Caracterização da secreção cutânea do anfíbio Trachycephalus nigromaculatus e investigação da atividade antimicrobiana

Trachycephalus nigromaculatus is a helmeted treefrog belonging to the Hylidae family, distinguished by its cranial apparatus adapted for phragmosis, a unique defense strategy. Its skin is notable for the abundance of poison glands, playing a crucial role in resistance against predators and pathogens in its natural habitat. However, the chemical composition of the venom and the effects of exposure to cutaneous secretion in humans and animals remain poorly understood. This study aimed to characterize the cutaneous secretion, assess its toxic effects, and explore the antimicrobial activity of Trachycephalus nigromaculatus secretion. We employed chromatographic analyses, SDS-PAGE gel electrophoresis, and mass spectrometry to identify the fractions of cutaneous secretion. The cell-inflammatory-epithelial interaction was examined through intravital microscopy, while the impact on cell viability was evaluated using the MTT assay. Antimicrobial activity was tested in vitro against S. aureus, S. coli, and C. albicans. Results revealed a complex mixture in T. nigromaculatus cutaneous secretion, with proteins ranging from 8.5 to 77 kDa. Noteworthy components include hyaluronidase, peroxiredoxin-6, and the toxin anntoxin. MALDI- TOF analysis indicated an abundant presence of peptides. Low concentrations (1μg/mL) of the secretion exhibited significant cytotoxic activity (76%) against VERO cells. In intravital microscopy, the secretion (3 μg and 30 μg) displayed relevant toxic activity, causing loss of elasticity, edema, infiltration, and hemorrhagic points, preventing the experiment's continuation after 2 hours of injection. Within 24 hours, signs of necrosis and infiltration were evident, accompanied by an increase in adhered and migrated cells. These effects were observed within just 5 minutes of topical application. Inhibition of microbial growth was observed in crude secretion against S. coli, S. aureus, and C. albicans. At least half of the fractions tested by HPLC exhibited relevant activity. Antimicrobial activity was particularly effective against gram-positive bacteria (S. aureus) and gram-negative bacteria (S. coli), demonstrating notable antifungal action against C. albicans. These results are significant as they represent the first description of the antimicrobial activity of T. nigromaculatus cutaneous secretion, highlighting substantial inhibition against fungi. Furthermore, this study provides the first detailed description of the biochemical content of cutaneous secretion, revealing a unique composition of compounds inducing notable toxic effects both in vivo and in vitro. These findings, coupled with the distinctive cranial apparatus of this species, reinforce its efficacy as an efficient defense mechanism.

Trachycephalus nigromaculatus é uma perereca-de-capacete da família Hylidae, destacada pelo seu aparelho craniano adaptado para a fragmose, uma estratégia de defesa peculiar. Sua pele é notória pela abundância de glândulas de veneno, desempenhando um papel crucial na resistência contra predadores e patógenos em seu habitat natural. No entanto, a composição química do veneno e os efeitos da exposição à secreção cutânea em humanos e animais permanecem pouco compreendidos. Este estudo objetivou caracterizar a secreção cutânea, avaliar seus efeitos tóxicos e explorar a atividade antimicrobiana da secreção de Trachycephalus nigromaculatus. Empregamos análises cromatográficas, eletroforese em gel SDS- PAGE e espectrometria de massa para identificar as frações da secreção cutânea. A interação célula-inflamatória-epitelial foi examinada por microscopia intravital, enquanto o impacto na viabilidade celular foi avaliado pelo ensaio MTT. A atividade antimicrobiana foi testada in vitro contra S. aureus, S. coli e C. albicans. Os resultados revelaram uma mistura complexa na secreção cutânea de T. nigromaculatus, com proteínas variando de 8,5 a 77 kDa. Destacam-se hialuronidase, peroxirredoxina-6 e a toxina anntoxina. A análise MALDI-TOF indicou uma presença abundante de peptídeos. Concentrações baixas (1μg/mL) da secreção apresentaram notável atividade citotóxica (76%) contra células VERO. Na microscopia intravital, a secreção (3 μg e 30 μg) exibiu atividade tóxica relevante, causando perda de elasticidade, edema, infiltração e pontos hemorrágicos, impedindo a continuidade após 2h de injeção. Em 24 horas, sinais de necrose e infiltração foram evidentes, juntamente com um aumento na contagem de células aderidas e migradas. Esses efeitos foram observados após apenas 5 minutos de aplicação tópica. Observou-se inibição do crescimento microbiano na secreção bruta para S. coli, S. aureus e C. albicans. Pelo menos metade das frações testadas por HPLC exibiram atividade relevante. A atividade antimicrobiana foi especialmente eficaz contra bactérias gram-positivas (S. aureus) e gram-negativas (S. coli), além de apresentar notável ação antifúngica contra C. albicans. Esses resultados são significativos, pois representam a primeira descrição da atividade antimicrobiana da secreção cutânea de T. nigromaculatus, destacando uma inibição importante contra fungos. Além disso, este estudo oferece a primeira descrição detalhada do conteúdo bioquímico da secreção cutânea, revelando uma composição singular de compostos que induzem efeitos tóxicos notáveis tanto in vivo quanto in vitro. Essas descobertas, aliadas ao distintivo aparelho ósseo na cabeça desta espécie, reforçam sua eficácia como uma eficiente arma de defesa.

