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

Toxic effects of glibenclamide in fetuses of normoglycemic rats: an alternative therapy for gestational diabetes mellitus

Abstract Gestational diabetes mellitus (GDM) is defined as glucose intolerance first diagnosed during the second or third trimester of pregnancy. The treatment aims at glycemic control through changes in the patient's diet with or without exercise, but some patients need insulin therapy. An alternative would be to use oral hypoglycemic agents such as glibenclamide (GLIB). The present study aims to analyze the toxic effects of GLIB in fetuses of pregnant rats which received 5 or 20mg/kg doses of GLIB. Glycemic dosage reveals no significant difference between control (deionized water) and treated groups, showing that these concentrations of GLIB were not effective to cause hypoglycemia in rats. The vitality of the fetuses in all groups was 100%. GLIB administration promoted increase in weight and significant changes in measures of external morphological parameters of treated fetuses. Histological analysis revealed that liver lobes, lobules and central lobular veins were well defined for all treatments. However, GLIB animals presented a light brownish precipitate into the center-lobular veins and in the liver parenchyma among the hepatocytes. These results indicated a possible passage of the drug through the blood-placental membrane, without serious changes that impair the development of neither bone tissue, nor the liver of these animals

Toxic effects of the administration of Mikania glomerata Sprengel during the gestational period of hypertensive rats

Herbal medicine is an ancient practice that has been gaining acceptance of the medical class through scientific studies that prove its effectiveness. However, its use should still be cautious. Medicinal plants have potential toxic effects not yet discovered, and may have unproven interactions with other medications. The use of drugs during pregnancy is still very dangerous and vigorously studied; however, there are few studies of herbal medicines in pregnant women. Existing studies prioritize on teratogenic or abortifacient effects. The aim of this study was to analyze the toxic effects of Mikania glomerata Sprengel administration, popularly known as “guaco” during the gestational period of hypertensive rats. For this experimental groups consisting of pregnant Wistar rats received treatments with guaco extract (1 to 2 mL). In order to analyze the possible toxic effects of guaco during pregnancy, weight gain of rats was assessed during pregnancy; reproductive performance of rats, morphological parameters, and fetal placental histology were compared. Although some parameters presented significant differences, we can conclude that changes prioritized by literature, such as toxicity, vasodilation and hypotension, have not been caused by guaco. The only fetal changes observed were due to the maternal hypertension. Some studies have reported vasodilator and hypotensive effects of guaco. However, only a few studies exist, and its actual effects remain unknown. Specific studies should be developed with higher doses of guaco for a definitive conclusion of its toxic and non-toxic effects.Keywords: Guaco, Hypertensive pregnant rats, Mikania glomerata, Perinatal toxicit

Toxic effects of the administration of Mikania glomerata Sprengel during the gestational period of hypertensive rats

Herbal medicine is an ancient practice that has been gaining acceptance of the medical class through scientific studies that prove its effectiveness. However, its use should still be cautious. Medicinal plants have potential toxic effects not yet discovered, and may have unproven interactions with other medications. The use of drugs during pregnancy is still very dangerous and vigorously studied; however, there are few studies of herbal medicines in pregnant women. Existing studies prioritize on teratogenic or abortifacient effects. The aim of this study was to analyze the toxic effects of Mikania glomerata Sprengel administration, popularly known as “guaco” during the gestational period of hypertensive rats. For this experimental groups consisting of pregnant Wistar rats received treatments with guaco extract (1 to 2 mL). In order to analyze the possible toxic effects of guaco during pregnancy, weight gain of rats was assessed during pregnancy; reproductive performance of rats, morphological parameters, and fetal placental histology were compared. Although some parameters presented significant differences, we can conclude that changes prioritized by literature, such as toxicity, vasodilation and hypotension, have not been caused by guaco. The only fetal changes observed were due to the maternal hypertension. Some studies have reported vasodilator and hypotensive effects of guaco. However, only a few studies exist, and its actual effects remain unknown. Specific studies should be developed with higher doses of guaco for a definitive conclusion of its toxic and non-toxic effects

