Evidence of caspase-mediated apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom.

The aim of this work was to investigate the involvement of caspases in apoptosis induced by l-amino acid oxidase isolated from Bothrops atrox snake venom. The isolation of LAAO involved three chromatographic steps: molecular exclusion on a G-75 column; ion exchange column by HPLC and affinity chromatography on a Lentil Lectin column. SDS-PAGE was used to confirm the expected high purity level of BatroxLAAO. It is a glycoprotein with 12% sugar and an acidic character, as confirmed by its amino acid composition, rich in "Asp and Glu" residues. It displays high specificity toward hydrophobic l-amino acids. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom LAAOs. This enzyme induces in vitro platelet aggregation, which may be due to H2O2 production by LAAOs, since the addition of catalase completely inhibited the aggregation effect. It also showed cytotoxicity towards several cancer cell lines: HL60, Jurkat, B16F10 and PC12. The cytotoxicity activity was abolished by catalase. A fluorescence microscopy evaluation revealed a significant increase in the apoptotic index of these cells after BatroxLAAO treatment. This observation was confirmed by phosphatidyl serine exposure and activation of caspases. BatroxLAAO is a protein with various biological functions that can be involved in envenomation. Further investigations of its function will contribute to toxicology advances.

A low molecular weight proinflammatory factor from rat spleen lymphocytes. Isolation and partial characterization.

A dialyzable low molecular weight proinflammatory factor (X (2)) from rat spleen lymphocytes was isolated through a combination of gel filtration and high voltage paper electrophoresis (HVE) and then partially characterized. It was able to potentiate the formation of carrageenin induced edema on the rat paw. Its amino acid analysis revealed Glu, Cys and Gly (1:1:1), but gammaGlu as N-terminal residue, initially suggesting oxidized glutathione (GSSG), since it showed exactly the same HV electrophoretic mobility as GSSG at pH 6.5. However, neither GSSG nor a synthetic homologue showed any proinflammatory activity. On basis of its infrared spectrum, HVE mobility and presence of a gamma-Glu-Cys-Gly (GSH) moiety, the hypothesis of identity of X (2) with leukotriene C(4) (LTC(4)) was raised. Once again it was not confirmed, since LTC(4) did not show any proinflammatory activity too, leading us to infer that, even excluding LTC(4), our data are consistent with a structure bearing a GSH moiety conjugated with a hydroxylated insaturated fatty acid chain which contributes a -COO(-) group, thus providing a final net charge of -2 at pH 6.5 and an Mr = 600-650.

The presynaptic activity of bothropstoxin-I, a myotoxin from Bothrops jararacussu snake venom.

Bothropstoxin-I from Bothrops jararacussu snake venom is a lysine-49 phospholipase A(2) with myotoxic and neurotoxic activities. In this study, we used mouse phrenic nerve-diaphragm preparations in the absence and presence of manganese (Mn(2+)), a presynaptic blocker, to investigate a possible presynaptic action of bothropstoxin-I. At concentrations of 0.9 mM and 1.8 mM, Mn(2+) produced 50% neuromuscular blockade in less than 4 min., which was spontaneously reversible at the lower concentration. Bothropstoxin-I (1.4 microM) irreversibly inhibited neuromuscular blockade by 50% in 31+/-4 min. (mean+/-S.E.M., n = 9). Pretreating preparations with 0.9 mM Mn(2+) prevented the blockade by bothropstoxin-I. When added after bothropstoxin-I, Mn(2+) produced its characteristic blockade and, after washing, the twitch tension returned to pre-Mn(2+) levels, indicating that bothropstoxin-I caused irreversible damage before the addition of Mn(2+). Electrophysiological measurements showed that a concentration of bothropstoxin-I (0.35 microM), which did not produce neuromuscular blockade, caused the appearance of giant miniature end-plate potentials with no change in the membrane resting potential but increased the quantal content. Preparations preincubated with Mn(2+) (0.9 mM, 30 min.) were protected against the depolarizing action of bothropstoxin-I (0.7 microM). These results show that, in addition to its well-known myotoxic effect, bothropstoxin-I also has a presynaptic action.

