induced blockage nor change KCl-induced contracture but partially reduced ACh-induced contracture. However, both Mn2+ concentrations partially hindered Bjssu venom-induced blockage after washing the preparations with Krebs solution, and only 1.6mM Mn2+ preparations significantly recovered ACh-induced contracture. The effect of Cdt venom myotoxicity on contractile responses was different from that of Bjssu venom myotoxicity. Pretreatment with 1.6mM Mn2+ partially reduced muscle damage percentage and creatine kinase (CK) activity (U/l) induced by both venoms. In conclusion, Mn2+ interfered in ACh-induced contracture of the nicotinic receptor; did not prevent Cdt venom neurotoxicity but partially reduced its myotoxicity in vitro due to the stabilizing action of this venom on the sarcolemmal membrane; and partially attenuated myotoxicity and neuromuscular blockage induced by Bjssu venom. The Mn2+ dual action (pre and postsynaptic) is useful to study snake venoms since most of them present one or both of these actions; besides, Mn2+ allowed recovering coherent interpretation of experimental versus clinical results.13247949

Neutralization of the neuromuscular activity of bothropstoxin-i, a myotoxin from Bothrops jararacussu snake venom, by a hydroalcoholic extract of Casearia sylvestris Sw. (guaçatonga)

Numerous plants are used as snakebite antidotes in Brazilian folk medicine, including Casearia sylvestris Swartz, popularly known as guaçatonga. In this study, we examined the action of a hydroalcoholic extract from C. sylvestris on the neuromuscular blockade caused by bothropstoxin-I (BthTX-I), a myotoxin from Bothrops jararacussu venom, in mouse isolated phrenic nerve-diaphragm (PND) preparations. Aqueous (8 and 12 mg/ml, n=4 and 5, respectively) and hydroalcoholic (12 mg/ml, n=12) extracts of the leaves of C. sylvestris caused facilitation in PND preparations followed by partial neuromuscular blockade. BthTX-I (20 µg/ml, n=4) caused 50% paralysis after 65±15 min (mean ± S.E.M). Preincubation (30 min at 37° C) of BthTX-I (20 µg/ml, n=4) with a concentration of the hydroalcoholic extract (4 mg/ml) that had no neuromuscular activity, such as the control (n=5), prevented the neuromuscular blockade caused by the toxin. This protection may be mediated by compounds such as flavonoids and phenols identified by thin-layer chromatography and colorimetric assays.465478Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Purification And N-terminal Sequencing Of Two Presynaptic Neurotoxic Pla2, Neuwieditoxin-i And Neuwieditoxin-ii, From Bothrops Neuwiedi Pauloensis (jararaca Pintada) Venom

