Edema Induced by sPLA2 from <em>Crotalus durissus terrificus</em> Involves PLC and PKC Signaling, Activation of cPLA2, and Oxidative Stress

sPLA2 from Crotalus durissus terrificus venom, free of crotapotin (Cdt sPLA2), purified and isolated sPLA2, was able to significantly increase lipid peroxidation, which occurred simultaneously with increased arachidonic acid (AA) metabolism. In addition, MDA and AA levels were elevated at 15 min after Cdt sPLA2 injection and after peak edema (negative control). Thus, oxidative stress and ROS play important roles in the inflammation induced by Cdt sPLA2. On the other hand, edema induced by sPLA2 involves the direct and indirect mobilization of arachidonic acid by the involvement of phosphokinase C (PKC) and phospholipase C (PLC), which indirectly stimulates cytosolic PLA2 (cPLA2). We also observed that the specific antivenin against Cdt venom had no significant effect on the neutralization of induced edema compared to the natural products 5-caffeine-linoleic acid (5CQA) and dexamethasone (AACOCF3). Our results also indicate that there was improvement in the inhibition of edema of natural polyphenolic compounds compared to antivenin or inhibition of the enzymatic activity of sPLA2 due to the fact that 5CQA is a potent antioxidant compound. Thus, our results show a clear correlation between increased arachidonic acid metabolism and oxidative stress

Structure of xanthan gum and cell ultrastructure at different times of alkali stress

The effect of alkali stress on the yield, viscosity, gum structure, and cell ultrastructure of xanthan gum was evaluated at the end of fermentation process of xanthan production by Xanthomonas campestris pv. manihotis 280-95. Although greater xanthan production was observed after a 24 h-alkali stress process, a lower viscosity was observed when compared to the alkali stress-free gum, regardless of the alkali stress time. However, this outcome is not conclusive as further studies on gum purification are required to remove excess sodium, verify the efficiency loss and the consequent increase in the polymer viscosity. Alkali stress altered the structure of xanthan gum from a polygon-like shape to a star-like form. At the end of the fermentation, early structural changes in the bacterium were observed. After alkali stress, marked structural differences were observed in the cells. A more vacuolated cytoplasm and discontinuities in the membrane cells evidenced the cell lysis. Xanthan was observed in the form of concentric circles instead of agglomerates as observed prior to the alkali stress.471102109CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQSem informaçã

Evaluation of Rhamnetin as an Inhibitor of the Pharmacological Effect of Secretory Phospholipase A2

Rhamnetin (Rhm), 3-O-methylquercetin (3MQ), and Rhamnazin (Rhz) are methylated derivatives of quercetin commonly found in fruits and vegetables that possess antioxidant and anti-inflammatory properties. Phospholipase A2 (PLA2) displays several important roles during acute inflammationtherefore, this study aimed at investigating new compounds able to inhibit this enzyme, besides evaluating creatine kinase (CK) levels and citotoxicity. Methylated quercetins were compared with quercetin (Q) and were incubated with secretory PLA2 (sPLA2) from Bothrops jararacussu to determine their inhibitory activity. Cytotoxic studies were performed by using the J774 cell lineage incubated with quercertins. In vivo tests were performed with Swiss female mice to evaluate decreasing paw edema potential and compounds' CK levels. Structural modifications on sPLA2 were made with circular dichroism (CD). Despite Q and Rhz showing greater enzymatic inhibitory potential, high CK was observed. Rhm exhibited sPLA2 inhibitory potential, no toxicity and, remarkably, it decreased CK levels. The presence of 3OH on the C-ring of Rhm may contribute to both its anti-inflammatory and enzymatic inhibition of sPLA2, and the methylation of ring A may provide the increase in cell viability and low CK level induced by sPLA2. These results showed that Rhm can be a candidate as a natural compound for the development of new anti-inflammatory drugs.Universidade Estadual Paulista (UNESP)Universidade Federal de São Paulo (UNIFESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed São Paulo, Postgrad Program Food Nutr & Hlth, BR-11015020 São Paulo, BrazilUniv Estadual Paulista, Biosci Inst, BR-11330900 São Paulo, BrazilBrazil Univ, Prorector Res, BR-08230030 São Paulo, BrazilUniv São Paulo, Pathol Lab Infect Dis LIM50, Dept Pathol, Sch Med, BR-01246903 São Paulo, BrazilUniv Fed São Paulo, Postgrad Program Food Nutr & Hlth, BR-11015020 São Paulo, BrazilWeb of Scienc

