Membrane-associated proteins of ejaculated sperm from Morada Nova rams

AbstractThe objective was to describe the profile of membrane proteins from sperm of tropically adapted Morada Nova rams (N = 5). Samples from protein-enriched fractions of ejaculated sperm (containing 400 μg of protein) were separated by two-dimensional electrophoresis and respective maps analyzed using PDQuest software (version 7.3.0; Bio-Rad). Proteins were identified using tandem mass spectrometry. Also, membrane proteins were incubated with antibodies against binder of sperm protein (BSP) 1 and bodhesin 2 (Bdh-2), components of vesicular gland secretion. For membrane proteins of ejaculated sperm, an average of 133 ± 4.6 spots were detected per gel, of which, 107 spots were consistently present on all gels. Sixty-eight spots and 37 proteins were identified using mass spectrometry, corresponding to 71.6% of the intensity of all spots detected. Three major spots identified as ram seminal vesicle protein (RSVP) 14 represented approximately 30% of the intensity of all spots. Two of the most intense spots in the gel reacted against anti-BSP1, at 14 kDa. In addition, four low molecular weight spots reacted with anti-Bdh-2 antibodies. Proteins RSVP and Bdh-2 belong to the BSP and spermadhesin families, respectively, and were previously reported as major components of ram seminal proteins. Additional proteins identified in the sperm membrane two-dimensional maps included alpha-2-heparan sulfate-glycoprotein, plasma glutamate carboxypeptidase, arylsulfatase A, cathelicidin, heat shock protein 70 kDa, angiotensin-converting enzyme, leucine aminopeptidase, and clusterin. Some proteins were present as multiple isoforms, such as tubulin (12), alpha-2-heparan sulfate-glycoprotein (5), ATP synthase (5), Bdh-2 (4) and RSVP14 (3). Based on gene ontology analysis, the most common biological processes associated with the membrane proteins were cellular processes (34%), response to stimulus (14%), and metabolic processes (11%). Binding (37%) and catalytic activity (32%) corresponded to the most frequent molecular functions for those proteins. In conclusion, we identified a diverse cohort of components of membrane proteins in ram sperm. Major proteins previously reported in seminal plasma, such as RSVP14 and Bdh-2, were also extracted from sperm membranes. Knowledge of sperm proteins is crucial for elucidating mechanisms underlying their association with sperm function

Software performance of the ATLAS track reconstruction for LHC run 3

Charged particle reconstruction in the presence of many simultaneous proton–proton (pp) collisions in the LHC is a challenging task for the ATLAS experiment’s reconstruction software due to the combinatorial complexity. This paper describes the major changes made to adapt the software to reconstruct high-activity collisions with an average of 50 or more simultaneous pp interactions per bunch crossing (pileup) promptly using the available computing resources. The performance of the key components of the track reconstruction chain and its dependence on pile-up are evaluated, and the improvement achieved compared to the previous software version is quantified. For events with an average of 60 pp collisions per bunch crossing, the updated track reconstruction is twice as fast as the previous version, without significant reduction in reconstruction efficiency and while reducing the rate of combinatorial fake tracks by more than a factor two

No Role For Enzymatic Activity Or Dantrolene-sensitive Ca2+ Stores In The Muscular Effects Of Bothropstoxin, A Lys49 Phospholipase A2 Myotoxin

