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. Trop. Dis, 11, pp. 22-33FONTES, M.R., SOARES, A.M., RODRIGUES, V.M., FERNANDES, A.C., DA SILVA, R.J., GIGLIO, J.R., Crystallization and preliminary X-ray diffraction analysis of a myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom (1999) Biochim. Biophys. Acta, 1432, pp. 393-395FRANCIS, B., GUTIERREZ, J.M., LOMONTE, B., KAISER II, Myotoxin II from Bothrops asper (Terciopelo) venom is a lysine-49 phospholipase A 2 (1991) Arch. Biochem. Biophys, 284, pp. 352-359GEOGHEGAN, P., ANGULO, Y., CANGELOSI, A., DIAZ, M., LOMONTE, B., Characterization of a basic phospholipase A2-homologue myotoxin isolated from the venom of the snake Bothrops neuwiedii (yarara chica) from Argentina (1999) Toxicon, 37, pp. 1735-1746GINSBORG, B.L., WARRINER, J., The isolated chick biventer cervicis nerve-muscle preparation (1960) Brit. J. Pharmacol, 15, pp. 410-411HALPERT, J., EAKER, D., Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake) (1975) J. Biol. Chem, 250, pp. 6990-6997HARVEY AL., BARFARAZ A., THOMPSON E., FAIZ A., PRESTON S., HARRIS JB. Screening of snake venoms for neurotoxic and myotoxic effects using simple in vitro preparations from rodents and chicks. Toxicon, 1994, 32, 257-65HELUANY, N.F., HOMSI-BRANDEBURGO, M.I., GIGLIO, J.R., PRADO-FRANCESCHI, J., RODRIGUES-SIMIONI, L., Effects induced by bothropstoxin, a component from Bothrops jararacussu snake venom, on mouse and chick muscle preparations (1992) Toxicon, 30, pp. 1203-1210HOLZER, M., MACKESSY, S.P., An aqueous endpoint assay of snake venom phospholipase A2 (1995) Toxicon, 35, pp. 1149-1155HOMSI-BRANDEBURGO, M.I., QUEIROZ, L.S., SANTO-NETO, H., RODRIGUES-SIMIONI, L., GIGLIO, J.R., Fractionation of Bothrops jararacussu snake venom: Partial chemical characterization and biological activity of bothropstoxin (1988) Toxicon, 26, pp. 615-627JOHNSON, E.K., OWNBY, C.L., Isolation of a myotoxin from the venom of Agkistrodon contortrix laticinctus (broad-banded copperhead) and pathogenesis of myonecrosis induced by it in mice (1993) Toxicon, 31, pp. 243-255KAISER II, Gutierrez, J.M., Plummer, D., Aird, S.D., Odell, G.V., AIRD SD., ODELL GV. The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper (1990) Arch. Biochem. Biophys, 278, pp. 319-325KATZ, B., MILEDI, R., The binding of acetylcholine to receptors and its removal from the synaptic cleft (1973) J. Physiol, 231, pp. 549-574KINI RM. Phospholipase A2 - A complex multifunctional protein puzzle. In: KINI RM. Ed., Venom phosholipase A2 enzymes. Structure, function and mechanism. New York: John Wiley &ampSons Inc Chichester, 1997, 1-28KONDO, K., NARITA, K., LEE, C.Y., Amino acid sequences of the two polypeptide chains in beta1-bungarotoxin from the venom of Bungarus multicinctus (1978) J. Biochem, 83, pp. 101-115KONDO, K., TODA, H., NARITA, K., LEE, C.Y., Amino acid sequence of β2-bungarotoxin from Bungarus multicinctus venom: The amino acid substitutions in the B chains (1982) J. Biochem, 91, pp. 1519-1530KONDO, K., ZHANG, J., XU, K., KAGAMIYAMA, H., Amino acid sequence of a presynaptic neurotoxin, agkistrodotoxin, from the venom of Agkistrodon halys pallas (1989) J. Biochem, 105, pp. 196-203KORDAS, M., On the role of junctional cholinesterase in determining the time course of the end-plate current (1977) J. Physiol, 270, pp. 133-150LEE, C.Y., HO, C.L., BOTES, D.P., Site of action of caudoxin, a neurotoxic phospholipase A2 from the horned puff adder (Bitis caudalis) venom (1982) Toxicon, 20, pp. 637-647LIND, P., EAKER, D., Amino-acid sequence of the alpha-subunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan (Oxyuranus s. scutellatus) (1982) European J. Biochem, 124, pp. 441-447MAGRO, A.J., SOARES, A.M., GIGLIO, J.R., FONTES, M.R., Crystal structures of BnSP-7 and BnSP-6, two Lys49 phospholipases A2: Quartenary structure and inhibition mechanism insights (2003) Biochem. Biophys. Res. Commun, 311, pp. 713-720MONTECUCCO, C., ROSSETTO, O., How do presynaptic PLA2 neurotoxins block nerve terminals? (2000) Trends Biochem. Sciences, 25, pp. 266-270OSHIMA-FRANCO, Y., HYSLOP, S., CINTRA, A.C., GIGLIO, J.R., DA CRUZ-HOFLING, M.A., RODRIGUES-SIMIONI, L., Neutralizing