Identificação e caracterização bioquímica de inibidores de serinopeptidases isolados a partir da peçonha das anêmonas-do-mar Anthopleura cascaia e Aulactinia veratra

Phylum Cnidaria comprises more than 10,000 species and around 10% of them are represented by sea anemones. These animals are underexplored sources of molecules, possessing structurally diverse toxins that can act over a diverse range of pharmacological targets, including enzymes. Sea anemones represent almost 96% of the manually annotated toxins from the phylum, but until now only 5% of its species have been studied about their toxin content. In the present work, the venoms of the sea anemones Anthopleura cascaia and Aulactinia veratra were studied and accessed through mass spectrometry analysis for searching serine peptidase inhibitors. The arsenal of toxins from both venoms was elucidated. Additionally, venom’s fractions were screened for inhibitory activity over trypsin, using time-course fluorescence-based kinetic assays or Mass spectrometry-based analysis. Beyond that, the spatial distribution of serine peptidase inhibitors in both sea anemones’ tissues were shown through Mass Spectrometry Imaging by MALDITOF. In the analysis of toxins composition, it was seen that A. cascaia venom presents at least three types of toxins: cytolysins, phospholipases and a toxin similar to natterin. For A. veratra, the classification based on blastp hit similarity and relying on domain architecture of the toxin’s sequences (translated transcripts) was performed. The thorough examination over toxins sequences led to the identification of 59 proteins and peptides belonging to 14 known toxin’s families of sea anemones and to the acknowledge of 20 peptides presenting 18 new cysteine scaffolds. The venom of this sea anemone mainly relies on neurotoxins from ShK-like, β-defensins, SCRiP, ICK, EGF-like types and on serine peptidase inhibitors from Kazal and Kunitz types. Furthermore, serine peptidase inhibitors from both venoms were isolated and present main distribution over tentacles, mesenterial filaments and pedal disc of these sea anemones, suggesting the preferential stock of these toxins. In conclusion, the methodological approaches applied in this study were able of identifying the presence of serine peptidase inhibitors on the venom and tissue of sea anemones through chromatographic techniques followed by enzymatic assays, and MALDI-Imaging.