Effects of Tityus serrulatus scorpion venom and its toxin TsTX-V on neurotransmitter uptake in vitro.

Scorpion neurotoxins targeting the Na(v) channel can be classified into two classes: alpha- and beta-neurotoxins and are reported as highly active in mammalian brain. In this work, we evaluate the effects of Tityus serrulatus venom (Ts venom) and its alpha-neurotoxin TsTX-V on gamma-aminobutyric acid (GABA), dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes. TsTX-V was isolated from Ts venom by ion exchange chromatography followed by reverse-phase (C18) high-performance liquid chromatography. Neither Ts venom nor TsTX-V was able to affect (3)H-Glu uptake. On the other hand, Ts venom (0.13 microg/mg) significantly inhibited both (3)H-GABA and (3)H-DA uptake ( approximately 50%). TsTX-V showed IC(50) values of 9.37 microM and 22.2 microM for the inhibition of (3)H-GABA and (3)H-DA uptake, respectively. These effects were abolished by pre-treatment with tetrodotoxin (TTX, 1 microM), indicating the involvement of voltage-gated Na(+) channels in this process. In the absence of Ca(2+), and at low Ts venom concentrations, the reduction of (3)H-GABA uptake was not as marked as in the presence of Ca(2+). TsTX-V did not reduce (3)H-GABA uptake in COS-7 cells expressing the GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced (3)H-GABA uptake by synaptosomes might be due to rapid cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes a reduction of (3)H-GABA and (3)H-DA uptake in a Ca(2+)-dependent manner, not directly affecting GABA transporters, but, in consequence of depolarization, involving voltage-gated Na(+) channels.

Effects of voltage-gated Na+ channel toxins from Tityus serrulatus venom on rat arterial blood pressure and plasma catecholamines.

Scorpion toxins interact with ionic channels of excitable cells, leading to a massive release of neurotransmitters. Voltage-gated Na+ channel toxins are mainly responsible for the toxic effects of scorpion envenoming and can be classified into two classes: alpha- and beta-neurotoxins. TsTX-V and TsTX-I from Tityus serrulatus venom (TsV) are, respectively, examples of these toxins. In this work, we compared the effects of these toxins on mean arterial pressure (MAP) and catecholamines release in rats. Toxins were isolated by ion exchange chromatography (TsTX-I) followed by RP-HPLC (TsTX-V). All experiments were performed on conscious unrestrained rats previously catheterised. The toxins (15 and 30 microg/kg) and TsV (50 and 100 microg/kg) were injected intravenously. MAP was continuously monitored through femoral catheter. Epinephrine (E) and norepinephrine (NE) levels were determined by RP-HPLC with electrochemical detection, at 10 min before and 2.5, 30 and 90 min after treatments. Maximal pressor effects were observed at 2.5-3.5 min. TsV induced intense long lasting increase in MAP, as did TsTX-I. TsTX-V showed the lowest pressor effects. TsV showed the highest effects on catecholamines release, followed by TsTX-I and TsTX-V with maximal effect at 2.5 min, followed by a gradual reduction, however remaining higher than controls. Although both toxins act on Na+ channels, TsTX-I displayed significant and more intense effects on catecholamines release and blood pressure than TsTX-V. It seems that the toxicity of TsTX-V is not related only with its ability to release catecholamines, indicating that other neurotransmitters, may be involved in its toxicity.

Further insights toward vitamin C determination and stability. Proposal of a new quantification method / Avaliações adicionais da determinação e estabilidade da vitamina C. Proposta de um novo método de quantificação

This work aimed at an evaluation of the classical iodine method for quantification of vitamin C (L-ascorbic acid) in fruit juices, as well as at a search into the stability of this so popular vitamin under different conditions of pH, temperature and light exposition, in addition to a proposal of a new quantification method. Our results point to the persistent reversibility of the blue color of the starch-triiodide complex at the end point when using the classical iodine titration, and the overestimation of the true vitamin concentration in fruit juices. A new quantification method is proposed in order to overcome this problem. Surprising conclusions were obtained regarding the controversial stability of L-ascorbic acid toward atmospheric oxygen, at low pH, even in fruit juice and at room temperature, showing that the major problem concerned with aging of fruit juices is proliferation of microorganisms rather than expontaneous oxidation of L-ascorbic acid.