Two presynaptic phospholipases A2 (PLA2), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (μBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10μg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0±8.0% (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71% homology with bothropstoxin-II and 54% homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92% homology with Basp-III and 62% homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA2.131103121AIRD, S.D., KAISER II, LEWIS RV., KRUGGEL WG. A complete amino acid sequence for the basic subunit of crotoxin (1986) Arch. Biochem. Biophys, 249, pp. 296-300AIRD, S.D., KRUGGEL, W.G., KAISER II, Amino acid sequence of the basic subunit of Mojave toxin from the venom of the Mojave rattlesnake (Crotalus s. scutulatus) (1990) Toxicon, 28, pp. 669-673BEGHINI, D.G., TOYAMA, M.H., HYSLOP, S., SODEK, L., NOVELLO, J.C., MARANGONI, S., Enzymatic characterization of a novel phospholipase A2 from Crotalus durissus cascavella rattlesnake (maracambóia) venom (2000) J. Protein Chem, 19, pp. 603-607BORJA-OLIVEIRA, C.R., DURIGON, A.M., VALLIN, A.C.C., TOYAMA, M.H., SOUCCAR, C., MARANGONI, S., RODRIGUES-SIMIONI, L., The pharmacological effects of Bothrops neuwiedi pauloensis (jararaca-pintada) snake venom on avian neuromuscular transmission (2003) Braz. J. Med. Biol. Res, 36, pp. 617-624BORJA-OLIVEIRA, C.R., SOARES, A.M., ZAMUNER, S.R., HYSLOP, S., GIGLIO, J.R., PRADO-FRANCESCHI, J., RODRIGUES-SIMIONI, L., Intraspecific variation in the neurotoxic and myotoxic activities of Bothrops neuwiedi snake venoms (2002) J. Venom. Anim. Toxins, 8, pp. 88-101BUCARETCHI, F., HERRERA, S.R.F., HYSLOP, S., BARACAT, E.C.E., VIEIRA, R.J., Snakebites by Bothrops spp in children in Campinas, São Paulo, Brazil (2001) Rev. Inst. Med. Trop. São Paulo, 43, pp. 329-333CHO, W., KEZDY, F.J., Chromogenic substrate and assay of phospholipase A 2 (1991) Meth. Enzymol, 197, pp. 75-79CINTRA, A.C., MARANGONI, S., OLIVEIRA, B., GIGLIO, J.R., Bothropstoxin-I: Amino acid sequence and function (1993) J. Protein Chem, 12, pp. 57-64COGO, J.C., PRADO-FRANCESCHI, J., CRUZ-HÖFLING, M.A., CORRADO, A.P., RODRIGUES-SIMIONI, L., Effects of Bothrops insularis on the mouse and chick nerve-muscle preparation (1993) Toxicon, 31, pp. 1237-1247COGO, J.C., PRADO-FRANCESCHI, J., GIGLIO, J.R., CORRADO, A.P., CRUZ-HÖFLING, M.A., DONATO, J.L., LEITE, G.B., RODRIGUES-SIMIONI, L., An unusual presynaptic action of Bothrops insularis snake venom mediated by phospholipase A2 fraction (1998) Toxicon, 36, pp. 1323-1332DE SOUSA, M.V., MORHY, L., ARNI, R.K., WARD, R.J., GUTIÉRREZ, J.M., Amino acid sequence of a myotoxic Lys-49-phospholipase A2 homologue from the venom of Cerrophidion (Bothrops) godmani (1998) Biochim. Biophys. Acta, 1384, pp. 204-208DURIGON, A.M., BORJA-OLIVEIRA, C.R., DAL, B.C., OSHIMA-FRANCO, Y., COGO, J.C., LAPA, A.J., SOUCCAR, C., RODRIGUES-SIMIONI, L., Neuromuscular activity of Bothrops neuwiedi pauloensis snake venom in mouse nerve-muscle preparations (2005) J. Venom. Anim. Toxins incl. 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Biochemical, Pharmacological, And Structural Characterization Of New Basic Pla2 Bbil-tx From Bothriopsis Bilineata Snake Venom

Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on μ7u-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. PLA 2 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 Cd2+, Zn2+, Mn 2+, and Mg2+ it was reduced in the presence or absence of Ca2+. 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. 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Purification and n-terminal sequencing of two presynaptic neurotoxic PLA2, neuwieditoxin-I and neuwieditoxin-II, from Bothrops neuwiedi pauloensis (jararaca pintada) venom

Two presynaptic phospholipases A2 (PLA2), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (µBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10µg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0&plusmn;8.0% (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71% homology with bothropstoxin-II and 54% homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92% homology with Basp-III and 62% homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA2

Measurement of the cross-section and charge asymmetry of WW bosons produced in proton-proton collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

This paper presents measurements of the $W^+ \rightarrow \mu^+\nu$ and $W^- \rightarrow \mu^-\nu$ cross-sections and the associated charge asymmetry as a function of the absolute pseudorapidity of the decay muon. The data were collected in proton--proton collisions at a centre-of-mass energy of 8 TeV with the ATLAS experiment at the LHC and correspond to a total integrated luminosity of 20.2~\mbox{fb^{-1}}. The precision of the cross-section measurements varies between 0.8% to 1.5% as a function of the pseudorapidity, excluding the 1.9% uncertainty on the integrated luminosity. The charge asymmetry is measured with an uncertainty between 0.002 and 0.003. The results are compared with predictions based on next-to-next-to-leading-order calculations with various parton distribution functions and have the sensitivity to discriminate between them.Comment: 38 pages in total, author list starting page 22, 5 figures, 4 tables, submitted to EPJC. All figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2017-13

Search for chargino-neutralino production with mass splittings near the electroweak scale in three-lepton final states in √s=13 TeV pp collisions with the ATLAS detector

A search for supersymmetry through the pair production of electroweakinos with mass splittings near the electroweak scale and decaying via on-shell W and Z bosons is presented for a three-lepton final state. The analyzed proton-proton collision data taken at a center-of-mass energy of √s=13  TeV were collected between 2015 and 2018 by the ATLAS experiment at the Large Hadron Collider, corresponding to an integrated luminosity of 139  fb−1. A search, emulating the recursive jigsaw reconstruction technique with easily reproducible laboratory-frame variables, is performed. The two excesses observed in the 2015–2016 data recursive jigsaw analysis in the low-mass three-lepton phase space are reproduced. Results with the full data set are in agreement with the Standard Model expectations. They are interpreted to set exclusion limits at the 95% confidence level on simplified models of chargino-neutralino pair production for masses up to 345 GeV