Umbelliferone induces changes in the structure and pharmacological activities of Bn IV, a phospholipase A(2) isoform isolated from Bothrops neuwiedi

In this paper was demonstrated that umbelliferone induces changes in structure and pharmacological activities of Bn IV, a lysine 49 secretory phospholipase A(2) (sPLA2) from Both tops neuwiedi. Incubation of Bn IV with umbelliferone virtually abolished platelet aggregation, edema, and myotoxicity induced by native Bn IV. The amino acid sequence of Bn IV showed high sequence similarities with other Lys49 sPLA2s from B. jararacussu (BthTx-I), B. pirajai (PrTx-I), and B. neuwiedi pauloensis (Bn SP6 and Bn SP7). This sPLA2 also has a highly conserved C-terminal amino acid sequence, which has been shown as important for the pharmacological activities of Lys49 sPLA2. Sequencing of Bn IV previously treated with umbelliferone revealed modification of S(1) and S(20). Fluorescent spectral analysis and circular dichroism (CD) studies showed that umbelliferone modified the secondary structure of this protein. Moreover, the pharmacological activity of Bn IV is driven by synergism of the C-terminal region with the a-helix motifs, which are involved in substrate binding of the Asp49 and Lys49 residues of 5PLA2 and have a direct effect on the Ca2+-independent membrane damage of some secretory snake venom PLA2. For Bn IV, these interactions are potentially important for triggering the pharmacological activity of this 5PLA2. (C) 2011 Elsevier Ltd. All rights reserved.

A Catalytically Inactive Lys49 PLA2 Isoform from Bothrops jararacussu venom that Stimulates Insulin Secretion in Pancreatic Beta Cells

A new secretory phospholipase A2 (sPLA2) isoform from Bothrops jararacussu venom (BjVIII) has been characterized by causing platelet aggregation, an absent activity in BthTx-I, Prtx-I and PrTx-II sPLA2s. According to our results, BjVIII also enhances insulin release by the pancreatic beta cells. The complete amino acid sequence of the new isoform was determined by Edman degradation and de novo peptide sequencing. These analyses showed a G35K amino acid modification for BjVIII in comparison with BthTx-I, PrTx-I and Prtx-II, a structural difference that has been related to the conflicting biological activities among BjVIII and other Lys49 sPLA2s. The whole set of evidences collected in this work indicates that, besides the C-terminal region and B-wing of PLA2, the calcium binding loop in BjVIII should be considered as an important region, involved in the pharmacological effects of Lys49-sPLA2 isoforms from the Bothrops genus.

Renal and cardiovascular effects of Bothrops marajoensis venom and phospholipase A(2)

Bothrops marajoensis is found in the savannah of Marajo Island in the State of Par S and regions of Amapa State, Brazil. The aim of the work was to study the renal and cardiovascular effects of the B. marajoensis venom and phospholipase A(2) (PLA(2)). The venom was fractionated by Protein Pack 5PW. N-terminal amino acid sequencing of sPLA(2) showed amino acid identity with other lysine K49sPLA(2)s of snake venom. B. marajoensis venom (30 mu g/mL) decreased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and sodium tubular transport. PLA(2) did not change the renal parameters. The perfusion pressure of the mesenteric bed did not change after infusion of venom. In isolated heart, the venom decreased the force of contraction and increased PP but did not change coronary flow. In the arterial pressure, the venom and PLA(2) decreased mean arterial pressure and cardiac frequency. The presence of atrial flutter and late hyperpolarisation reversed, indicating QRS complex arrhythmia and dysfunction in atrial conduction. In conclusion, B. marajoensis venom and PLA(2) induce hypotension and bradycardia while simultaneously blocking electrical conduction in the heart. Moreover, the decrease in glomerular filtration rate, urinary flow and electrolyte transport demonstrates physiological changes to the renal system. (C) 2009 Elsevier Ltd. All rights reserved.

Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A(2) from Bothrops pirajai venom

Background: Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A(2) are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A(2) drugs.Methods: HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated.Results: HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 +/- 0.28 mu g/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA(2) inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic acid and p-BPB. HP-2 previous incubated with the platelets inhibits the aggregation induced by untreated PrTX-III as well as arachidonic acid.Conclusion: HP-2 changes the structure of PrTX-III, inhibiting the enzymatic activity of this enzyme. In addition, PrTX-III platelet aggregant activity was inhibited by treatment with HP-2, p-BPB and aristolochic acid, and these results were corroborated by docking scores.

Isolamento e caracterização biológica e bioquímica de um activador do factor x da coagulação presente no veneno da serpente Bothrops erythromelas

Dissertação de Mestrado Integrado em Medicina Veterinária

Crystal structure of Bn IV in complex with myristic acid: A Lys49 myotoxic phospholipase A(2) from Bothrops neuwiedi venom

The LY549-PLA(2)s myotoxins have attracted attention as models for the induction of myonecrosis by a catalytically independent mechanism of action. Structural studies and biological activities have demonstrated that the myotoxic activity of LYS49-PLA(2) is independent of the catalytic activity site. The myotoxic effect is conventionally thought to be to due to the C-terminal region 111-121, which plays an effective role in membrane damage. In the present study, Bn IV LYS49-PLA(2) was isolated from Bothrops neuwiedi snake venom in complex with myristic acid (CH3(CH2)(12)COOH) and its overall structure was refined at 2.2 angstrom resolution. The Bn IV crystals belong to monoclinic space group P2(1) and contain a dimer in the asymmetric unit. The unit cell parameters are a = 38.8, b = 70.4, c = 44.0 angstrom. The biological assembly is a "conventional dimer" and the results confirm that dimer formation is not relevant to the myotoxic activity. Electron density map analysis of the Bn IV structure shows clearly the presence of myristic acid in catalytic site. The relevant structural features for myotoxic activity are located in the C-terminal region and the Bn IV C-terminal residues NKKYRY are a probable heparin binding domain. These findings indicate that the mechanism of interaction between Bn IV and muscle cell membranes is through some kind of cell signal transduction mediated by heparin complexes. (C) 2010 Elsevier Masson SAS. All rights reserved.

Influência da temperatura na germinação de sementes de Digitaria insularis, Leptochloa virgata, Pennisetum setosum e Sorghum halepense

Objetivou-se com este trabalho avaliar a influência de temperaturas constantes na germinação de sementes de espécies de plantas daninhas da família Poaceae que apresentam ocorrência em áreas de pastagem, para isto, foram analisadas as sementes de Digitaria insularis, Leptochloa virgata, Pennisetum setosum e Sorghum halepense coletadas manualmente de diversas plantas em áreas de pastagem. As sementes foram dispostas em caixas gerbox sobre folha dupla de papel "germitest", umedecido com água destilada equivalente a 2,5 vezes a massa do papel seco. As caixas foram incubadas em três germinadores verticais tipo MANGELSDORF com as temperaturas de 25ºC, 30ºC e 35ºC e fotoperíodo de 12 horas. As avaliações foram realizadas, efetuando-se a contagem diária das ocorrências germinativas, a partir da protrusão da radícula, durante 28 dias após o início do estudo. Os parâmetros avaliados foram porcentagem e índice de velocidade de germinação (IVG), efetuando-se a análise estatística pela análise de variância com comparação de médias pelo teste de Tukey a 5% de probabilidade e transformação dos resultados de porcentagem de germinação. As temperaturas que proporcionam os melhores resultados de porcentagem e velocidade de germinação são 30 e 35°C, para as espécies de D. insularis, L. virgata e S. halepense, por outro lado, a espécie de P. setosum não apresenta germinação em nenhuma das temperaturas avaliadas (25º 30º e 35ºC) não são adequadas para a sua germinação.

Associação de adjuvantes ao hervicida clethodim e o controle de Digitaria insularis.