Este trabalho teve como objetivo uma avaliação do método clássico do iodo para a quantificação da vitamina C (ácido L-ascórbico) em sucos de frutas, assim como uma pesquisa da estabilidade desta vitamina tão popular sob diferentes condições de pH, temperatura e exposição à luz, além de uma proposta de novo método de quantificação. Nossos resultados indicam uma persistente reversibilidade da cor azul do complexo amido-triiodeto no ponto final, quando usamos a clássica titulação com iodo, e uma super-estimação da verdadeira concentração da vitamina em sucos de frutas. Um novo método de dosagem é proposto a fim de superar este problema. Conclusões surpreendentes foram obtidas em relação à controvertida estabilidade do ácido L-ascórbico frente ao oxigênio atmosférico, em pH baixo, mesmo no suco da fruta e em temperatura ambiente, mostrando que o maior problema em relação ao envelhecimento de sucos de frutas é a proliferação de microorganismos e não a oxidação espontânea do ácido L-ascórbico.

Anticrotalic and antitumoral activities of gel filtration fractions of aqueous extract from Tabernaemontana catharinensis (Apocynaceae).

The high mortality caused by Crotalus durissus terrificus snake venom is mainly due to crotoxin, which acts on the neuromuscular junction inhibiting the mechanism mediating acetylcholine release, thus leading to motor and respiratory paralysis and subsequently to animal death. We recently demonstrated that the aqueous extract (AE) of Tabernaemontana catharinensis can inhibit the lethal activity of C. d. terrificus venom. Eight fractions, PI to PVIII, were obtained by gel filtration of the extract on Sephadex G-10, and assayed for lethality and cytotoxicity. Fraction PVII [2.0 mg/100 g rat/50 microl saline solution (ss)] injected intramuscularly (i.m.) 20 s after the venom (240 microg) or crotoxin (200 microg/50 microl ss) neutralized the lethal activity of 2 LD50 of both. Fractions PI, PVI and PVIII (5.0 mg/100 g rat/50 microl ss) presented potent antitumoral activity in vitro against cells from human breast carcinoma (SK-BR-3) after 24 h incubation, as measured by Mosmann colorimetric method. Fraction PVII contains 12-methoxy-4-methylvoachalotine as its major component. These results demonstrate that the antivenom and antitumoral activities of the AE of T. catharinensis are exerted by different substances present in fraction PVII and fractions PI, PVI and PVIII, respectively, whose characteristics are distinct in terms of staining and Rf when analyzed by thin layer chromatography. The results also show that a preliminary fractionation by Sephadex G-10 gel filtration is a good option as a first step for isolation of biologically active substances from T. catharinensis.

Adoçantes dietéticos na alimentação de ratos: uma abordagem histológica e bioquímica / Dietary sweeteners in rat nourishment: a histological and biochemical approach

Ratos Wistar foram submetidos a uma dieta composta de ração e: 1. água; 2. sacarose a 10 por cento; 3. Aspartame a 1 por cento; 4. ciclamato de sódio a 1 por cento; e 5. sacarina a 1 por cento ad libitum, a partir dos 15 dias de idade. Na fase adulta (5,5 meses), cortes histológicos do duodeno, fígado, baço, bexiga e pâncreas desses animais não revelaram alterações anátomo-patológicas. A evolução da massa corporal foi também muito próxima entre os cinco grupos. Detectou-se menor nível de triglicérideos séricos nos grupos Sacarose e Aspartame, assim como de colesterol no grupo Água. Essas variações foram aparentemente indiretas e ligadas à maior ou menor ingestão de ração e à sua composição.

Wistar rats were fed a diet composed of chow and: 1. water; 2. 10 percent sacarose; 3. 1 percent aspartame; 4. 1 percentsodium cyclamate and 5. 1 percent saccharin ad libitum, from the age of 15 days. At the adult phase (5.5months), histologic cuts of these animals did not show anatomo-morphologic alterations. Evolution ofbody weight was also very close to one another among the five groups. Lower serum triglyceride levelswere detected in the Sucrose and Aspartame groups, the same occurring with cholesterol in the Watergroup. These alterations were apparently indirect and due to a higher or lower ingestion of chow and its composition.