The role of low levels of phospholipase A2 (PLA2) activity and intracellular Ca2+ stores in the pharmacological action of bothropstoxin (BthTX), a myotoxic Lys49 PLA2 homologue isolated from the venom of Bothrops jararacussu, was investigated. We examined the muscular effects of BthTX in the mouse diaphragm and its PLA2 activity in radiolabeled human and rat primary cultures of skeletal muscle. Although it is a Lys49 PLA2 homologue, BthTX had a low, but easily detectable, level of enzymatic activity relative to two Asp49 PLA2 enzymes from Naja naja kaouthia and Naja naja atra venoms, and this activity was reduced by about 85% in the presence of Sr2+ (4.0 mM). However, the replacement of 1.8 mM Ca2+ by 4 mM Sr2+ did not alter the BthTX-induced contracture and blockade of the muscle twitch tension. In addition, Sr2+ decreased by 50% the time required to cause 50% paralysis, and evoked approximately a four-fold increase in the number of spontaneous spikes. In isolated sarcoplasmic reticulum preparations, BthTX opened the intracellular Ca2+ release channel (ryanodine receptor) and lowered the threshold of Ca2+-induced Ca2+ release by a second, as yet unidentified, mechanism. However, in intact muscle, dantrolene, an antagonist of some forms of intracellular Ca2+ release, had no effect on the actions of BthTX. These findings do not support any role for the low levels of PLA2 activity, or dantrolene-sensitive intracellular Ca2+ stores, in the action of BthTX. The mechanism whereby Sr2+ stimulates the pharmacological activity of BthTX remains to be clarified. © 1995.331114791489Cintra, Marangoni, Oliveira, Giglio, Bothropstoxin-I: amino acid sequence and function (1993) J. Prot. Chem., 12, pp. 57-64Condrea, Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A2 from Agkistrodon piscivorus piscivorus snake venom. A reconsideration (1989) Toxicon, 27, pp. 705-706Dettbarn, Palade, Arachidonic acid-induced Ca2+ release from isolated sarcoplasmic reticulum (1993) Biochem. Pharmac., 45, pp. 1301-1309Dhillon, Condrea, Maraganore, Heinrikson, Benjamin, Rosenberg, Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A2 from Agkistrodon piscivorus piscivorus snake venom (1987) Biochem. Pharmac., 36, pp. 1723-1730Díaz, Gutiérrez, Lomonte, Nüñez, P-Bromophenacyl bromide modification of Bothrops asper myotoxin II, a lysine-49 phospholipase A2, affects its pharmacological activities (1993) Toxicon, 31, pp. 1202-1206Fletcher, Rapuano, Condrea, Yang, Rosenberg, Relationship between catalysis and toxicoloical properties of three phospholipases A2 from elapid snake venoms (1981) Toxic. appl. Pharmac., 59, pp. 375-388Fletcher, Tripolitis, Erwin, Hanson, Rosenberg, Conti, Beech, Fatty acids modulate calcium-induced calcium release from skeletal muscle heavy sarcoplasmic reticulum fractions: implications for malignant hyperthermia (1990) Biochem. cell. Biol., 68, pp. 1195-1201Fletcher, Jiang, Gong, Smith, Snake venom cardiotoxins and bee venom melittin activate phospholipase C activity in primary cultures of skeletal muscle (1991) Biochem. cell. Biol., 69, pp. 274-281Fletcher, Trioplitis, Beech, Species difference in modulation of calcium release by Naja naja kaouthia snake venom cardiotoxin in terminal cisternae from human and equine skeletal muscle (1993) Toxicon, 31, pp. 43-51Fletcher, Jiang, Middlebrook, Antibodies having markedly different effects in enzymatic activity and induction of acetylcholine release by two presynaptically-acting phospholipase A2 neurotoxins (1995) Biochem. Pharmac., 49, pp. 381-388Ghassemi, Dhillon, Rosenberg, β-Bungarotoxin-induced phospholipid hydrolysis in rat brain synaptosomes: effect of replacement of calcium by strontium (1988) Toxicon, 26, pp. 509-514Harris, Johnson, Further observations on the pathological responses of rat skeletal muscle to toxins isolated from the venom of the Australian tiger snake Notechis scutatus scutatus (1975) Clinical and Experimental Pharmacology and Physiology, 5, pp. 587-600Hawgood, Smith, The mode of action at the mouse neuromuscular function of the phopholipase A-crotapotin complex isolated from venom of the South American rattlesnake (1977) Br. J. Pharmac., 61, pp. 597-606Heluany, Homsi-Brandeburgo, Giglio, Prado-Franceschi, Rodrigues-Simioni, Effects induced by bothropstoxin, a component from Bothrops jararacussu snake venom on mouse and chick muscle preparations (1992) Toxicon, 30, pp. 1203-1210Homsi-Brandeburgo, Queiroz, Santo Neto, Rodrigues-Simioni, Giglio, Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin (1988) Toxicon, 26, pp. 615-627Kini, Evans, A model to explain the pharmacological effects of snake venom phospholipases A2 (1989) Toxicon, 27, pp. 613-635Li, Yu, Zhu, Jain, Tsai, Phospholipase A2 engineering: structural and functional roles of the highly conserved active site residue aspartate-49 (1994) Biochemistry, 33, pp. 14,714-14,722Lomonte, Gutiérrez, A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo) (1989) Toxicon, 27, pp. 725-733Melo, Suarez-Kurtz, Release of sarcoplasmic enzymes from skeletal muscle by Bothrops jararacussu venom: antagonism by heparin and by the serum of South American marsupials (1988) Toxicon, 26, pp. 87-95Nelson, Nelson, Intra- and extraluminal sarcoplasmic reticulum membrane regulatory sites for Ca2+-induced release (1990) FEBS Lett., 263, pp. 292-294Ownby, Fletcher, Colberg, Cardiotoxin 1 from cobra (Naja naja atra) venom causes necrosis of skeletal muscle in vivo (1993) Toxicon, 31, pp. 697-709Van den Bergh, Slotboom, Verheij, De Haas, The role of aspartic acid-49 in the active site of phospholipase A2 (1988) Eur. J. Biochem., 176, pp. 353-357Wieland, Fletcher, Rosenberg, Gong, Malignant hyperthermia slow sodium current in cultured human muscle cells (1989) Am J Physiol, 257, pp. C759-C765Yudkowsky, Beech, Fletcher, Mytoxin a reduces the threshold for calcium-induced calcium release in skeletal muscle (1994) Toxicon, 32, pp. 273-27