O filo Cnidaria é composto por mais de 10.000 espécies e cerca de 10% destas são anêmonas-do-mar. Estes animais são considerados fontes subexploradas de moléculas, possuindo um diverso arsenal de toxinas que podem agir sobre diferentes alvos farmacológicos, incluindo enzimas. Toxinas de anêmonas-do-mar representam cerca de 96% das toxinas anotadas para o filo Cnidaria, embora apenas 5% de suas espécies tenham sido estudadas quanto à composição de toxinas até o momento. Neste trabalho elucidamos por espectrometria de massas a composição da peçonha das anêmonas Anthopleura cascaia e Aulactinia veratra, buscando a identificação de inibidores de serinopeptidases. O arsenal de toxinas para ambas anêmonas foi elucidado. Ainda, descrevemos as etapas de purificação envolvidas na busca de inibidores e a seleção destes candidatos por meio da inibição da atividade da tripsina, avaliada por duas técnicas distintas։ Cinética enzimática e Espectrometria de massas. Adicionalmente, descrevemos a localização de candidatos a inibidores no tecido das anêmonas através do Imageamento por espectrometria de massas. Na análise sobre a composição de toxinas destas anêmonas, vimos que a peçonha da A. cascaia apresentou a existência três tipos de toxinas incluindo citolisinas, fosfolipases e naterinas. Para a espécie A. veratra, a classificação de toxinas baseadas no blastp hit e na arquitetura de domínios das toxinas foi realizada. Esta análise revelou a presença de 59 proteínas e peptídeos pertencentes a 14 famílias de toxinas de anêmonasdo-mar; além do reconhecimento de 20 peptídeos apresentando 18 novos scaffolds de cisteínas. A peçonha desta anêmona é principalmente composto por neurotoxinas do tipo ShK-like, β-defensinas, SCRiP, ICK, EGF-like e inibidores de serinopeptidases. Os dados obtidos mostram que ambas anêmonas são ricas fontes de inibidores de serinopeptidases, especialmente tipo Kunitz e Kazal. Tais inibidores apresentam distribuição na região dos tentáculos, mesentério e disco pedal das anêmonas, o que pode indicar o estoque preferencial destas toxinas. E conclusão, o conjunto de abordagens metodológicas empregadas neste trabalho foi capaz de atender os objetivos propostos: identificar a presença de inibidores de serinopeptidases na peçonha e tecido de anêmonas, tanto por fracionamento cromatográfico seguido de ensaio enzimático, quanto por MALDI-Imaging.

Estudo da ação de inibidores peptídicos de enzimas trombina-símiles na coagulação sanguínea humana / Study of the action of thrombin-like enzymes peptides inhibitors in human blood coagulation

Ophidism is considered the most important cause of accidents by venomous animals around the world. Botropic venom is a highly complex mixture of proteins, peptides and enzymes that, when interacting with human proteins, triggers several local symptoms, such as edema, ecchymosis, pain, blisters and, in more severe cases, necrosis and limb amputation. It also causes systemic symptoms, such as blood incoagulability. The metalloproteases (SVMPs) and the serine proteases (SVSPs) are the major components of Bothrops jararaca venom and are correlated with local and systemic effects. The treatment for poisoning indicated by the Ministry of Health is the administration of botropic antivenom, which despite being highly effective, has certain limitations, such as the partial neutralization of SVSPs. Among the strategies that aim to improve the antivenom and, thus, the treatment of envenomation, is the search for selective inhibitors for SVSPs, which can act simultaneously to the antivenom. Considering the similarity of the catalytic triad between human serine proteases and SVSPs, and since inertia against human serine proteases is a prerequisite for the association of any molecule with antivenom, the present project aimed at the “Study of the action of thrombin-like enzymes peptide inhibitors in human blood coagulation”. The peptide inhibitors, selective for the inhibition of SVSPs, were designed based on a substrate hydrolyzed by these enzymes from Bothrops jararaca venom and showed different inhibition efficacy. In silico analyzes suggest that the inhibitors bound in regions distant from the catalytic triad (His, Asp and Ser) of batroxobin, a thrombin-like enzyme used as a model in studies of enzyme/peptide inhibitor interaction.

O ofidismo é considerado a causa mais importante de acidentes por animais peçonhentos em todo o mundo. O veneno botrópico é uma mistura altamente complexa de proteínas, peptídeos e enzimas que, quando interagem com as proteínas humanas, desencadeiam vários sintomas locais, como edema, equimose, dor, bolhas e, em casos mais graves, necrose e amputação do membro; e sistêmicos, como a incoagulabilidade sanguínea. As metaloproteinases (SVMPs) e as serinoproteinases (SVSPs) são os componentes majoritários do veneno de Bothrops jararaca e estão correlacionadas com os efeitos locais e sistêmicos. O tratamento para o envenenamento indicado pelo Ministério da Saúde é a administração do antiveneno botrópico, que apesar de ser altamente eficaz, apresenta certas limitações, como a parcial neutralização das SVSPs. Dentre as estratégias que visam à melhoria do antiveneno e, assim, o tratamento, está a pesquisa por inibidores seletivos de SVSPs, que possam atuar de forma sinérgica ao antiveneno. Considerando a similaridade da tríade catalítica entre serinoproteinases humanas e as SVSPs, e visto que a inércia contra serinoproteinases humanas é um pré-requisito para a associação de qualquer molécula ao antiveneno, o presente projeto objetivou o “Estudo da ação de inibidores peptídicos de enzimas trombina-símiles na coagulação sanguínea humana”. Os inibidores peptídicos, seletivos quanto à inibição de SVSPs, foram desenhados com base em substrato hidrolisado por estas enzimas do veneno de Bothrops jararaca, e apresentaram diferentes eficácias de inibição. Análises in silico sugerem que os inibidores se ligaram em regiões distantes da tríade catalítica (His, Asp e Ser) da batroxobina, uma enzima trombina-símile usada como modelo em estudos de interação da enzima/inibidor peptídico.