Estudo das frações proteicas derivadas do veneno de serpentes "crotalicas" e "bothropicas" com atividade antibacteriana, isolamento, purificação e caracterização bioquimica e biologica

Orientador: Sergio MarangoniTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Nesta tese foram apresentados os resultados da purificação de várias proteínas biologicamente ativas do veneno de serpentes crotálicas (CrotaJus durissus temfjcus, Crotalus durissus cascavel Ia, Crotalus durissus ruruima e CrotaJus durissus colliJineatus); botrópica (Bothrops pirajal) e coral (Mícrurus dumerilíi carínícauda). Muitas destas frações foram purificadas e caracterizadas do ponto de vista biológico, bioquímico e farmacológico. FraçOes como: crotamina, fosfolipase A2 (PLA2), crotapotinas e outras mostraram uma significativa atividade antibacteriana contra bactérias frtopatogênicas e patogênicas. As análises ultraestruturais e outros dados bioquímicos indicam que estas frações exercem essa atividade de formas diferenciadas. Foram observados quatro mecanismos de ação: 1.interação das frações com a membrana bacteriana, induzindo a ruptura e desorganização da mesma com posterior lise celular, 2. desorganização citoplasmática através da entrada das frações por poros na membrana causando lise celular, 3. digestão da membrana com posterior extravasamento do conteúdo interno bacteriano com lise celular, 4.1ise da membrana através dos radicais livres gerados pela reação enzimática do veneno com a membrana bacteriana. Durante o desenvolvimento da tese foram publicados e submetidos à publicação os seguintes artigos: 1.0liveira et aI., 2002 apresentaram o fracionamento do veneno de CrotaJus durissus terrificus em HPLC de fase reversa, com obtenção das frações: crotapotinas (F5 e F7) e PLA2 (F15, F16 e F17). As frações apresentaram atividade antibacteriana contra Xanthomonas axonopodis pv. passiflorae. A fração F17 apresentou atividade anticoagulante e foi seqüenciada apresentando uma homologia estrutural com outras PLA2 em tomo de 60-90%. Sua massa molecular de 14,6kDa foi determinada através de tricina SDS-PAGE, eletroforese de duas dimensões e MALDI TOFF. 2. T oyama at aI., 2003 verificaram a atividade enzimática, neurotóxica e antibacteriana da fração PLA2 (F15) isolada do veneno de CrotaJus dirissus teTTificus. 3.0liveira et aI., 2003 isolaram, purificaram, seqüenciaram a fração crotapotina, um componente ácido da crotoxina do veneno de Crotalus duríssus cascavel Ia, e realizaram testes para verificar a atividade antibacteriana em bactérias fitopatogênicas Gram-positivas e Gram-negativas, além de testes inflamatórios e nefrotóxicos. 4.Havt et aI., 2005 isolaram uma nova lectina tipo C (BPL) de Bothrops pirajai que foi caracterizada biológica e bioquimicamente. Esta fração também mostrou um efeito nefrotóxico em perfusão em rins isolados de ratos. 5. Toyama et al.,2004 isolaram, purificaram e caracterizaram bioquimicamente uma nova crotamina símile do veneno de Crotalus durissus cascavel/a. Foram identificados os peptídeos biologicamente ativos da crotamina, os quais foram responsáveis pela estimulação de secreção de insulina. . 6.Gandhi et aI., 2004 purificaram uma nova lectina tipo C (crotacetin), obtida do veneno de Crotalus durissus cascavel/a. Esta nova lectina induziu a agregação plaquetária, a aglutinação eritrocitária e também mostrou uma atividade antibacteriana. 7. Cavada et aI., 2004 purificaram e caracterizaram uma nova lectina da semente de Lonchocarpus sericeus, com alta especificidade por N-acetilglicosamina. 8. Cháriston et ai, 2004 isolaram e determinaram a estrutura primária de uma nova PLA2 de Micrurus dumerilli carinicauda. 9.Toyama et aI., 2004 isolaram, purificaram e caracterizaram uma nova isoforma da crotamina do veneno de Crotalus durissus ruruima e mostraram seu efeito antibacteriano. 10.Toyama et aI., 2004 isolaram, purificaram e caracterizaram uma nova PLA2 do veneno de Crotalus durissus collilineatus. Nestes trabalhos foram determinadas as regiões moleculares responsáveis pela atividade antibacteriana. 