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

Evaluation of the Inhibitory Potential of Casuarictin, an Ellagitannin Isolated from White Mangrove (Laguncularia racemosa) Leaves, on Snake Venom Secretory Phospholipase A2

Ellagitannins constitute the largest group of hydrolyzable tannins of plants, and, from this group, casuarictin (Casu) was identified in some plant species. However, to our knowledge, no investigation of secretory phospholipase A2 (sPLA2) inhibition by Casu has been performed yet. Casuarictin was isolated by chromatography n-butanol (n-BuOH) partition of Laguncularia racemosa leaves. The pharmacological and biological effects of Casu were evaluated on isolated sPLA2 from the rattlesnake (Crotalus durissus terrificus) and using a plant bacterial strain. The compound was able to form a protein complex consisting of a stable sPLA2 + Casu complex. Analyses carried out with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF) revealed that the molecular mass of sPLA2 increased from 14,425.62 to 15,362.74 Da. The enzymatic activity of the sPLA2 + Casu complex was significantly lower than that of native sPLA2. Besides, molecular interactions of Casu with sPLA2 were able to virtually abolish the native edematogenic effect as well as myonecrosis induced by the protein when injected 10 min after sPLA2. Therefore, Casu may be considered a potential anti-inflammatory that can be used to treat edema and myonecrosis induced by serine-secreting phospholipase A2. In addition, the compound also showed great antimicrobial potential

Identification, isolation and characterization of the PLA2 plasma inhibitor from the non-venomous snake Boa constrictor serum

O plasma de uma série de animais possui componentes que promovem uma resistência natural ao envenenamento, através da inibição de proteínas específicas que compõem o veneno das serpentes, como as fosfolipases A2 (PLA2s). A hipótese mais consistente para a presença de tais inibidores seria a proteção contra o auto envenenamento. Porém, a ocorrência de inibidores no plasma de serpentes não peçonhentas, bem como em outras espécies de animais, abriu novas perspectivas em relação à presença de tais moléculas. Deste modo, o presente trabalho tem como objetivo a identificação, o isolamento e a caracterização de um inibidor de PLA2 (PLI) presente no soro da Boa constrictor (BoaPLI), serpente não peçonhenta que habita extensamente o território brasileiro. Para tal, o inibidor foi isolado do soro de B. constrictor através de dois passos cromatográficos, tendo 0,63% de recuperação. A partir de então, foi realizada sua caracterização estrutural por SDS-PAGE, sendo identificada uma banda principal a 25 kDa, em condições redutoras, e 20 kDa, em condições não redutoras. Sua estrutura primária parcial foi identificada, possuindo alta homologia com outros PLIγs, como o de Lachesis muta. Sua estrutura secundária também é semelhante a outros PLIγs. Além disso, quando incubado com a Asp-49 e Lys-49 PLA2s, em análise por dicroísmo circular, observou-se que não houve alteração severa de seu espectro. As análises cromatográficas de exclusão molecular (SEC) corroboraram a hipótese de formação de oligômeros do inibidor isolado. Para investigar o tipo de interação entre o PLI e PLA2s, uma varredura de fluorescência foi realizada, mostrando uma diminuição no pico da fluorescência quando incubadas. A interação entre inibidor e PLA2 também foi evidenciada por western blotting. Uma vez a interação confirmada, foi realizado o ensaio de inibição enzimática com a Asp-49 PLA2, uma vez que a substituição no resíduo 49 (Lys-49 PLA2) leva a perda de atividade enzimática. O BoaγPLI apresentou inibição de caráter dose-dependente, chegando a ∼48% de inibição. Seu potencial inibitório também foi certificado por ensaios farmacológicos de edema e miotoxicidade. Não houve diferença entre o potencial inibitório com as duas isoformas de PLA2s testadas. Para averiguar o envolvimento do inibidor com tal atividade, foi realizado o ensaio de tromboelastometria, no qual o tempo de coagulação foi diminuído quando a crotoxina foi incubada com o inibidor. Ademais, para investigação do potencial inibitório do BoaγPLI em relação ao veneno total de uma serpente ofiófaga, foi testada a sua ação inibitória da atividade anticoagulante do veneno de Micrurus laticollaris. O BoaγPLI foi eficaz na inibição da atividade anticoagulante, evidenciado pelo retorno em tempo de coagulação e da força do coágulo comparado ao controle. Deste modo, o presente estudo pode contribuir introduzindo novas perspectivas para inibidores de PLA2 provenientes de plasma de serpentes não peçonhentas.Plasma in several organisms has components that promote a natural resistance to envenomation by inhibiting specific proteins that constitute snake venoms, such as phospholipases A2 (PLA2s). The most consistent hypothesis for the presence of such inhibitors would be protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in the plasma of non-venomous snakes, as well as in other species of animals, has opened new perspectives for the presence of these molecules. Thus, the work aimed to identify, isolate, and characterize a PLA2 inhibitor (PLI) present in the serum of Boa constrictor, a non-venomous snake that dwells extensively in the Brazilian territory. For such purpose, the inhibitor was isolated from B. constrictor serum by two chromatographic steps, with 0.63% of recovery. From this point on, the structural characterization was performed by SDS-PAGE, which showed a major band at 25 kDa under reducing conditions and 20 kDa under non-reducing conditions. Its partial primary structure was identified, possessing high homology with other PLIγs, such as Lachesis muta. Its secondary structure is also similar to other PLIγs. In addition, when incubated with Asp-49 and Lys-49 PLA2s, analyzed by circular dichroism, showed that there was no severe change in its spectrum. The chromatographic analysis of molecular exclusion corroborated the presence of oligomers in the isolated inhibitor. To investigate the interaction between the PLI and the PLA2, a fluorescence scan was performed, showing a decrease in the fluorescence peak when incubated. The interaction between inhibitor and PLA2 was also confirmed by western blotting. Once the interaction was established, an enzymatic inhibition assay was performed only with Asp-49 PLA2, since the substitution on the 49 residue (Lys-49) leads to loss of enzymatic activity. The BoaPLI showed a dose-dependent inhibition, reaching at ~48% when the inhibitor was incubated with PLA2. Its inhibitory potential was also confirmed by pharmacological tests of edema and myotoxicity. There was no difference between the inhibitory potential with the two isoforms of PLA2s tested. To investigate the involvement of the inhibitor with such activity, a thromboelastometric assay was performed in which the coagulation time was decreased when the crotoxin was incubated with the inhibitor. Furthermore, to investigate the inhibitory potential of BoaγPLI in relation to the whole venom of an ophiophage snake, the inhibitory action of the anticoagulant activity of the venom of Micrurus laticollaris, ophiophage snake and sympatric to B. constrictor was also investigated, whereas the BoaγPLI was efficient on returning the clotting time and strength. Thus, the present work may provide new insights into protein inhibitors acting on envenomation in non-venomous snakes