Caracterização bioquímica e farmacológica de compostos analgésicos presentes na secreção das glândulas parotóides dos sapos Rhinella Icterica e Rhinella marina / Biochemical and pharmacological characterization of analgesic compounds from the toads Rhinella icterica and Rhinella marina parotoids glands secretion

Currently, natural products are considered an important source of molecules with therapeutic potential. Literature demonstrates that the secretion components of frogs belonging to the Bufonidae family induce antinociceptive effect. Thus, the objectives of this study are to identify and characterize antinociceptive molecules in the secretion of parotid glands of Rhinella icterica and Rhinella marina frogs and to determine their action movements. After an initial screening, we detected the presence of four compounds with analog activity. These compounds were purified by RP-HPLC and reproduced by mass spectrometry such as Dehydrobufotenin, Bufalin, Morinobufagin and Telecinobufagin. The antinociceptive effect of these compounds was applied by the electronic Frey test using the carrageenan induced hyperalgesia model. The results showed that Bufotenin, Telecinobufagin, Marinobufagin and Bufalin at doses of 50 μg, 100 μg and 200 μg after oral administration were able to reverse carrageenan-induced hyperalgesia 30 minutes after treatment, with a peak of professional activity within 1 hour. (p < 0.001). Additionally, no motor alterations were observed for any of the four compounds, which were applied through open field test, elevated cross maze or cytotoxic activity in cardiomyocytes. The mechanisms of action involved in the analytical effect of these compounds are still available. Our results demonstrated treatment with selected antagonist for receptor 5 -HT1a (300 nmol/paw), 5-HT2a (300 nmol/paw), delta opioid (90 μg/paw) and various kappa opioid (75 μg/paw) involved in the treatment. bufotenine alkaloid-induced antinociceptive effect (200 μg/100 μL). Animals composed of steroid compounds telecinobufagin (200 μg/100μL) and marinobufagin (200 μg/100μL) suffered reversible hyperalgesia when injected with selective antagonists AM251 (40 μg/paw) and AM630 (25 μg/paw) revealing or executing receptors. CB1 and CB2, respectively, without antinociceptive effect (p < 0.001). One bufalin (200 μg/100μL) was the only steroid compound that did not involve any tested mechanism, being opioids, cannabinoids and serotoninergics. Therefore, we performed an analysis in molecules databases, considering a bufalin structure in search of action targets. The results obtained from this analysis show that bufalin is activated by chloride channels in vitro. Based on this, the evaluations or involvement of calcium-activated chloride channels and type 2 channels without antinociceptive effect due to their presence in peripheral nociceptive fibers. The results revealed or the involvement of calcium-activated chloride channels (300 μM/paw) without buffalo effect. With these data, we can confirm the antinocieptive activity of the four compounds of the paranoid secretion of the toads R. icterica and R. marina - Bufotenine, Bufalin, Marinobufagin and Telecinobufagin.

