One-dimensional Dirac oscillator in a thermal bath

We analyze the one-dimensional Dirac oscillator in a thermal bath. We found that the heat capacity is two times greater than the heat capacity of the one-dimensional harmonic oscillator for higher temperatures.Comment: 4 pages, 3 figures, to appear in Physics Letters

The Structure Of The D49 Phospholipase A2 Piratoxin Iii From Bothrops Pirajai Reveals Unprecedented Structural Displacement Of The Calcium-binding Loop: Possible Relationship To Cooperative Substrate Binding

Snake venoms are rich sources of phospholipase A2 homologues, both active calcium-binding Asp49 enzymes and essentially inactive Lys49 proteins. They are responsible for multiple pharmacological effects, some of which are dependent on catalytic activity and others of which are not. Here, the 2.4 Å X-ray crystal structure of an active Asp49 phospholipase A2 from the venom of the snake Bothrops pirajai, refined to conventional and free R values of 20.1 and 25.5%, respectively, is reported. Unusually for phospholipases A2, the dependence of the enzyme rate on the substrate concentration is sigmoidal, implying cooperativity of substrate binding. The unprecedented structural distortion seen for the calcium-binding loop in the present structure may therefore be indicative of a T-state enzyme. An explanation of the interaction between the substrate-binding sites based on the canonical phospholipase A2 dimer is difficult. 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Purification And Preliminary Crystallographic Analysis Of A New Lys49-pla2 From B. Jararacussu

BjVIII is a new myotoxic Lys49-PLA2 isolated from Bothrops jararacussu venom that exhibits atypical effects on human platelet aggregation. To better understand the mode of action of BjVIII, crystallographic studies were initiated. Two crystal forms were obtained, both containing two molecules in the asymmetric unit (ASU). Synchrotron radiation diffraction data were collected to 2.0 Å resolution and 1.9 Å resolution for crystals belonging to the space group P2 12 12 1 (a = 48.4 Å, b = 65.3 Å, c = 84.3 Å) and space group P3 121 (a = b = 55.7 Å, c = 127.9 Å), respectively. Refinement is currently in progress and the refined structures are expected to shed light on the unusual platelet aggregation activity observed for BjVIII. © 2008 by the authors; licensee Molecular Diversity Preservation International.95736750van Deenen, L., de Haas, G., Heemskerk, C.H., Hydrolysis of synthetic mixed-acid phosphatides by phospholipase A from human pancreas (1963) Biochim Biophys Acta, 67, pp. 295-304Higuchi, D.A., Barbosa, C.M.V., Bincoletto, C., Chagas, J.R., Magalhaes, A., Richardson, M., Sanchez, E.F., Pesquero, J., L.. Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom (2007) Biochimie, 89, pp. 319-328Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kézdy, F.J., Heinrikson, R.L., A new class of phospholipases A2 with lysine in place of aspartate 49. 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Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom (2002) Biochem Pharmacol, 64, pp. 723-732Fuly, A.L., Soares, A.M., Marcussi, S., Giglio, J.R., Guimarães, J.A., Signal transduction pathways involved in the platelet aggregation induced by a D-49 phospholipase A2 isolated from Bothrops jararacussu snake venom (2004) Biochimie, 86, pp. 731-739Magro, A.J., Murakami, M.T., Marcussi, S., Soares, A.M., Arni, R.K., Fontes, M.R.M., Crystal structure of an acidic platelet aggregation inhibitor and hypotensive phospholipase A2 in the monomeric and dimeric states: Insights into its oligomeric state (2004) Biochem Biophys Res Commun, 323, pp. 24-31Toyama, M.H., Carneiro, E.M., Marangoni, S., Barbosa, R.L., Corso, G., Boschero, A.C., Biochemical characterization of two crotamine isoforms isolated by a single step RP-HPLC from Crotalus durissus terrificus (South American rattlesnake) venom and their action on insulin secretion by pancreatic islets (2000) Biochim Biophys Acta, 1474, pp. 56-60Fonseca, F.V., Antunes, E., Morganti, R.P., Monteiro, H.S.A., Martins, A.M.C., Toyama, D.O., Marangoni, S., Toyama, M., H.. 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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±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. 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Biological And Structural Characterization Of A New Pla 2 From The Crotalus Durissus Collilineatus Venom