capacity of commercial bothropic antivenom against Bothrops jararacussu venom and bothropstoxin-I (2000) Muscle Nerve, 23, pp. 1832-1839OSHIMA-FRANCO, Y., LEITE, G.B., SILVA, G.H., CARDOSO, D.F., HYSLOP, S., GIGLIO, J.R., DA CRUZ-HOFLING, M.A., RODRIGUES-SIMIONI, L., Neutralization of the pharmacological effects of bothropstoxin-I from Bothrops jararacussu (jararacuçu) venom by crotoxin antiserum and heparin (2001) Toxicon, 39, pp. 1477-1485PEREIRA, M.F., NOVELLO, J.C., CINTRA, A.C., GIGLIO, J.R., LANDUCCI, E.T., OLIVEIRA, B., MARANGONI, S., The amino acid sequence of bothropstoxin-II, an Asp-49 myotoxin from Bothrops jararacussu (jararacuçu) venom with low phospholipase A2 activity (1998) J. Protein Chem, 17, pp. 381-386RE, L., BAROCCI, S., CAPITANI, C., VIVANI, C., RICCI, M., RINALDI, L., PAOLUCCI, G., MORALES, M.A., Effects of some natural extracts on the acetylcholine release at the mouse neuromuscular junction (1999) Pharmacol. Res, 39, pp. 239-245RE, L., COLA, V., FULGENZI, G., MARINELLI, F., CONCETTONI, C., ROSSINI, L., Postsynaptic effects of methoctramine at the mouse neuromuscular junction (1993) Neuroscience, 57, pp. 451-457RE, L., GIUSTI, P., CONCETTONI, C., DI SARRA, B., Computerized estimation of spontaneous and evoked acetylcholine release at the neuromuscular junction (1989) J. Pharmacol. Meth, 22, pp. 233-242RE, L., MORETTI, V., ROSSINI, L., GIUSTI, P., Sodium-activated potassium current in mouse diaphragm (1990) FEBS Lett, 270, pp. 195-197RIBEIRO, L.A., ALBUQUERQUE, M.J., PIRES DE CAMPOS VAF., KATZ G., TAKAOKAM NY., LEBRÃO ML., JORGE MT. Óbitos por serpentes peçonhentas no Estado de São Paulo: Avaliação de 43 casos, 1988/98 (1998) Rev. Assoc. Méd. Bras, 44, pp. 312-318RITONJA A., GUBENSEK F. Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom. Biochim. Biophys. Acta, 1985, 828, 306-12RODRIGUES-SIMIONI, L., BORGESE, N., CECCARELLI, B., The effects of Bothrops jararacussu venom and its components on frog nerve-muscle preparation (1983) Neuroscience, 10, pp. 475-489RODRIGUES-SIMIONI, L., ZAMUNER, S.R., COGO, J.C., BORJA-OLIVEIRA, C.R., PRADO-FRANCESCHI, J., CRUZ-HOFLING, M.A., CORRADO, A.P., Pharmacological evidence for a presynaptic action of venoms from Bothrops insularis (jararaca ilhoa) and Bothrops neuwiedi (jararaca pintada) (2004) Toxicon, 43, pp. 633-638SCHAGGER, H., VON JAGOW, G., Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kDa (1987) Anal. Biochem, 166, pp. 368-379SOARES, A.M., GUERRA-SÁ, R., BORJA-OLIVEIRA, C.R., RODRIGUES, V.M., RODRIGUES-SIMIONI, L., RODRIGUES, V., FONTES, M.R.M., GIGLIO, J.R., Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A2 homologue from Bothrops neuwiedi venom (2000) Arch. Biochem. Biophys, 378, pp. 201-209STÜHMER, W., ROBERTS, W.M., ALMERS, W., The loose patch clamp (1983) Single Channel Recording, p. 123. , SAKMANN B AND NEHER E, Eds, Plenum Press, New York;TOYAMA, M.H., SOARES, A.M., WEN-HWA, L., POLIKARPOV, I., GIGLIO, J.R., MARANGONI, S., Amino acid sequence of piratoxin-II, a myotoxic lys49 phospholipase A 2 homologue from Bothrops pirajai venom (2000) Biochimie, 82, pp. 245-250TSAI, I.H., LU, P.J., WANG, Y.M., HO, C.L., LIAW, L.L., Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus) (1995) Biochem. J, 311, pp. 895-900VAN DEN BERGH, C.J., SLOTBOOM, A.J., VERHEIJ, H.M., DE HAAS, G.H., The role of aspartic acid-49 in the active site of phospholipase A2. A site-specific mutagenesis study of porcine pancreatic phospholipase A 2 and the rationale of the enzymatic activity of Lys-49 phospholipase A2 from Agkistrodon piscivorus piscivorus venom (1988) European J. Biochem, 176, pp. 353-357VAN DEENEN LL, D.H.G., The substrate specificity of phospholipases A2 (1963) Biochim. Biophys. Acta, 70, pp. 538-553VILJOEN, C.C., BOTES, D.P., KRUGER, H., Isolation and amino acid sequence of caudoxin, a presynaptic acting toxic phospholipase A2 from the venom of the horned puff adder (Bitis caudalis) (1982) Toxicon, 20, pp. 715-737ZAMUNER, S.R., PRADO-FRANCESCHI, J., RODRIGUES-SIMIONI, L., The screening of Bothrops venoms for neurotoxic activity using the chick biventer cervicis preparation (1996) Toxicon, 34, pp. 314-31