Structural Characterization and Neuromuscular Activity of a New Lys49 Phospholipase A(2) Homologous (Bp-12) Isolated from Bothrops pauloensis Snake Venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Bp-12 was isolated from Bothrops pauloensis snake venom in only one chromatographic step in reverse phase HPLC on mu-Bondapack C-18. The molecular mass of 13,789.56 Da was determined by mass spectrometry. The amino acids composition showed that Bp-12 presented high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). The sequence of Bp-12 contains 122 amino acid residues: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKEL CECDKAVAICLRENL NTYNKKYRYFLKPLCKKADA AC, with a pI value of 8.55 and with a high homology with Lys49 PLA(2) from other snake venoms. In mouse phrenic nerve-diaphragm, the time needed for 50% paralysis was: 45 +/- 6 min (1.4 mu M) and 16 +/- 6 min (3.6 mu M). Bp-12 can induce indirect and directly blocked evoked twitches, even in the preparations in which Ca2+ is replaced by Sr2+, being the addition of d-tubocurarine required for direct blocking. These results identify Bp-12 as a new member of the Lys49 PLA(2) family and shows that this toxin might contribute to the effects of the crude venom on the neuromuscular junction.276355362Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Priscila Randazzo-Moura PhD thesisConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Toxic effects of glibenclamide in fetuses of normoglycemic rats: an alternative therapy for gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is defined as glucose intolerance first diagnosed during the second or third trimester of pregnancy. The treatment aims at glycemic control through changes in the patient's diet with or without exercise, but some patients need insulin therapy. An alternative would be to use oral hypoglycemic agents such as glibenclamide (GLIB). The present study aims to analyze the toxic effects of GLIB in fetuses of pregnant rats which received 5 or 20mg/kg doses of GLIB. Glycemic dosage reveals no significant difference between control (deionized water) and treated groups, showing that these concentrations of GLIB were not effective to cause hypoglycemia in rats. The vitality of the fetuses in all groups was 100%. GLIB administration promoted increase in weight and significant changes in measures of external morphological parameters of treated fetuses. Histological analysis revealed that liver lobes, lobules and central lobular veins were well defined for all treatments. However, GLIB animals presented a light brownish precipitate into the center-lobular veins and in the liver parenchyma among the hepatocytes. These results indicated a possible passage of the drug through the blood-placental membrane, without serious changes that impair the development of neither bone tissue, nor the liver of these animals.Keywords: Gestational diabetes mellitus, Glibenclamide, Toxicit

Shared Structural Determinants For The Calcium-independent Liposome Membrane Permeabilization And Sarcolemma Depolarization In Bothropstoxin-i, A Lys49-pla2 From The Venom Of Bothrops Jararacussu

The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A2 from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93. These results expand the known structural determinants of the biological activity as evaluated by previous creatine kinase release experiments. Furthermore, a strong correlation is observed between the structural determinants of sarcolemma depolarization and calcium-independent disruption of liposome membranes, suggesting that a common mechanism of action underlies the permeabilization of the biological and model membranes. © 2009 Elsevier Ltd. 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Rodrigues-Simioni, L., Purification and N-terminal sequencing of two presynaptic neurotoxic PLA2s, Neuwieditoxin-I and Neuwieditoxin II, from Bothrops neuwiedi pauloencis (jararaca pintada) venom (2007) J Venom Anim Toxins Trop Dis, 13, pp. 103-121Bortoleto-Bugs, R.K., Bugs, M.R., Neto, A.A., Ward, R.J., A micelle nucleation model for the interaction of dodecyl sulphate with Lys49-phospholipases A(2) (2007) Biophys Chem, 125, pp. 213-220Bülbring, E., Observations on the isolated phrenic nerve diaphragm preparation of the rat (1946) Br J Pharmacol, 1, pp. 38-61Chacur, M., Longo, I., Picolo, G., Gutierrez, J.M., Lomonte, B., Guerra, J.L., Teixeira, C.F., Cury, Y., Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: pharmacological mediation and molecular determinants (2003) Toxicon, 41, pp. 667-678Chacur, M., Milligan, E.D., Sloan, E.M., Wieseler-Frank, J., Barrientos, R.M., Martin, D., Poole, S., Watkins, L.R., Snake venom phospholipase A2s (Asp49 and Lys49) induce mechanical allodynia upon peri-sciatic administration: involvement of spinal cord glia, proinflammatory cytokines and nitric oxide (2004) Pain, 108, pp. 180-191Chioato, L., Aragao, E.A., Lopes Ferreira, T., Ivo de Medeiros, A., Faccioli, L.H., Ward, R.J., Mapping of the structural determinants of artificial and biological membrane damaging activities of a Lys49 phospholipase A(2) by scanning alanine mutagenesis (2007) Biochim Biophys Acta, 1768, pp. 1247-1257Chioato, L., De Oliveira, A.H., Ruller, R., Sa, J.M., Ward, R.J., Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2 (2002) Biochem J, 366, pp. 971-976Chioato, L., Ward, R.J., Mapping structural determinants of biological activities in snake venom phospholipases A2 by sequence analysis and site directed mutagenesis (2003) Toxicon, 42, pp. 869-883da Silva Giotto, M.T., Garratt, R.C., Oliva, G., Mascarenhas, Y.P., Giglio, J.R., Cintra, A.C., de Azevedo 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W.A., Beiboer, S.H., van Kampen, M.D., Verheij, H.M., Slotboom, A.J., Egmond, M.R., Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K (1999) Protein Eng, 12, pp. 497-503Kaiser, I.I., Gutierrez, J.M., Plummer, D., Aird, S.D., Odell, G.V., The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper (1990) Arch Biochem Biophys, 278, pp. 319-325Kini, R.M., (1997) Venom Phospholipase A2 Enzymes, , John Wiley & Sons, ChichesterKini, R.M., Iwanaga, S., Structure-function relationships of phospholipases. 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Protection By Mikania Laevigata (guaco) Extract Against The Toxicity Of Philodryas Olfersii Snake Venom