11. Toyama et a/., 2004 purificaram e caracterizaram uma nova L-amino oxidase do veneno de Crotalus durissus cascavel Ia (Casca LAO) através de exclusão molecular e HPLC de troca iônica. Casca LAO apresentou uma grande inibição bacteriana em bactérias Gram-negativas (Xanthomonas axonopodis pv. passiflorae) e Gram-positivas (Streptococcus mutans). Os estudos destas frações apresentam perspectivas futuras na aplicação terapêutica, como agentes antibacterianos, de novos peptídeos sintéticos ou naturaisAbstract: In this dissertation we present the results of purification of several biologically active protein from the crotalic rattlesnake venom (Crotalus durissus terrificus, Crotalus durissus cascavel/a, Crotalus durissus ruruima e Crotalus durissus collilineatus); from bothropic (Bothrops piraja) and coral (Micrurus dumerilli camicauda). Many of these fractions were purified and biochemical, biologically and pharmacologically characterized, some were reported and other submitted for publication in specialized joumals. Some fraction as crotamine, phospholipase A2 (PLA2), crotapotins and other showed a significant antibacterial activity against phytopatogenical and pathogenic bacterium. Ultra structural and biochemical analyses suggest that this fraction exert this antibacterial activity by different ways, which are grouped according to the mode action into four different mechanisms: 1.interaction of some fractions with the bacterial membrane inducing cellular Iyses by membrane rupture and disorganization. 2. Bacterial cell Iyses by action of fraction from the venom that form pore on the membrane and consequently cytoplasmatic disorganization and membrane rupture. 3. Membrane cell digestion induced by protein from the venom and consequently lost of cytoplasmatic content and cellular Iyses. 4. Membrane cel! Iyses induced by free radical generated by enzymatic action of protein form the venom. During the development of this thesis we reported and submitted some articles listed below: 1. Oliveira et aI., 2002 presents the purification of crotapotins (F5 and F7) and PLA2 (F15, F16 and F17)from the Crotalus durissus terrificus venom by reverse phase HPLC. These fraction showed significant antibacterial activity against Xanthomonas axonopodis pv. Passiflorae. Fraction F17 also showed an anticoagulant activity and its amino acid sequence showed high amino acid sequence identity with.60-90% with other PLA2 from different sources. Its molecular mass was 14,6kDa determined by Tricine PAGE-SDS, two dimensional electrophoresis and MALDI TOFF. 2. Toyama et aI., 2003 showed the enzymatic, neurotoxic and antibacterial activities of PLA2 isoform (F15) from the Crotalus durissus terrificus venom. 3. Oliveira et aI., 2003 isolated, purified and determined the amino acid sequence of crotapotin fraction, an acid compound of crotoxin from the Crotalus durissus cascavel/a, and detennined its antibacterial activity against Gram-positive and Gram-negative phytopathogenic baderium, intlammatory and nephrotoxicity. 4. Havt et aI., 2005 isolated a new type C-Iectin (SPL) trom the Bothrops pirajaithat was biologically and biochemical characterized. This fraction also showed a nephrotoxic effect on the isolated perfused kidney. 5. Toyama et aI., 2004 isolated, purified and characterized biochemical a new crotamine like from Crotalus durissus cascavella venom. In this work we identified the biologically active peptide from this crotamine, which were responsible for increasing insulin secretion. 6. Gandhi et aI., 2004 purified a new lectin type C (crotocetin) trom the Crotalus durissus cascavella venom. This lectin induced platelet aggregation, erythrocyte agglutination and also showed an antibacterial activity. 7. Cavada et aI., 2004 purified and characterized a new lectin from the Lonchocarpus serieus seed that showed high specific binding against N-acetyl glucosamine. 8. Cháriston et aI., 2004 isolated and determined the amino acid sequence of new PLA2 from Micrurus dumerilli carinicauda. 9. Toyama et aI., 2004 isolated, purified and characterized a new crotamine isofonn from the Crotalus durissus ruruima venom and showed its antibacterial activity. 10. Toyama et aI., 2004 isolated, purified and charaderized a new PLA2 trom the Crotalus durissus collilineatus venom. In this work we determined the molecular region of the PLA2 responsible for the antibacterial activity. 11. Toyama et aI., 2004 purified and characterized a new L-amino acid oxidase from the Crotalus durissus cascavella whole venom (Casca LAAO) by molecular exdusion and cation exchange HPLC. Casca LAAO inhibited strongly the bacterial growth rate of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (Streptococcus mutans). These studies presented here showed a future perspective to therapeutic application of these peptides, synthetical/y or natural/y obtained as potential antibacterial substancesDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Edema Induced by a Crotalus durissus terrificus Venom Serine Protease (Cdtsp 2) Involves the PAR Pathway and PKC and PLC Activation

Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze N&alpha;-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis

Identification of two novel cytolysins from the hydrozoan Olindias sambaquiensis (Cnidaria)

Background: Although the hydrozoan Olindias sambaquiensis is the most common jellyfish associated with human envenomation in southeastern and southern Brazil, information about the composition of its venom is rare. Thus, the present study aimed to analyze pharmacological aspects of O. sambaquiensis venom as well as clinical manifestations observed in affected patients. Crude protein extracts were prepared from the tentacles of animals; peptides and proteins were sequenced and submitted to circular dichroism spectroscopy. Creatine kinase, cytotoxicity and hemolytic activity were evaluated by specific methods.Results: We identified two novel cytolysins denominated oshem 1 and oshem 2 from the tentacles of this jellyfish. The cytolysins presented the amino acid sequences NEGKAKCGNTAGSKLTFKSADECTKTGQK (oshem 1) and NNSKAKCGDLAGWSKLTFKSADECTKTGQKS (oshem 2) with respective molecular masses of 3.013 kDa and 3.375 kDa. Circular dichroism revealed that oshem 1 has random coils and small a-helix conformation as main secondary structure whereas oshem 2 presents mainly random coils as its main secondary structure probably due to the presence of W (13) in oshem 2. The hemolysis levels induced by oshem 1 and oshem 2 using a peptide concentration of 0.2 mg/mL were, respectively, 51.7 +/- 6.5% and 32.9 +/- 8.7% (n = 12 and p <= 0.05). Oshem 1 and oshem 2 showed significant myonecrotic activity, evaluated by respective CK level measurements of 1890.4 +/- 89 and 1212.5 +/- 103 (n = 4 and p <= 0.05). In addition, myonecrosis was also evaluated by cell survival, which was measured at 72.4 +/- 8.6% and 83.5 +/- 6.7% (n = 12 and p <= 0.05), respectively. The structural analysis showed that both oshem 1 and oshem 2 should be classified as a small basic hemolytic peptide.Conclusion: The amino acid sequences of two peptides were highly similar while the primary amino acid sequence analysis revealed W (22th) as the most important mutation. Finally oshem 1 and oshem 2 are the first cytolytic peptides isolated from the Olindias sambaquiensis and should probably represent a novel class of cytolytic peptides