2016

Evaluación del protectante nufilm-17 en la efectividad de los insecticidas botánicos y biológicos sobre la entomofauna en el cultivo del repollo (Brassica oleraceae L.) hibrido izalco en época de apante

Con el objetivo de evaluar el efecto del protectante Nufilm-17 sobre la efectividad de los productos Botánicos y Biológicos contra Plutella xylostella L. en el cultivo de repollo se llevó a cabo el presente trabajo en el Centro Experimental del valle de Sébaco "Raúl Gonzalez”, de Dic.91 - Marzo 92. El diseño utilizado fué de Bloques Completos al Azar (BCA), con 4 repeticiones y 7 tratamientos que fueron distribuidos al azar en el campo: las variables a medir fueron: número de larvas de P. xylostella números de arañas, números de polybias porcentaje de parasitismo de Diadegma insularis sobre larvas de P. xylostella números de cabezas/ha. precio/cabeza, daño foliar. Además se hizo análisis económico para conocer la rentabilidad del uso de las alternativas en el control de las principales plaga en el cultivo. Los recuento se hicieron cada 8 días hasta el final de la cosecha. El comportamiento de P. xylostella fue diferente en la distintas etapas del cultivo siendo la etapa de crecimiento vegetativo y formación de cabeza (0-30 y 30-60 DDT) donde se presentaron las mayores poblaciones. Siendo notorio el control que efectuo el tratamiento Nim + Nufilm-17 sobre P. xylostella que logró mantener las poblaciones bajas. El porcentaje de parasitismo observado en larvas de P. xylostella registró valores entre 5 y 30 %. El análisis económico, demostró que los tratamientos Nim + Nufilm-17 y B. thuringiesis presentaron mayor rentabilidad teniendo una tasa de retorno marginal superior a la tasa comparativa que es de 125 %.

Evaluación de productos botánicos y biológicos para el control de Plutella xylostella en el cultivo del repollo (Brassica oleraceae L.) hibrido Izalco en época de apante

Con el fin de encontrar el mejor producto para el control de Plutella xylostella en el cultivo del repollo, se llevó a cabo un ensayo a nivel de campo en época seca (Die 91-Mar 92) en el valle de Sébaco-Matagalpa donde se evaluaron los tratamientos Bacillus thuringiensis nacional, Dipel comercial, Javelin, Nim, Mamey y Júpiter, con un criterio de aplicación de 0.5 larvas por plantas. Las poblaciones de Plutella xylostella fueron relativamente bajas en la etapa de crecimiento vegetativo disminuyendo en las últimas dos etapas formación y llenado de cabeza. Durante las tres fases del cultivo las poblaciones de Plutella xylostella fueron menores en los tratamientos Javelin Mamey y Júpiter. Los insecticidas Dipel. Javelin y Mamey fueron diferentes estadísticamente del testigo (sin aplicación), según el análisis económico los primeros dos insecticidas son rentables económicamente sucediendo lo contrario con el insecticida Mamey por lo tanto su uso no es recomendado para el manejo de la plaga. El insecticida Júpiter puede ser utilizado contra la plaga ya que muestra una tasa de retorno marginal superior a la tasa comparativa. La presencia de enemigos naturales (Araña, Polybia sppy el parasitoide Diadegma insularis) fue notoria en el cultivo, cabe señalar su importancia ya que intervienen en el equilibrio del ecosistema dentro del cultivo (plaga-repollo). Estos predadores no se vieron afectados por las aplicaciones de los insecticidas botánicos y biológicos evaluados en el cultivo. El uso de estos productos parecen ser una alternativa de manejo de las plagas del cultivo a largo plazo ya que el establecimiento de la fauna benéfica en el sistema y el no uso de productos sintéticos evitan los problemas de resistencia en las plagas y la contaminación ambiental.

Evaluación de hongos entomopatogenos sobre plagas claves en el cultivo del arroz (Julio a octubre del 2000)