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A 2 (PLA 2) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80 with crotalic PLA 2s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50 if compared to other sources of PLA 2s such as from the Bothrops snake venom. Also, this PLA 2 induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and q-BPB showed a similar reduction. The q-BPB did not reduce significantly the myotoxic activity induced by the PLA 2, but the anhydrous acetic acid treatment and the pre-incu-bation of PLA 2 with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiflorae (Gram-negative), which was drastically reduced by incubation of this PLA 2 with q-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or b-wing region may be involved in the binding of this PLA 2 to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein. © 2005 Springer Science+Business Media, Inc.242103112Aguiar, A.S., Alves, C.R., Melgarejo, A., Giovanni-De-Simone, S., (1996) Toxicon, 34, pp. 555-565Babu, A.S., Gowda, T.V., (1994) Toxicon, 32, pp. 749-752Bon, C., Changeux, J.P., Jeng, T.W., Fraenkel-Conrat, H., (1979) Eur. J. Biochem., 99, pp. 471-481Breithaupt, H., (1976) Toxicon, 14, pp. 221-233Condrea, E., Fletcher, J.E., Rapuano, B.E., Yang, C.C., Rosenberg, P., (1981) Toxicon, 19, pp. 705-720Dennis, E.A., (1997) Trends Biochem. Sci., 22, pp. 1-12Faure, G., Bon, C., (1988) Biochemistry, 27, pp. 730-738Fuentes, L., Hernandez, M., Nieto, M.L., Sanchez-Crespo, M., (2002) FEBS Lett., 531, pp. 7-11Gowda, T.V., Middlebrook, J.L., (1994) Toxicon, 32, pp. 955-964Habermann, E., Breithaupt, H., (1978) Toxicon, 16, pp. 19-30Holzer, M., MacKessy, S.P., (1996) Toxicon, 34, pp. 1149-1155Kudo, I., Murakami, M., (2002) Prostag. Oth. Lipid Mediat., 68, pp. 3-58. , 69Lambeau, G., Lazdunski, M., (1999) TIPS, 20, pp. 162-170Landucci, E.C., Condino-Neto, A., Perez, A.C., Hyslop, S., Corrado, A.P., Novello, J.C., Marangoni, S., Denucci, G., (1994) Toxicon, 32, pp. 217-226Lomonte, B., Angulo, Y., Calderon, L., (2003) Toxicon, 42, pp. 885-901Murakami, M., Kudo, I., (2002) J. Biochem (Tokyo), 131, pp. 285-292Oliveira, D.G., Toyama, M.H., Novello, J.C., Beriam, L.O., Marangoni, S., (2002) J. Protein Chem, 21, pp. 161-168Prijatelj, P., Sribar, J., Ivanovski, G., Krizaj, I., Gubensek, F., Pungercar, J., (2003) Eur. J. Biochem., 270, pp. 3018-3025Schagger, H., Von Jagow, G., (1987) Anal. Biochem., 166, pp. 368-379Scott, D.L., Achari, A., Vidal, J.C., Sigler, P.B., (1992) J. Biol. Chem., 267, pp. 22645-22657Soares, A.M., Marcussi, S., Stabeli, R.G., Franca, S.C., Giglio, J.R., Ward, R.J., Arantes, E.C., (2003) Biochem. Biophys. Res. Commun., 302, pp. 193-200Soares, A.M., Andrião-Escaso, S.H., Angulo, Y., Lomonte, B., Gutierrez, J.M., Marangoni, S., Toyama, M.H., Giglio, J.R., (2000) Arch. Biochem. Biophys., 373, pp. 7-15Toyama, M.H., Costa, P.D., Novello, J.C., Oliveira, B., Giglio, J.R., Cruz-Höfling, M.A., Marangoni, S., (1999) J. Protein Chem, 18, pp. 371-378Toyama, M.H., Deoliveira, D.G., Beriam, L.O., Novello, J.C., Rodrigues-Simioni, L., Marangoni, S., (2003) Toxicon, 41, pp. 1033-1038Verheij, H.M., Volwerk, J.J., Jansen, E.H., Puyk, W.C., Dijkstra, B.W., Drenth, J., De Haas, G.H., (1980) Biochemistry, 19, pp. 743-750Yang, C.C., (1997) Venom Phospholipase A2 Enzymes: Structure, Function and Mechanism, pp. 185-204. , Kini, R. M. (ed.), Wiley, Chichester U

