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

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

Metabolismo energético do camarão da amazonia, Macrobrachium amazonicum

O Camarão da Amazônia, Macrobrachium amazonicum, tem um grande potencial para a aquicultura e possui uma ampla distribuição na América do Sul oriental. Seu cultivo envolve a utilização de rações comerciais, mas até o presente momento não existem rações balanceadas para essa espécie devido à falta de informações sobre o metabolismo desses animais. A qualidade das rações, não está associada diretamente a quantidade de proteínas, e lipídeos presentes, mas sim ao balanço de aminoácidos e ácidos graxos que as compõem. Ainda, estudos sugerem que os lipídios, colesterol, e carboidratos complexos sejam nutrientes importantes já que estão relacionados à maturação gonadal, ao desenvolvimento e crescimento desses animais. Alguns estudos indicam que a proteína é um dos principais nutrientes orgânicos para os Penaeus, porém o mesmo não ocorre para camarões dulcícolas. Dessa maneira, o presente estudo tem como objetivo compreender de uma melhor forma o metabolismo energético de M. amazonicum pela avaliação dos níveis de colesterol, triglicerídeos, proteínas, lipídios e carboidratos quando alimentados com dois tipos diferentes de rações para camarão Penaeus disponíveis no mercado (P38 e V35) e uma ração alternativa de menos custo usada na criação de tilápias (T32). Machos acima de três gramas do morfotipo Pinça cor-de-canela (CC) de M. amazonicum foram coletados de viveiros, transferidos para aquários e foram submetidos a três tipos de dietas com diferentes rações comerciais P38, V35 e T32 durante nove dias. Foram realizados testes para averiguar as concentrações totais de proteínas e carboidratos solúveis, lipídeos e umidade dos camarões e das rações. Foram verificados também os níveis de colesterol, triglicerídeos, carboidratos, proteínas plasmáticas e os principais lipídeos, triglicerídeos e fosfolipídios presentes...The Amazon Shrimp, Macrobrachium amazonicum has great potential for aquaculture and has a wide distribution in eastern South America. Its farming involves the use of commercial feeds, but to date there are no balanced feeds for this species due to lack of information about these animals metabolism. The quality of the feed is not directly associated with the total amount of protein or lipids, but the amount of each amino acids and fatty acids that compose them. On the other hand, studies suggest the lipids, cholesterol, and carbohydrates as the major nutrients as they are related to gonadal maturation development and growth of these animals. Studies suggest that proteins are the main energy source for Penaeus shrimps however this is not wholly true for praws. Thus, this study aims to understand in a better way the energetic metabolism of M. amazonicum evaluated by the levels of cholesterol, triglycerides, carbohydrates, proteins, while feed with commercial ration, two used in Penaues aqualculture (P38 and V35) and one alternative cheaper feed used in tilapia aquaculture (T32). Cinnamon Claw (CC) males above three grams of M. amazonicum ware colected from farms, transferred to tanks and ware feed with three diets P38, V35 and T32 for nine days. Total soluble carbohydrates, proteins lipids and moisture content tests of the shrimp and rations ware conducted. Also ware checked cholesterol, triglycerides, carbohydrates, proteins levels from hemolymph and major fat acids, triglycerides and phospholipids in the abdominal muscle and hepatopancreas. Diets V35 and T32 showed the best responses to mass gain and metabolic pattern. Despite feed T32 ware formulated for fishes aquaculture the environment they live are similar to those macrobrachiuns lives, which may have resulted in a better carbohydrate and fat acids, as ω-6/ω-3, balance. Proteins ware apparently well absorbed in ration with... (Complete abstract click electronic access below

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

“METABOLIC RIDE” a conceptual evaluation tool for metabolic biochemistry teaching for graduate and postgraduate students in biological sciences and related areas

Biochemistry as a discipline have a high degree of difficulty. Otherwise, application of creative games as teaching methodology has spread in various disciplines. "METABOLIC RIDE" board game is a conceptual and perceptual evaluation tool for biochemistry teaching, aiming to review concepts transmitted in classroom, promoting a competitive challenge to students without denying tools that are at their disposal, stimulating their skills such as their creativity. Further, it makes possible to correlate metabolic routes and their interconnections to establish that metabolic pathways are not separated, such as a railway map. In addition, this game proved to be an excellent tool for student’s complementary evaluation, which allowed to analyze the student’s perception and thus realize that when properly stimulated some groups could show a great productive and creative capacity. However, this game demonstrated to students new ways to approach complex subjects in biochemistry using creativity.A disciplina de bioquímica no geral é considerada de alto grau de dificuldade. Contudo, a aplicação de jogos lúdicos como metodologia de ensino vem se disseminando em várias disciplinas. “METABOLIC RIDE” é uma ferramenta de avaliação conceitual e de percepção para o ensino de bioquímica, visando rever conceitos difundidos em sala de aula, promovendo um desafio competitivo aos estudantes sem negar as ferramentas que estão a sua disposição, estimulando diversas aptidões dos mesmos, como a sua criatividade. Ainda, possibilita correlacionar a importância das rotas metabólicas e suas interligações a fim de sedimentar que os caminhos metabólicos não estão separados, como um mapa ferroviário. Ademais, este jogo se mostrou uma excelente ferramenta de avaliação complementar, mostrando que quando devidamente estimulados alguns grupos foram capazes de mostrar uma capacidade produtiva e criativa. Além disso, mostrou-se novas formas de abordar temas complexos em bioquímica com práticas lúdicas