Con el objetivo de evaluar el efecto de tres cepas de hongos entomopatógenos formuladas en polvo y aceite, sobre las poblaciones del chinche de la espiga del arroz Oebalus insularis (Stal), la novia del arroz Rupela albinella (Cramer) y el saltamonte Conocephalus sp se realizó un experimento en condiciones de campo y laboratorio durante Agosto a Diciembre del 2000. A nivel de laboratorio se evaluaron las cepas 38, 64, 114 y 121 de Beauveria bassiana (Bals), sobre la mortalidad del chinche, las cepas evaluadas a nivel de campo fueron 114 y 121 también de Beauveria bassiana(Bals) y la cepa Niña bonita de Metarhizium anisopliae (Metch); se evaluó además la mezcla de las cepas Ma-NB más Bb-121, comparadas con una parcela sin aplicación de hongos y uso de Metamidofos. Se utilizó el umbral de 0.6 chinches en 10 redadas para realizar las aplicaciones, los muestreos se realizaron cada se mana, iniciando antes de la floración hasta finalizar el período de protección de espiga. Se seleccionaron cinco sitios al azar en cada tratamiento y se registró el número de plagas capturadas. Se inició con una aplicación preventiva del hongo, cuando se observó entre el 5 y 10% de floración, posteriormente fueron aplicados cuando se alcanzó el umbral de daño. Los resultados obtenidos indican que los tratamientos tuvieron un comportamiento similar, se encontró diferencias significativas entre las poblaciones de plagas en las fechas de muestreo (P:0.0001); sin embargo, se pudo observar que las cepas Ma-N B y Bb-114 formuladas en polvo iniciaron con la mayor población y al final del ciclo la incidencia de la plagas en dichas parcelas fue un poco menor que en las demás. En cambio los tratamientos Ma-NB + Bb-121 y Bb-114 formuladas en aceite presentaron poblaciones de la plaga ligeramente mayores que los demás tratamientos. Las cepas Bb-114 y Ma-NB, así como la mezcla de ambas, formuladas en polvo, en concentraciones de 10 12 conidias/ml son promisorias para el manejo de las plagas objeto de estudio. A nivel de laboratorio las cepas que se comportaron mejor fueron la 64 y 114 de Beauveria bassiana.

Parasitoides de huevos de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: noctuidae) Dalbulus maidis (Delong y wolcott) (Homoptera: cicadellidae) y Peregrinus maidis (Ashm) (Homoptera: delphacidae) en maíz (Zea mays)

Durante la época seca bajo riego y la época lluviosa de 1988 se llevó a cabo un sondeo de los parasitoides de huevos de spodoptera frugiperda en maíz, en el centro nacional de investigación de granos básicos “Humberto tapia” (depto. De Managua) y en la estación experimental “Raúl González” del valle de sebaco (depto. de Matagalpa). También se hizo una evaluación de la presencia de telenomus remus en alguno de los sitios de liberación de estas especies en el año 1976. En la calera (depto. De Managua), la trinidad (depto. De Estelí), valle de sebaco (depto. De Matagalpa) y camoapa (depto. De Boaco); en 1984 en la cartonera y santa clara (depto. De león). Durante el ciclo de postrera del año 1988 se llevó a cabo un sondeo de los parasitoides de huevos de dalbulus maidis y peregrinus maidis en el centro experimental “ Humberto tapia”, y en una finca en el km 93 y 98 de la carretera león- Chinandega, km 40 de la carretera Managua-león y en san juan de la concepción (depto. De Masaya). En la estación experimental “Humberto tapia” no se encontraron parasitoides de huevos ni parasitoides ovo-larvales de s. frugiperda. en la “Raúl González” tampoco se encontraron parasitoides de huevos sensuestricto de s. frugiperda, pero se detectó la presencia de parasitoide ovo-larval, chelonus insularis en las dos épocas de siembra se comprobó que en el estado fenológico de la planta durante las dos épocas ni la época de siembra influyeron significativamente sobre la tasa de parasitismo de c. insularis dentro y entre las masas de huevos de s. frugiperda ya no se encontró diferencias significativa en el porcentaje de masas de huevos parasitadas 27 días después de germinación (DDG) (45%) y 55 DDG (50%) en la época seca, ni en la lluviosa 26 DDG (20%), 43 DDG (29%) y 57DDG (0%). Tampoco hubo efecto de la época de siembra (seca, 47% y lluviosa, 19%) sobre el porcentaje de masas de huevos parasitadas. En el porcentaje promedio de huevos por masa parasitados a los 27 DDG (19%) y 55 DDG (8%) durante la época seca y la lluviosa 26 DDG (18%), 43 DDG (9%) y 57 DDG (0%) tampoco se encontró una diferencia significativa. La época (seca 17% y la lluviosa 19%) tampoco influyo sobre el porcentaje promedio de huevos por masas parasitadas. La edad de la masa de huevos de s. frugiperda (5-6 días de edad, 33.33. % y 3-4 días de edad 12.12%) influyo significativamente sobre el porcentaje de masas de huevos parasitadas por c.insulularis el cual afecto las poblaciones de s. frugiperda en la época seca y lluviosa en un 8.35 % y 5.32% respectivamente. Los anteriores resultados más el no aparente establecimiento aparente de t.remus en los sitio donde fue liberado, permiten recomendar la introducción nuevamente de raza especifica de este parasitoide para nuestra condiciones de clima, debiéndose realizar las liberaciones preferiblemente en sitio montañosos y de clima fresco. La presencia de parasitoides de D. maidis fue nula. Las cuatro especies parasíticas de huevos de p.maidis encontradas en esta investigación (anagrus spp, gonatocerus spp, paracentrobia spp y oligosita oophagus), se consideran como un nuevo hallazgo para Nicaragua. Estos resultados insinúan la necesidad de conocer los periodo crítico de infestación de d. maidis en el maíz , para realizar aplicaciones mínima de insecticida con el fin de controlarlo sin afectar las poblaciones de benéficos que atacan a p.maidis, evitando así que este insecto se convierta en una plaga peligrosa para el cultivo de maíz.