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOIn this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA(2), crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 mu M/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA(2), thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this proteinSpringer253183192FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2004/00040-3300211.2004New York, N

Structural And Functional Characterization Of Basic Pla2 Isolated From Crotalus Durissus Terrificus Venom

The venom of Crotalus durissus terrificus was fractionated by reverse-phase HPLC to obtain crotapotins (F5 and F7) and PLA2 (F15, F16, and F17) of high purity. The phospholipases A2 (PLA2s) and crotapotins showed antimicrobial activity against Xanthomonas axonopodis pv. passiflorae, although the unseparated crotoxin did not. The F17 of the PLA 2 also revealed significant anticoagulant activity, althrough for this to occur the presence of Glu 53 and Trp 61 is important. The F17 of the PLA2 showed allosteric behavior in the presence of a synthetic substrate. The amino acid sequence of this PLA2 isoform, determined by automatic sequencing, was HLLQFNKMLKFETRK NAVPFYAFGCYCGWGGQRRPKDATDRCCFVHDCCYEKVTKCNTKWDFYRYSLKSGY ITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPDSRCREPSETC. Analysis showed that the sequence of this PLA2 isoform differed slightly from the amino acid sequence of the basic crotoxin subunit reported in the literature. The homology with other crotalid PLA2 cited in the lit-erature varied from 60% to 90%. The pL was estimated to be 8.15, and the calculated molecular weight was 14664.14 as determined by Tricine SDS-PAGE, two-dimensional electrophoresis, and MALDI-TOFF. These results also suggested that the enzymatic activity plays an important role in the bactericidal effect of the F17 PLA2 as well as that of anticoagulation, although other regions of the molecule may also be involved in this biological activity. © 2002 Plenum Publishing Corporation.213161168Aird, S.D., Kaiser, I.I., (1985) Biochemistry, 24, pp. 7054-7058Aird, S.D., Kruggel, W.G., Kaiser, I.I., (1985) Toxicon, 28, pp. 669-673Anderson, N.L., Anderson, N.G., (1991) Electrophoresis, 12, pp. 883-906Beghini, D.G., Toyama, M.H., Hyslop, S., Sodek, L., Novello, J.C., Marangoni, S., (2000) J. Prot. Chem., 19, pp. 603-607Breithaupt, H., (1976) Toxicon, 14, pp. 221-233Carredano, B., Westerlind, B., Persson, M., Saareinen, S., Ramaswamy, D., Eaker, H., Eklund, M.W., (1998) Toxicon, 36, pp. 75-92Cho, W., Kezdy, F.J., (1991) Methods Enzymol., 23, pp. 75-79Faure, G., Choumet, V., Bouchier, C., Camoin, L., Guillaume, J.L., Monegier, B., Vuilhorgne, M., Bon, C., (1994) Eur. J. Biochem., 223, pp. 161-164Faure, G., Guillaume, J.L., Camoin, L., Saliou, B., Bon, C., (1991) Biochemistry, 30, pp. 8074-8083Gutierrez, J.M., Lomonte, B., (1995) Toxicon., 33, pp. 1405-1424Habermann, E., Breithaupt, H., (1978) Toxicon., 16, pp. 19-30Hendon, R.A., Fraenkel-Conrat, H., (1976) Toxicon., 14, pp. 283-289Holzer, M., Mackessy, S.P., (1996) Toxicon., 34, pp. 1149-1155Kini, R.M., Evans, H.J., (1989) Toxicon., 27, pp. 613-635Kini, R.M., Evans, H.J., (1987) J. Biol. Chem., 262, pp. 14402-14407Lambeau, G., Ancian, P., Nicolas, J.P., Cupillard, L., Zvaritch, E., Lazdunski, M., (1996) Seances Soc. Biol. Fil., 190, pp. 425-435Lomonte, B., Moreno, E., Tarkowski, A., Hanson, L.A., Maccarana, M., (1994) J. Biol. Chem., 269, pp. 29867-29873Paramo, L., Lomonte, B., Pizarro-Cerda, J., Bengoechea, J.A., Gorvel, J.P., Moreno, E., (1998) Eur. J. Biochem., 253, pp. 452-461Pieterson, W.A., Volwerk, J.J., Haas, G.H., (1974) Biochemistry, 13, pp. 1439-1445Rubsamen, K., Breithaupt, H., Habermann, E., (1971) Arch. Pharmacol., 270, pp. 274-288Schagger, H., Von Jagow, G., (1987) Anal. Biochem., 166, pp. 368-379Selistre De Araujo, H.S., White, S.P., Ownby, C.L., (1996) Arch. Biochem. Biophys., 326, pp. 21-30Shiomi, K.A., Kazama, A., Shimakura, K., Nagashima, Y., (1998) Toxicon, 36, pp. 589-599Soares, A.M., Andrião-Escaso, S.H., Bortoleto, R.K., Rodrigues-Simioni, L., Arni, R.K., Ward, R.J., Gutierrez, J.M., Giglio, J.R., (2001) Arch. Biochem. Biophys., 387, pp. 188-196Toyama, M.H., Soares, A.M., Wen-Hwa, L., Polikarpov, I., Giglio, J.R., Marangoni, S., (2000) Biochimie, 82, pp. 245-250Verheij, H.M., Boffa, M.C., Rothen, C., Bryckaert, M.C., Verger, R., De Hass, G.H., (1980) Eur. J. Biochem., 112, pp. 25-32Zhao, K., Zhou, Y., Lin, Z., (2000) Toxicon., 38, pp. 901-91