Atualmente, os produtos naturais são considerados uma importante fonte de moléculas com potencial terapêutico. A Literatura demonstra que os componentes da secreção de sapos pertencentes à família Bufonidae induzem efeito antinociceptivo. Assim, os objetivos deste estudo são identificar e caracterizar moléculas antinociceptivas na secreção das glândulas parotoides de sapos Rhinella icterica e Rhinella marina e determinar seus mecanismos de ação. Após um screening inicial detectamos a presença de quatro compostos com atividade analgésica. Estes compostos foram purificados por RP-HPLC e identificados por espectrometria de massa como Dehidrobufotenina, Bufalina, Morinobufagina e Telecinobufagina. O efeito antinociceptivo destes compostos foram avaliados pelo teste de von Frey eletrônico, utilizando o modelo de hiperalgesia induzida por carragenina. Os resultados demonstraram que a Bufotenina, Telecinobufagina, Marinobufagina e Bufalina nas doses de 50 μg, 100 μg e 200 μg após administração por via oral, conseguiram reverter a hiperalgesia induzida pela carragenina 30 minutos após o tratamento, com um pico de atividade observado em 1 hora (p<0,001). Adicionalmente, não foram observadas alterações motoras para nenhum dos quatro compostos, que foram avaliados através do teste de campo aberto, labirinto em cruz elevado ou atividade citotóxica em cardiomiócitos. Avaliamos ainda os mecanismos de ação envolvidos no efeito analgésico destes compostos. Nossos resultados demonstraram que o tratamento com o antagonista seletivo para receptores 5- HT1a (300 nmol/pata), 5-HT2a (300 nmol/pata), delta opióide (90 μg/pata) e kappa opióide (75 μg/pata) estão envolvidos no efeito antinociceptivo induzido pelo alcaloide bufotenina (200 μg/100 μL). Já os animais tratados com os compostos esteroides telecinobufagina (200μg/100μL) e marinobufagina (200μg/100μL) tiveram a hiperalgesia revertida quando foram injetados com os antagonistas seletivos AM251 (40 μg/pata) e AM630 (25 μg/pata) revelando o envolvimento de receptores CB1 e CB2 respectivamente no efeito antinociceptivo (p<0,001). A bufalina (200μg/100μL) foi o único composto esteroide que não apresentou envolvimento de nenhum dos mecanismos testados, sendo eles opióides, canabinóides e serotoninérgicos. Portanto, realizamos análise em bancos de dados de moléculas considerando a estrutura da bufalina na busca por alvos de ação. Os resultados obtidos a partir dessa análise mostraram que a bufalina é ativadora de canais de cloreto in vitro. Com base nisso, avaliamos o envolvimento dos canais de cloreto ativados por cálcio e dos canais tipo 2 no efeito antinociceptivo, devido sua presença em fibras nociceptivas periféricas. Os resultados revelaram o envolvimento de canais de cloreto ativados por cálcio (300 μM/pata) no efeito da bufalina. Com esses dados, podemos confirmar a atividade antinocieptiva dos quatro compostos isolados da secreção das parotóides dos sapos R. icterica e R. marina - Bufotenina, Bufalina, Marinobufagina e Telecinobufagina.

Estabelecimento de metodologia de diferenciação de células tronco embrionárias de camundongo em células precursoras de oligodendrócitos: uma plataforma para triagem de novas drogas remielinizantes / Establishment of a mouse embryonic stem cell line differentiation methodology into oligodendrocyte precursor cells: a platform for remyelinating drug screening.

The myelin sheath plays a crucial role in nerve function. In the Central Nervous System, the myelin sheath is produced by oligodendrocytes (OL), which are differentiated from oligodendrocyte precursor cells (OPC). Several neurological disorders, including multiple sclerosis, display damage in the myelin sheath and failure in the remyelination. Therefore, researching for new compounds that act in the OPCs differentiation to OLs is a promising strategy to treat demyelinating diseases. In this study, we established a methodology that allows the generation of OPCs in large quantities from two lineages of mouse embryonic stem cells (mESCs; lineages HM-1 and USP-1). Initially, mESCs were induced to differentiate into neural precursors and, later, to OPCs by adding growth factors such as FGF, EGF and PDGF. The characteristic and predominant expression of Olig2, Sox-10, PDGFRα and NG2 observed by immunofluorescence, and the double labeling of PDGFRα and NG2 by flow cytometry, confirmed the OPCs generation from mESCs. Therefore, this study shows an efficient and reproducible methodology for generating OPCs capable of being used in screening assays or in OLs lineage biology studies.