Evaluation of Macroalgae Sulfated Polysaccharides on the Leishmania (L.) amazonensis Promastigote

The sulfated polysaccharides from Solieria filiformis (Sf), Botryocladia occidentalis (Bo), Caulerpa racemosa (Cr) and Gracilaria caudata (Gc) were extracted and extensively purified. These compounds were then subjected to in vitro assays to evaluate the inhibition of these polysaccharides on the growth of Leishmania (L.) amazonensis promastigotes. Under the same assay conditions, only three of the four sulfated polysaccharides were active against L. amazonensis, and the polysaccharide purified from Cr was the most potent (EC50 value: 34.5 μg/mL). The polysaccharides derived from Bo and Sf demonstrated moderate anti-leishmanial activity (EC50 values of 63.7 μg/mL and 137.4 μg/mL). In addition, we also performed in vitro cytotoxic assays toward peritoneal macrophages and J774 macrophages. For the in vitro cytotoxicity assay employing J774 cells, all of the sulfated polysaccharides decreased cell survival, with CC50 values of 27.3 μg/mL, 49.3 μg/mL, 73.2 μg/mL, and 99.8 μg/mL for Bo, Cr, Gc, and Sf, respectively. However, none of the sulfated polysaccharides reduced the cell growth rate of the peritoneal macrophages. These results suggest that macroalgae contain compounds with various chemical properties that can control specific pathogens. According to our results, the assayed sulfated polysaccharides were able to modulate the growth rate and cell survival of Leishmania (L.) amazonensis promastigotes in in vitro assays, and these effects involved the interaction of the sulfated polysaccharides on the cell membrane of the parasites

“Biotecnological war” uma ferramenta de avaliação conceitual e de percepção para o ensino de biotecnologia e química de proteínas para os alunos de graduação em ciências biológicas

Biochemistry in general is practically unanimous as a discipline with a high degree of difficulty, complex and "boring". So, practical and creative play games as teaching methodology has been disseminated in several disciplines of biological sciences. “Biotecnological war” board game is a proposal that was initially conceived as an alternative complementary tool for biochemistry teaching of proteins and peptides, challenging students, aiming to review concepts transmitted in classroom, stimulating student’s abilities, such as their creativity, competitiveness, resource management and making possible to correlate biochemistry importance of proteins and peptides as new products. This game proved to be an excellent tool for complementary evaluation of students, which besides stimulating teamwork, also stimulated "a strong competitive spirit" within the classroom, which allowed to analyze students' perception in relation to the theme and mainly articulated group work.A bioquímica no geral é taxada como uma disciplina com alto grau de dificuldade, complexa e “chata”. Por outro lado, a aplicação de jogos lúdicos práticos e criativos como metodologia de ensino vem se disseminando em várias disciplinas em ciências biológicas. O jogo de tabuleiro “Biotecnological war” é uma proposta pensada como uma ferramenta complementar para o ensino de bioquímica de proteínas e peptídeos, desafiando os discentes, visando rever conceitos transmitidos em sala de aula, e estimulando aptidões dos estudantes, como sua criatividade, competitividade e gestão de recursos. Possibilitando ainda correlacionar a importância da bioquímica de proteínas e peptídeos com o desenvolvimento de produtos. Este jogo se mostrou uma excelente ferramenta de avaliação complementar dos alunos, e além de estimular o trabalho em equipe, também estimulou “um forte espírito competitivo”, o que permitiu analisar a percepção dos alunos em relação ao tema e principalmente o trabalho em grupo