“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

“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

Clinical and Evolutionary Implications of Dynamic Coagulotoxicity Divergences in Bothrops (Lancehead Pit Viper) Venoms

Despite coagulotoxicity being a primary weapon for prey capture by Bothrops species (lancehead pit vipers) and coagulopathy being a major lethal clinical effect, a genus-wide comparison has not been undertaken. To fill this knowledge gap, we used thromboelastography to compare 37 venoms, from across the full range of geography, taxonomy, and ecology, for their action upon whole plasma and isolated fibrinogen. Potent procoagulant toxicity was shown to be the main venom effect of most of the species tested. However, the most basal species (B. pictus) was strongly anticoagulant; this is consistent with procoagulant toxicity being a novel trait that evolved within Bothrops subsequent to their split from anticoagulant American pit vipers. Intriguingly, two of the arboreal species studied (B. bilineatus and B. taeniatus) lacked procoagulant venom, suggesting differential evolutionary selection pressures. Notably, some terrestrial species have secondarily lost the procoagulant venom trait: the Mogi Mirim, Brazil locality of B. alternatus; San Andres, Mexico locality of B. asper; B. diporus; and the São Roque of B. jararaca. Direct action on fibrinogen was extremely variable; this is consistent with previous hypotheses regarding it being evolutionary decoupled due to procoagulant toxicity being the primary prey-capture weapon. However, human patients live long enough for fibrinogen depletion to be clinically significant. The extreme variability may be reflective of antivenom variability, with these results thereby providing a foundation for such future work of clinical relevance. Similarly, the venom diversification trends relative to ecological niche will also be useful for integration with natural history data, to reconstruct the evolutionary pressures shaping the venoms of these fascinating snakes

BoaγPLI: Structural and functional characterization of the gamma phospholipase A2 plasma inhibitor from the non-venomous Brazilian snake Boa constrictor.

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor's presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely

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