A bainha de mielina desempenha um papel crucial nas funções nervosas. No sistema nervoso central, a bainha de mielina é produzida por oligodendrócitos (OLs), os quais são diferenciados a partir de células precursoras de oligodendrócitos (OPCs). Em várias desordens neurológicas, entre elas, a Esclerose Múltipla, a bainha de mielina é danificada e a remielinização deixa de acontecer. Desse modo, a busca por novos compostos que atuem na diferenciação de OPCs a OLs, é um caminho promissor na terapia de doenças desmielinizantes. Neste estudo, estabelecemos uma metodologia que permitiu gerar OPCs a partir de duas linhagens de células tronco embrionárias de camundongos (mESCs – linhagens USP-1 e HM-1). Inicialmente, as mESCs foram induzidas a diferenciar em precursores neurais e, posteriormente, a OPCs, pela adição de fatores de crescimento como FGFb, EGF e PDGF-AA. A expressão característica e predominante de Olig2, Sox10, PDGFRα e NG2 observada por imunofluorescência e a dupla marcação de PDGFRα e NG2 por citometria de fluxo (com rendimentos de 88,3-87,3%) confirmaram a obtenção de OPCs a partir de mESCs. Estas células podem ser expandidas por 5 passagens adicionais e ainda serem capazes de gerar bilhões de OPC puros. Portanto, o estudo apresenta uma metodologia eficiente e reprodutível que permite a geração de um modelo a ser empregado em triagem de novas drogas ou no estudo da biologia da linhagem de OLs.

Avaliação de atividades antimicrobianas e antiparasitárias dos extratos alcoólico e aquoso da própolis de Scaptotrigona affinis postica / Evaluation of antimicrobial and antiparasitic activities of alcoholic and aqueous extracts from Scaptotrigona affinis postica propolis

Propolis is a natural product made by bees and mainly composed by resins, which are chemical compounds derived from plants with pharmacological potential. Antimicrobial, antifungic and antiparasitic potential has already been demonstrated in the literature. Propolis from Scaptotrigona aff. postica bee (indigenous stingless bee) is traditionally used in the Brazilian State of Maranhão countryside for the treatment of wounds, but few studies were conducted to understand the biological mechanism behind this activity. Micro-organisms resistance to antibiotics is growing, arising interest in investigating new antimicrobial compounds discovery. The Chagas disease (tropical disease neglected) also demands more effective medicines, causing thousands of deaths annually, and reducing productive capacity. The objective of the present study was evaluate S. postica propolis potential. For this purpose, two extraction steps were held using water and methanol. The extracts were tested against bacterial strains, fungal strains, and Trypanossoma cruzi. The extracts did not present cytotoxicity for mammalian cells, in the concentration which showed microbial activity. One step purification was carried out, through extraction in solid phase, and the fractions eluted. Then antimicrobial activity was evaluated against bacterial strains. Inhibition in the growth of Staphylococcus aureus, Bacillus megaterium, Microccocus luteus and Escherichia coli strains was observed. These fractions were submitted to MS/MS analysis to identification from compounds. The substances identified were polyphonic, flavonoids, and alkaloids. This study is a pioneer in identifying biological activities from propolis S. postica fractions extracts and search for antiparasiticactivity.

A própolis é produto natural produzido pelas abelhas. Seus componentes majoritários são as resinas, compostos químicos derivados de plantas, o que lhes confere potencial farmacológico. Já foi demostrado que as moléculas encontradas nas própolis têm grande potencial antimicrobiano, antifúngico e antiparasitário. No Brasil, a própolis da abelha Scaptotrigna aff. postica (abelha indígena sem ferrão) é utilizada no interior do estado do Maranhão no tratamento de feridas pela população, mas poucos são os estudos realizados para melhor compreender suas atividades biológicas. Com o surgimento de microrganismos resistentes à ação dos antibióticos, há interesse em investigar novas substâncias que possam vir a ser novas descobertas de antimicrobianos. A doença de Chagas (doença tropical negligenciada), também necessita de medicamentos mais eficazes, pois acarretam milhares de mortes anualmente, além de redução da capacidade produtiva. Desta forma, o objetivo do trabalho foi avaliar o potencial biológico da própolis de S. aff. postica. Para tanto, foram realizadas duas etapas de extrações, com água e metanol. Os extratos foram testados contra cepas bacterianas, fúngicas e contra o protozoário Trypanosoma cruzi. Os extratos testados não apresentaram citotoxicidade para células de mamíferos, nas concentrações em que exibiram atividade antimicrobiana. Após uma etapa de purificação, observou-se que as frações eluídas inibiram o crescimento das bactérias Staphylococcus aureus, Bacillus megaterium, Microccocus luteus e de duas cepas de Escherichia coli. Essas frações foram submetidas a análise por MS/MS para identificação dos compostos presentes. As principais substâncias identificadas foram: compostos polifenólicos, flavonoides e majoritariamente alcalóides. O trabalho é pioneiro no estudo e identificação de frações de extratos da própolis S. postica, bem como, no teste antiparasitário.