Ação de vários agentes sobre o efeito paralisante dos membros posteriores induzido pela crotamina em camundongos / Action of several agents on the paralyzing effect of the hind legs induced by crotamine in mice

A crotamina, neurotoxina isolada a partir da peçonha de Crotalus durissus terrificus (cascavel sul-americana), foi a primeira proteína estudada no Brasil sob o aspecto bioquímico e farmacológico. O quadro clínico mais evidente e característico por ela induzido é a intensa paralisação dos membros posteriores quando injetada por via i.v. (este trabalho) ou mesmo i.p. (tradicional) em camundongos ou ratos. Vários fármacos, inclusive aqueles descritos como antagonistas da crotamina em diafragma isolado de rato, não foram eficientes para reverter seu efeito in vivo. Somente alterações químicas como acetilação de aminogrupos ou redução/carboximetilação das pontes dissulfeto conseguiram abolir completamente esse efeito.

Valores normais de temperatura na face póstero-superior do trapézio por avaliaçäo termográfica / Titulo normal temperature at the posterior superior aspect of the trapezius by thermographic assessment

Este trabalho teve como objetivo verificar, por medidas termográficas, as temperaturas médias na região póstero-superior do músculo trapézio, em indivíduos normais (mão portadores de qualquer sintomatologia na região), na expectativa de padronizar valores normais de temperatura nos lados direito e esquerdo, para que, em estudos posteriores, possamos compará-los com os achados de indivíduos portadores de fibromialgia, na tentativa de se estabelecer mais um método de diagnóstico da patologia. Constatou-se que, num total de 13 indivíduos estudados, em apenas 1 (7,74 por cento) houve igualdade (35,8 C) de temperatura entre os lados direito e esquerdo naquela região do trapézio. Nos 12 restantes (92,3 por cento) o lado direito apresentou temperaturas mais elevadas ( T = 0,2 a 1,7 C) do que o esquerdo. Essa diferença mostrou-se independente da faixa etária, sexo e lado predominante de atividade física e é aparentemente decorrente de assimetria circulatória dos grandes vasos, não caracterizando, portando, nenhuma patologia ou síndrome fibromiálgica

Convulxin does not induce convulsions when injected into the rat dorsal hippocampus

La convulxina, una neurotoxina extraída del veneno de Crotalus durissus terrificus, ejerce un efecto convulsivante cuando se inyecta por vía endovenosa en ratones y gatos. En la búsqueda de nuevos compuestos más efectivos selectivos, diversos estudios se han desarrollado en el campo de las neurotoxinas. En consecuencia, el objetivo de este trabajo fue analizar los efectos comportamentales, electroencefalograficos y neuropatológicos desencadenados por la inyección intrahipocampal de convulxina. En otra secuencia experimental, fue utilizada una mexcla de convulxina y plasma rico en plaquetas, como tentativa de testar la hipótesis de la acción indirecta de la convulxina. Los resultados han demostrado que tanto la convulxina como la mexcla de convulxina y plasma rico en plaquetas fueron incapaces de desencadenar convulsione o de causar lesiones celulares específicas (AU)

Convulxin does not induce convulsions when injected into the rat dorsal hippocampus

La convulxina, una neurotoxina extraída del veneno de Crotalus durissus terrificus, ejerce un efecto convulsivante cuando se inyecta por vía endovenosa en ratones y gatos. En la búsqueda de nuevos compuestos más efectivos selectivos, diversos estudios se han desarrollado en el campo de las neurotoxinas. En consecuencia, el objetivo de este trabajo fue analizar los efectos comportamentales, electroencefalograficos y neuropatológicos desencadenados por la inyección intrahipocampal de convulxina. En otra secuencia experimental, fue utilizada una mexcla de convulxina y plasma rico en plaquetas, como tentativa de testar la hipótesis de la acción indirecta de la convulxina. Los resultados han demostrado que tanto la convulxina como la mexcla de convulxina y plasma rico en plaquetas fueron incapaces de desencadenar convulsione o de causar lesiones celulares específicas