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. © 2012 Elsevier Ltd.604614622Abbas, A.K., Lichtman, A.H., (2005) Imunologia Celular e Molecular, , Elsevier, Rio de JaneiroAbreu, V.A., Dal-Belo, C.A., Hernandes-Oliveira, S.S., Borja-Oliveira, C.R., Hyslop, S., Furtado, M.F., Rodrigues-Simioni, L., Neuromuscular and phospholipase activities of venoms from three subspecies of Bothrops neuwiedi (B. n. goyazensis, B. n. paranaensis and B. n. diporus) (2007) Comp. Biochem. Physiol. A Mol. Integr. 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Protection by Mikania laevigata (guaco) extract against the toxicity of Philodryas olfersii snake venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)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 mu l/ml and 118.9 +/- 9.3% with 40 mu l/ml in PND, and 120.7 +/- 7.7% with 40 mu l/ml and 114.5 +/- 4.4% with 50 mu l/ml in BC after 120 min; n = 4-6, mean +/- SEM). In PND, the ethanol alone (40 mu l/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 mu l/ml) the extract caused total and reversible blockade in both preparations. Venom (50 mu g/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 mu l/ml) for 30 min before incubation with venom (50 mu g/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 TNF alpha and IFN gamma. 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 TNF alpha, and IFN gamma expression in both preparations as observed in control. Venom (50 mu g/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 TNF alpha and IFN gamma. 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 TNF alpha and IFN gamma expression. These results indicate that the M. laevigata extract protected nerve-muscle preparations against the myotoxic, neurotoxic and inflammatory effects of P. olfersii venom. (c) 2012 Elsevier Ltd. All rights reserved.604SI614622Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Cross-neutralization Of The Neurotoxicity Of Crotalus Durissus Terrificus And Bothrops Jararacussu Venoms By Antisera Against Crotoxin And Phospholipase A2 From Crotalus Durissus Cascavella Venom

We have previously demonstrated that rabbit antisera raised against crotoxin from Crotalus durissus cascavella venom (cdc-crotoxin) and its PLA 2 (cdc-PLA2) neutralized the neurotoxicity of this venom and its crotoxin. In this study, we examined the ability of these antisera to neutralize the neurotoxicity of Crotalus durissus terrificus and Bothrops jararacussu venoms and their major toxins, cdt-crotoxin and bothropstoxin-I (BthTX-I), respectively, in mouse isolated phrenic nerve-diaphragm preparations. Immunoblotting showed that antiserum to cdc-crotoxin recognized cdt-crotoxin and BthTX-I, while antiserum to cdc-PLA2 recognized cdt-PLA 2 and BthTX-I. ELISA corroborated this cross-reactivity. Antiserum to cdc-crotoxin prevented the neuromuscular blockade caused by C. d. terrificus venom and its crotoxin at a venom/crotoxin:antiserum ratio of 1:3. Antiserum to cdc-PLA2 also neutralized the neuromuscular blockade caused by C. d. terrificus venom or its crotoxin at venom or toxin:antiserum ratios of 1:3 and 1:1, respectively. The neuromuscular blockade caused by B. jararacussu venom and BthTX-I was also neutralized by the antisera to cdc-crotoxin and cdc-PLA 2 at a venom/toxin:antiserum ratio of 1:10 for both. Commercial equine antivenom raised against C. d. terrificus venom was effective in preventing the neuromuscular blockade typical of B. jararacussu venom (venom:antivenom ratio of 1:2), whereas for BthTX-I the ratio was 1:10. These results show that antiserum produced against PLA2, the major toxin in C. durissus cascavella venom, efficiently neutralized the neurotoxicity of C. d. terrificus and B. jararacussu venoms and their PLA2 toxins.466604611Beghini, 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. 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São Paulo, 19, pp. 21-22Breithaupt, H., Enzymatic characteristics of Crotalus phospholipase A2 and the crotoxin complex (1976) Toxicon, 14, pp. 221-233Bülbring, E., Observation on the isolated phrenic nerve diaphragm preparation of the ratr (1946) Br. J. Pharmacol., 1, pp. 38-61Chavez-Olórtegui, C., Silva Lopes, C., Drumond Cordeiro, F., Granier, C., Diniz, C.R., An enzyme linked immunosorbent assay (ELISA) that discriminates between Bothrops atrox and Lachesis muta muta venoms (1993) Toxicon, 31, pp. 417-425Choumet, V., Jiang, M.S., Radvanyi, F., Ownby, C., Bon, C., Neutralization of lethal potency and inhibition of enzymatic activity of a phospholipase A2 neurotoxin, crotoxin, by non-precipitating antibodies (Fab) (1989) Fed. Eur. Biochem. Soc. Lett., 244, pp. 167-173Choumet, V., Faure, G., Robbe-Vincent, A., Saliou, B., Mazié, J.C., Bon, C., Immunochemical analysis of a snake venom phospholipase A2 neurotoxin, crotoxin, with monoclonal antibodies (1992) Mol. 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