Papel da enzima aldeído desidrogenase-2 e de aldeídos reativos na progressão da encefalomielite autoimune experimental / Gonçalves. Role of aldehyde dehydrogenase-2 enzyme and reactive aldehydes in the progression of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease characterized by chronic inflammation and demyelination of the central nervous system. The neuroinflammatory response is a combination of factors such as oxidative stress, activation of cells from the immune system and axonal demyelination. MS is an incurable and disabling disease that affects more than 2.5 million people worldwide and induces motor, cognitive and sensory changes, including persistent chronic pain. Considering the progressive characteristic of MS and the lack of an effective a deeper understanding of this pathology and the discovery of new therapies are necessary. The aldehyde dehydrogenase-2 (ALDH2) is a mitochondrial enzyme responsible for controlling the aldehydic load, such as the 4-hydroxynonenal (4-HNE). The 4-HNE is widely involved in nociception and neurodegeneration by forming adducts with receptors located in the nociceptive neurons or myelin proteins. We previously demonstrated that Alda-1, a small molecule that selectively activates ALDH2, induces analgesia in inflammatory and neuropathic pain models. However, the role role of ALDH2 in the neurodegeneration-induced nociception is unknown. The aim of this project was to evaluate the role of ALDH2 in the hypernociception and the disease progression in a model of experimental autoimune encefalomyielitis (EAE). In orderto achieve this goal, we used two strategies: a) gain of ALDH2 function, by using a pharmacological treatment with Alda-1 and b) loss of function, by using a carrier mutation mice that confers a loss of about 90% in its activity (ALDH2 *2). The long lasting treatment with Alda-1 reduces hypernociception and improves the motor clinical signs. This effect is accompanied by a reduction in the central neuroinflammatory response, since we detected less immunostaining for glial cells (microglia and astrocytes), as well as lower neuronal activation in the dorsal horn of the spinal cord. Although ALDH2*2 do not display worse clinical signs, these animals present higher spinal neuroinflammation and 4-HNE adducts with proteins of the spinal cord and peripheral blood when compared to wild animals. These phenomenons are blocked by Alda-1. Taken together, our results indicate that ALDH2 participates, at least in part, in hypernociception and motor clinical signs in EAE, by controlling neuroinflammation, particularly 4-HNE levels. Alda-1 confers neuroprotection and contributes for the improvement of EAE-induced motor impairment. Therefore, ALDH2 may be a novel therapeutic target for multiple sclerosis.

A esclerose múltipla (EM) é uma doença autoimune neurodegenerativacaracterizada por inflamação crônica e desmielinização do sistema nervoso central. A resposta neuroinflamatória é um conjunto de fatores como estresse oxidativo, ativação de células do sistema imune e desmielinização axonal. A EM é uma doença incurável, incapacitante, que acomete mais de 2,5 milhões de pessoas no mundo e induz alterações motoras, cognitivas e sensoriais, incluindo dor crônica persistente. Considerando a característica progressiva da EM e a falta de uma terapia eficaz, tanto frente à melhora do quadro doloroso como frente à evolução da doença, faz-se necessário um entendimento mais aprofundado dessa patologia assim como a busca de novas terapias. A aldeído desidrogenase-2 é uma enzima mitocondrial responsável pela eliminação de aldeídos reativos produzidos pelo seu metabolismo, como o 4- hidroxinonenal (4-HNE). O 4-HNE é um aldeído amplamente envolvido na nocicepção e neurodegeneração, por formar ligações de alta afinidade com receptores expressos em neurônios nociceptivos ou proteínas que compõem a mielina, alterando a sua função. Demonstramos anteriormente que a Alda-1, uma pequena molécula que ativa seletivamente ALDH2 induz analgesia em modelos de dor inflamatória e neuropática. Contudo, o papel desta enzima no desenvolvimento da dor de origem neurodegenerativa ainda é desconhecido. Assim, o objetivo desse projeto foi avaliar o papel da ALDH2 na hipernocicepção e progressão da encefalomielite autoimune experimental (EAE), um modelo animal que mimetiza a EM. Para tanto, utilizamos duas estratégias: a) ganho de função da ALDH2, por meio do tratamento farmacológicco com a Alda-1 e b) perda de função, utilizando camundongos transgênicosportadores de uma mutação que confere perda de cerca de 90% na sua atividade (ALDH2*2). A progressão doença foi avaliada por meio do desenvolvimento de hipernocicepção, ou seja, diminição do limiar nociceptivo dos animais e o comprometimento motor por meio de escores. Os resultados mostraram que o tratamento prolongado com a Alda-1 reduziu a hipernocicepção e melhorou os sinais clínicos motores induzidos pela doença. Este efeito foi acompanhado de redução na resposta neuroinflamatória central, uma vez que detectamos menor imunomarcação para células da glia (micróglia e astrócitos), bem como menor ativação neuronal no corno posterior da medula espinal, importante região envolvida na nocicepção. Apesar de não apresentarem piora no quadro clínico com relação aos selvagens, os animais ALDH2*2 apresentaram neuroinflamação espinal acentuada e maior formação de mais adutos de 4-HNE com proteínas da medula espinal e do sangue periférico. Esses fenômenos foram bloqueados pela Alda-1. Em conjunto, nossos resultados indicam que a ALDH2 participa, pelo menos em parte, na hipernocicepção e nos sinais clínicos motores na EAE, por controlar a neuroinflamação, particularmente pelo controle dos níveis de 4-HNE, conferindo neuroproteção e contribuindo para a melhora do quadro da EAE. Portanto, a ALDH2 pode ser um novo alvo terapêutico para a esclerose múltipla.