Antibothropic action of Casearia sylvestris Sw. (flacourtiaceae) extracts

Casearia sylvestris Sw., popularly known in Brazil as 'guacatonga', has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects22678479

Antibothropic Action Of Casearia Sylvestris Sw. (flacourtiaceae) Extracts

Casearia sylvestris Sw., popularly known in Brazil as 'guaçatonga', has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C. sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects. Copyright © 2008 John Wiley & Sons, Ltd.226784790Bjarnason, J.B., Fox, J.W., Hemorrhagic metalloproteinases from snake venoms (1994) Pharmacol Ther, 62, pp. 325-372Borges, M.H., Soares, A.M., Rodrigues, V.M., Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A 2 (2000) Comp Biochem Physiol B Biochem Mol Biol, 127, pp. 21-30Borges, M.H., Soares, A.M., Rodrigues, V.M., Neutralization of proteases from Bothrops snake venoms by aqueous extract from Casearia sylvestris (Flacourtiaceae) (2001) Toxicon, 39, pp. 1863-1869Bülbring, E., Observation on the isolated phrenic nerve diaphragm preparation of the rat (1946) Br J Pharmacol, 1, pp. 38-61De Oliveira, M., Cavalcante, W.L., Arruda, E.A., Melo, P.A., Dal-Pai Silva, M., Gallaci, M., Antagonism of myotoxic and paralyzing activities of bothropstoxin-l by suramin (2003) Toxicon, 42, pp. 373-379Girish, K.S., Kemparaju, K., Inhibition of Naja naja venom hyaluronidase by plant-derived bioactive components and polysaccharides (2005) Biochemistry (Moscow), 70, pp. 948-952Gutierrez, J.M., Romero, M., Díaz, C., Borkow, G., Ovadia, M., Isolation and characterization of a metalloproteinase with weak hemorrhagic activity from the venom of the snake Bothrops asper (terciopelo) (1995) Toxicon, 33, pp. 19-29Harbone, J.B., (1998) Phytochemical Methods: A Guide to Modern Techniques of Plants Analysis, , 3rd edn, Chapman & Hall: LondonHavsteen, B., Flavonoids, a class of natural products of high pharmacological potency (1983) Biochem Pharmacol, 32, pp. 1141-1148Heluany, N.F., Homsi-Brandeburgo, M., Giglio, J.R., Prado-Franceschi, J., Rodrigues-Simioni, L., Effects induced by bothrop-stoxin, a component from Bothrops jararacussu snake venom, on mouse and chick muscle preparations (1992) Toxicon, 30, pp. 1203-1210Iwanaga, S., Suzuki, T., Enzymes in snake venoms (1979) Snake Venoms. Handbook of Experimental Pharmacology, pp. 61-158. , Lee CY ed, Springer: New YorkJin, G.Z., Yamagata, Y., Tomita, K., Structure of rutin pentamethanol (1990) Chem Pharm Bull, 38, pp. 297-300Khan, A.A., Ashfaq, A., Ali, M.N., (1979) Pharmacognostic Studies of Selected Indigenous Plants of Pakistan, , Pakistan Forest Institute. Spinezer Printers: PeshawarMahmood A, Ahmad M, Jabeen A, Zafar M, Nadeem S. 2005. Pharmacognostic studies of some indigenous medicinal plants of Pakistan. Available in http://www.siu.edu/∼ebl/leaflets/abid.htm. Access in 06/03/2005Maistro, E.L., Carvalho, J.C., Mantovani, M.S., Evaluation of the genotoxic potential of the Casearia sylvestris extract on HTC and V79 cells by the comet assay (2004) Toxicol In Vitro, 18, pp. 337-342Markland, F.S., Snake venoms and the hemostatic system (1998) Toxicon, 36, pp. 1749-1800Matrisian, L.M., The matrix-degrading metalloproteinases (1992) BioEssays, 14, pp. 455-463Milani Junior, R., Jorge, M.T., de Campos, F.P., Snake bites by the jararacuçu (Bothrops jararacussu): Clinicopatho-logical studies of 29 proven cases in Sao Paulo State, Brazil (1997) Q J Med, 90, pp. 323-334(2001) Manual de Diagnóstico e Tratamento de Acidentes por Animais Pegonhentos, , Ministério da