Centrality dependence of charged particle production at large transverse momentum in Pb-Pb collisions at sNN=2.76\sqrt{s_{\rm{NN}}} = 2.76 TeV

The inclusive transverse momentum ($p_{\rm T}$) distributions of primary charged particles are measured in the pseudo-rapidity range $|\eta|<0.8$ as a function of event centrality in Pb-Pb collisions at $\sqrt{s_{\rm{NN}}}=2.76$ TeV with ALICE at the LHC. The data are presented in the $p_{\rm T}$ range $0.15<p_{\rm T}<50$ GeV/$c$ for nine centrality intervals from 70-80% to 0-5%. The Pb-Pb spectra are presented in terms of the nuclear modification factor $R_{\rm{AA}}$ using a pp reference spectrum measured at the same collision energy. We observe that the suppression of high-$p_{\rm T}$ particles strongly depends on event centrality. In central collisions (0-5%) the yield is most suppressed with $R_{\rm{AA}}\approx0.13$ at $p_{\rm T}=6$-7 GeV/$c$. Above $p_{\rm T}=7$ GeV/$c$, there is a significant rise in the nuclear modification factor, which reaches $R_{\rm{AA}} \approx0.4$ for $p_{\rm T}>30$ GeV/$c$. In peripheral collisions (70-80%), the suppression is weaker with $R_{\rm{AA}} \approx 0.7$ almost independently of $p_{\rm T}$. The measured nuclear modification factors are compared to other measurements and model calculations.Comment: 17 pages, 4 captioned figures, 2 tables, authors from page 12, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/284

Measurement of the polarisation of W bosons produced with large transverse momentum in pp collisions at sqrt(s) = 7 TeV with the ATLAS experiment

This paper describes an analysis of the angular distribution of W->enu and W->munu decays, using data from pp collisions at sqrt(s) = 7 TeV recorded with the ATLAS detector at the LHC in 2010, corresponding to an integrated luminosity of about 35 pb^-1. Using the decay lepton transverse momentum and the missing transverse energy, the W decay angular distribution projected onto the transverse plane is obtained and analysed in terms of helicity fractions f0, fL and fR over two ranges of W transverse momentum (ptw): 35 < ptw < 50 GeV and ptw > 50 GeV. Good agreement is found with theoretical predictions. For ptw > 50 GeV, the values of f0 and fL-fR, averaged over charge and lepton flavour, are measured to be : f0 = 0.127 +/- 0.030 +/- 0.108 and fL-fR = 0.252 +/- 0.017 +/- 0.030, where the first uncertainties are statistical, and the second include all systematic effects.Comment: 19 pages plus author list (34 pages total), 9 figures, 11 tables, revised author list, matches European Journal of Physics C versio

Observation of a new chi_b state in radiative transitions to Upsilon(1S) and Upsilon(2S) at ATLAS