Sulfated polysaccharide extracted of the green algae Caulerpa racemosa increase the enzymatic activity and paw edema induced by sPLA2 from Crotalus durissus terrificus venom

Sulfated polysaccharides derived from seaweed have shown great potential for use in the development of new drugs. In this study, we observed that a low-molecular-weight sulfated polysaccharide from Caulerpa racemosa, termed CrSP, could interact with secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus terrificus venom. When native sPLA2 (14 kDa) was incubated with CrSP, they formed a molecular complex (sPLA2:CrSP) with a molecular mass of 32 kDa, approximately. Size exclusion chromatography experiments suggested that CrSP formed a stable complex with sPLA2. We belived that sPLA2 and SPCr are involved an ionic interaction between negatively charged CrSP and the positively charged basic amino acid residues of sPLA2, because this interaction induced significant changes in sPLA2 enzymatic and pharmacological activities. CrSP caused a significant increase in sPLA2 enzymatic and bactericidal activity and increased its edematogenic effect. A pharmacological assay showed that the myotoxic activity of sPLA2:CrSP is unrelated to its enzymatic activity and that sPLA2:CrSP may have a practical application as a natural antibacterial agent for use in humans and commercially raised animals

An Evaluation of 3-Rhamnosylquercetin, a Glycosylated Form of Quercetin, against the Myotoxic and Edematogenic Effects of sPLA2 from Crotalus durissus terrificus

This paper shows the results of quercitrin effects on the structure and biological activity of secretory phospholipase (sPLA2) from Crotalus durissus terrificus, which is the main toxin involved in the pharmacological effects of this snake venom. According to our mass spectrometry and circular dichroism results, quercetin was able to promote a chemical modification of some amino acid residues and modify the secondary structure of C. d. terrificus sPLA2. Moreover, molecular docking studies showed that quercitrin can establish chemical interactions with some of the crucial amino acid residues involved in the enzymatic activity of the sPLA2, indicating that this flavonoid could also physically impair substrate molecule access to the catalytic site of the toxin. Additionally, in vitro and in vivo assays showed that the quercitrin strongly diminished the catalytic activity of the protein, altered its Vmax and Km values, and presented a more potent inhibition of essential pharmacological activities in the C. d. terrificus sPLA2, such as its myotoxicity and edematogenic effect, in comparison to quercetin. Thus, we concluded that the rhamnose group found in quercitrin is most likely essential to the antivenom activities of this flavonoid against C. d. terrificus sPLA2

Purification and characterization of the first γ-phospholipase inhibitor (γPLI) from Bothrops jararaca snake serum.

Phospholipases A2 (PLA2) are enzymes acting on the cell membrane phospholipids resulting in fatty acids and lysophospholipids and deconstructing the cell membrane. This protein is commonly found in snake venoms, causing tissue inflammation in the affected area. Evidence indicates that snakes have natural resistance to their own venom due to protective properties in plasma, that inhibit the action of proteins present in their venom. Given that, this study aimed to purify and characterize a γPLI from Bothrops jararaca serum, named γBjPLI. PLA2 inhibitor was isolated using two chromatographic steps: an ion exchange column (DEAE), followed by an affinity column (crotoxin coupled to a CNBr-activated Sepharose resin). The purity and biochemical characterization of the isolated protein were analyzed by RP-HPLC, SEC, SDS-PAGE, circular dichroism and mass spectrometry. The ability to inhibit PLA2 was determined by enzymatic activity, neutralization of paw edema and myonecrosis. The protein purity was confirmed by RP-HPLC and SEC, whilst an apparent molecular mass of 25 kDa and 20 kDa was obtained by SDS-PAGE, under reducing and non-reducing conditions, respectively. According to mass spectrometry analysis, this protein showed 72% and 68% of coverage when aligned to amino acid sequences of two proteins already described as PLIs. Thus, the inhibitory activity of enzymatic, edema and myonecrotic activities by γBjPLI suggests a role of this inhibitor for protection of these snakes against self-envenomation