Saúde do Brasil, 2nd edn. Fundação Nacional da Saúde: BrasíliaMors, W.B., Nascimento, M.C., Pereira, B.M., Pereira, N.A., Plant natural products active against snake bite - the molecular approach (2000) Phytochemistry, 55, pp. 627-642Oshima-Franco, Y., Alves, C.M.V., Andréo Filho, N., Neutralization of the neuromuscular activity of bothropstoxin-l, a myotoxin from Bothrops jararacussu snake venom, by a hydroalcoholic extract of Casearia sylvestris Sw. (guaçatonga) (2005) J Venom Anim Toxins Trop Dis, 11, pp. 465-478Oshima-Franco, Y., Hyslop, S., Cintra, A.C.O., Giglio, J.R., Cruz-Höfling, M.A., Rodrigues-Simioni, L., Neutralizing capacity of commercial bothropic antivenom against Bothrops jararacussu venom and bothropstoxin-l (2000) Muscle Nerve, 23, pp. 1832-1839Oshima-Franco, Y., Leite, G.B., Dal Belo, C.A., The presynaptic activity of bothropstoxin-l, a myotoxin from Bothrops jararacussu snake venom (2004) Basic Clin Pharmacol Toxicol, 95, pp. 175-182Oshima-Franco, Y., Leite, G.B., Silva, G.H., Neutralization of the pharmacological effects of bothropstoxin-l from Bothrops jararacussu (jararacuçu) venom by crotoxin antiserum and heparin (2001) Toxicon, 39, pp. 1477-1485Oshima-Franco, Y., Leite, G.B., Valério, A.A., Rabbit antivenom efficacy against myotoxic and neurotoxic activities of Bothrops jararacussu venom and bothropstoxin-l (2002) J Venom Anim Toxins, 8, pp. 226-243Pessini, A.C., Takao, T.T., Cavalheiro, E.C., A hyaluronidase from Tityus serrulatus scorpion venom: Isolation, characterization and inhibition by flavonoids (2001) Toxicon, 39, pp. 1495-1504Randazzo-Moura, P., Leite, G.B., Silva, G.H., Study of myotoxicity of bothropstoxin-l (BthTX-l) using manganese (Mn 2+) in mouse phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations (2006) Braz J Morphol Sci, 23, pp. 171-184Rates, S.M.K., Plants as source of drugs (2001) Toxicon, 39, pp. 603-613Rodrigues-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., Prado-Franceschi, J., Cintra, A.C.O., Giglio, J.R., Jiang, M.S., Fletcher, J.E., No role for enzymatic activity or dantrolene-sensitive Ca 2+ stores in the muscular effects of bothropstoxin, a Lys49 phospholipase A 2 myotoxin (1995) Toxicon, 33, pp. 1479-1489Rosenfeld, G., Symptomatology, pathology and treatment of snakebites in South America (1971) Venomous Animals and their Venoms, pp. 345-384. , Bucherl W, Buckley E eds, Academic Press: New YorkRucavado, A., Escalante, T., Gutiérrez, J.M., Effect of the metalloproteinase inhibitor batimastat in the systemic toxicity induced by Bothrops asper snake venom: Understanding the role of metalloproteinases in envenomation (2004) Toxicon, 43, pp. 417-424Selistre, H.S., Queiroz, L.S., Cunha, O.A., De Souza, G.E., Giglio, J.R., Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) snake venom (1990) Toxicon, 28, pp. 261-273Simōes CMO, Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR. 2004. Farmacognosia: da Planta ao Medicamento, 5th edn. UFRGS/UFSC: Porto Alegre/FlorianópolisSoares, A.M., Oshima-Franco, Y., Vieira, C.A., Mn 2+ ions reduce the enzymatic and pharmacological activities of bothropstoxin-l, a myotoxic Lys49 phospholipase A 2 homologue from Bothrops jararacussu snake venom (2002) Int J Biochem Cell Biol, 34, pp. 668-677Yingprasertchai, S., Bunyasrisawt, S., Ratanabanangkoon, K., Hyaluronidase inhibitors (sodium cromoglycate and sodium auro-thiomalate) reduce the local tissue damage and prolong the survival time of mice injected with Naja kaouthia and Calloselasma rhodostoma venoms (2003) Toxicon, 42, pp. 635-64