The chi_b(nP) quarkonium states are produced in proton-proton collisions at the Large Hadron Collider (LHC) at sqrt(s) = 7 TeV and recorded by the ATLAS detector. Using a data sample corresponding to an integrated luminosity of 4.4 fb^-1, these states are reconstructed through their radiative decays to Upsilon(1S,2S) with Upsilon->mu+mu-. In addition to the mass peaks corresponding to the decay modes chi_b(1P,2P)->Upsilon(1S)gamma, a new structure centered at a mass of 10.530+/-0.005 (stat.)+/-0.009 (syst.) GeV is also observed, in both the Upsilon(1S)gamma and Upsilon(2S)gamma decay modes. This is interpreted as the chi_b(3P) system.Comment: 5 pages plus author list (18 pages total), 2 figures, 1 table, corrected author list, matches final version in Physical Review Letter

Search for displaced vertices arising from decays of new heavy particles in 7 TeV pp collisions at ATLAS

We present the results of a search for new, heavy particles that decay at a significant distance from their production point into a final state containing charged hadrons in association with a high-momentum muon. The search is conducted in a pp-collision data sample with a center-of-mass energy of 7 TeV and an integrated luminosity of 33 pb^-1 collected in 2010 by the ATLAS detector operating at the Large Hadron Collider. Production of such particles is expected in various scenarios of physics beyond the standard model. We observe no signal and place limits on the production cross-section of supersymmetric particles in an R-parity-violating scenario as a function of the neutralino lifetime. Limits are presented for different squark and neutralino masses, enabling extension of the limits to a variety of other models.Comment: 8 pages plus author list (20 pages total), 8 figures, 1 table, final version to appear in Physics Letters

Measurement of the inclusive isolated prompt photon cross-section in pp collisions at sqrt(s)= 7 TeV using 35 pb-1 of ATLAS data

A measurement of the differential cross-section for the inclusive production of isolated prompt photons in pp collisions at a center-of-mass energy sqrt(s) = 7 TeV is presented. The measurement covers the pseudorapidity ranges |eta|<1.37 and 1.52<=|eta|<2.37 in the transverse energy range 45<=E_T<400GeV. The results are based on an integrated luminosity of 35 pb-1, collected with the ATLAS detector at the LHC. The yields of the signal photons are measured using a data-driven technique, based on the observed distribution of the hadronic energy in a narrow cone around the photon candidate and the photon selection criteria. The results are compared with next-to-leading order perturbative QCD calculations and found to be in good agreement over four orders of magnitude in cross-section.Comment: 7 pages plus author list (18 pages total), 2 figures, 4 tables, final version published in Physics Letters

Measurement of D*+/- meson production in jets from pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

This paper reports a measurement of D*+/- meson production in jets from proton-proton collisions at a center-of-mass energy of sqrt(s) = 7 TeV at the CERN Large Hadron Collider. The measurement is based on a data sample recorded with the ATLAS detector with an integrated luminosity of 0.30 pb^-1 for jets with transverse momentum between 25 and 70 GeV in the pseudorapidity range |eta| < 2.5. D*+/- mesons found in jets are fully reconstructed in the decay chain: D*+ -> D0pi+, D0 -> K-pi+, and its charge conjugate. The production rate is found to be N(D*+/-)/N(jet) = 0.025 +/- 0.001(stat.) +/- 0.004(syst.) for D*+/- mesons that carry a fraction z of the jet momentum in the range 0.3 < z < 1. Monte Carlo predictions fail to describe the data at small values of z, and this is most marked at low jet transverse momentum.Comment: 10 pages plus author list (22 pages total), 5 figures, 1 table, matches published version in Physical Review

Search for scalar top quark pair production in natural gauge mediated supersymmetry models with the ATLAS detector in pp collisions at sqrt(s) = 7 TeV

The results of a search for pair production of the lighter scalar partners of top quarks in 2.05 fb-1 of pp collisions at sqrt(s) =7 TeV using the ATLAS experiment at the LHC are reported. Scalar top quarks are searched for in events with two same flavour opposite-sign leptons (electrons or muons) with invariant mass consistent with the Z boson mass, large missing transverse momentum and jets in the final state. At least one of the jets is identified as originating from a b-quark. No excess over Standard Model expectations is found. The results are interpreted in the framework of R-parity conserving, gauge mediated Supersymmetry breaking `natural' scenarios, where the neutralino is the next-to-lightest supersymmetric particle. Scalar top quark masses up to 310 GeV are excluded for the lightest neutralino mass between 115 GeV and 230 GeV at 95% confidence level, reaching an exclusion of the scalar top quark mass of 330 GeV for the lightest neutralino mass of 190 GeV. Scalar top quark masses below 240 GeV are excluded for all values of the lightest neutralino mass above the Z boson mass.Comment: 7 pages plus author list (20 pages total), 4 figures, 1 table, matches published PLB versio