Status of the gill net fishery for the small pelagic fish species in western, south-western and southern coastal waters of Sri Lanka during 1995-1997

Small pelagic fish species are mainly caught by gill nets operated by fibre reinforced plastic boats fitted with 8-25hp out board engines, traditional crafts fitted with 8-1hp out board engines and non mechanised traditional crafts. Around 28 to 55% of the small pelagic catch in the study area consisted of trenched sardine Amblygaster sirm during 1995-1997 period. Another 26-36% of the catch composed of other Sardinella species such as Sardinella gibbosa, S. albella, S. sindensis and S. longiceps. Engraulids such as Encrasicholina heteroloba, Stolephorus insularis and Stolephorus indicus and Thryssa spp formed around 3-5% of the catch. The major component of this fishery consisted of Clupeids and Engrauhds and over 65 species ranged between smaller Engraulids to incidental rock fish, sail fish, seer fish, sharks, skates and rays. Around 1.4 to 1.9% of the catch consisted of Chirocentrus dorab, Sphyraenaspp, Scomberomorus spp, Lepturcanthus sp and Megalaspis cordyla. Around 1-11% of the catch consisted of incidentally catches of sharks, rays, skates and sail fish. Another 1.6 to 6% of the catch consisted of Selar crumenophthalamus and Rastrelliger kanagurta. The best fishing season appeared to be from June to October in the west coast and August to December in the south coast. The major components of Amblygaster sirm, Sardinella albella and Sardinella gibbosa were caught within the size ranges of 10.0-22.5 cm, 11.0-13.0 cm and 11.0-15.0 cm respectively. However, smaller sized fish of above species of sizes between 6.9 cm to 9.7 cm total length were incidentally caught in the gill nets operated for small Engraulids with a stretched mesh size of 1.6cm. The overall catch rate for the major fish landing centre at Negombo indicated an increase from 38.5 kg/boat trip during 1984-1990 period to 49.5 kg/boat trip during 1995-1997 period. The catch rate for the dominant species Amblygaster sirm has decreased from 28.17 kg/boat trip during 1983-1990 period to 17.47 kg/boat trip during 1995-1997 period at Negombo. The paper also discusses the changing overall catch rates, change in species abundance and possible management consequences that should be considered.

AN ACTIVATOR OF BLOOD-COAGULATION FACTOR-X FROM THE VENOM OF BUNGARUS-FASCIATUS

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mel. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

Trimeresurus stejnegeri snake venom plasminogen activator - Site-directed mutagenesis and molecular modeling

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L,, and Bon, C, (1995) J. Biol. Chem. 270, 10246-10255), To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site, When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity, Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the K-m for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.