An Evaluation Of 3-rhamnosylquercetin, A Glycosylated Form Of Quercetin, Against The Myotoxic And Edematogenic Effects Of Spla 2 From Crotalus Durissus Terrificus

This paper shows the results of quercitrin effects on the structure and biological activity of secretory phospholipase (sPLA2) from Crotalus durissus terrificus, which is the main toxin involved in the pharmacological effects of this snake venom. According to our mass spectrometry and circular dichroism results, quercetin was able to promote a chemical modification of some amino acid residues and modify the secondary structure of C. d. terrificus sPLA2. Moreover, molecular docking studies showed that quercitrin can establish chemical interactions with some of the crucial amino acid residues involved in the enzymatic activity of the sPLA2, indicating that this flavonoid could also physically impair substrate molecule access to the catalytic site of the toxin. Additionally, in vitro and in vivo assays showed that the quercitrin strongly diminished the catalytic activity of the protein, altered its Vmax and Km values, and presented a more potent inhibition of essential pharmacological activities in the C. d. terrificus sPLA2, such as its myotoxicity and edematogenic effect, in comparison to quercetin. Thus, we concluded that the rhamnose group found in quercitrin is most likely essential to the antivenom activities of this flavonoid against C. d. terrificus sPLA2. © 2014 Daniela de Oliveira Toyama et al

Renal Effects Of Supernatant From Macrophages Activated By Crotalus Durissus Cascavella Venom: The Role Of Phospholipase A2 And Cyclooxygenase

In Brazil, the genus Crotalus is responsible for approximately 1500 cases of snakebite annually. The most common complication in the lethal cases is acute renal failure, although the mechanisms of the damaging effects are not totally understood. In this work, we have examined the renal effects caused by a supernatant of macrophages stimulated by Crotalus durissus cascavella venom as well as the potential role of phospholipase A2 and cyclo-oxygenase. Rat peritoneal macrophages were collected and placed in a RPMI medium and stimulated by crude Crotalus durissus cascavella venom (1, 3 or 10 μg/ml) for 1 hr. They were then washed and kept in a culture for 2 hr. The supernatant (1 ml) was tested in an isolated perfused rat kidney. The first 30 min. of each experiment were used as an internal control, and the supernatant was added to the system after this period. All experiments lasted 120 min. A study of toxic effect on perfusion pressure, glomerular filtration rate, urinary flow, percent of sodium tubular transport and percent of proximal tubular sodium transport was made. The lowest concentration of venom (1 μg/ml) was not statistically different from the control values. The most intense effects were seen at 10 μg/ml for all renal parameters. The infusion of the supernatant of macrophages stimulated with crude venom (3 or 10 μg/ml) increased the perfusion pressure, glomerular filtration rate and urinary flow, decreased the percent of sodium tubular transport and percent of proximal tubular sodium transport. Dexamethasone (10 μM) and quinacrine (10 μM) provided protection against the effect of the venom on glomerular filtration rate, urinary flow, percent of sodium tubular transport, percent of proximal tubular sodium transport and perfusion pressure. 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