Papel da enzima aldeído desidrogenase-2 e de aldeídos reativos na dor neuropática / The role of Aldehyde Dehydrogenase-2 enzyme and reactive Aldehydes in neuropathie pain

Neuropathie pain control is a chall.enge and unmet clinical need. Aldehyde dehydrogenase-2 (ALOH2) is a mitochondrial enzyme responsible for the metabolism of reactive aldehydes, such as acetaldehydes, 4-hidroxinonenal (4-HNE) and malondialdehyde (MOA). The endogenous generation of reactive aldehydes occurs upon oxidative stress and lipid peroxidation. Our group demonstrated that reactive aldehydes are directly involved with the genesis of acute pain, once that Alda-1, a small molecule that selectively activates the ALOH2 induces analgesia by reducing the levels of these reactive aldehydes in the inflamed tissue. However, the role of this enzyme in neuropathie pain is still unknow. Therefore, the aim of this project was to investigate if the sustained activation of ALOH2 is able to control the development of neuropathie pain in mice. Thus, we used two strategies: (a) ALOH2 loss of function, by using a transgenic mice that carriers an inactivating point mutation in ALOH2 (ALOH2*2) or pharmacological inhibition of ALOH2 (Aldi) and (b) gain of function, by giving Alda-1 to mice. Wild type mice C57/BL were used as control group. Neuropathy was induced by the chronic constriction of the sciatic nerve (CCI). The nociceptive threshold was determined before and during 28 days after surgery, by the von-Frey filaments method. After the nociceptive evaluation, samples of tissues involved in the nociceptive pathway, such as spinal cord, dorsal root ganglia (ORG), sciatic nerve and blood were obtained for the detection of 4-HNE adducts by Western Blot or competitive ELISA assay. MOA was quantified by detection of reactive substances to thiobarbituric acid (TSARS). Our results showed that the CCI reduces the nociceptive threshold (hyperalgesia), both on wild type mice and ALOH2*2, for the entire period of observation (28 days). Alda-1 blocks the hyperalgesia-induced by CCI in the wild type mice. This effect persists for 14 days and is no longer detected on days 21th and 28th. Regarding the ALOH2*2 animals, Alda-1 induces analgesia for 14 days. This effect is partial on 21th and 28th days. The biochemical assays demonstrated that the CCI did not alter the levels of 4-HNE adducts with proteins from the spinal cord, sciatic nerve or ORG. Interestingly, elevation on the levels of 14 these aldehyde were evaluated in serum, 7 days after CCI. The systemic increasing in 4-HNE is accompanied by the elevation of MD.A in the sciatic nerve. These alterations are controlled by the sustained activation of ALDH2 by Alda-1. Alterations of this aldehydes were not detected in ALDH2*2 mice. On the other hand, the inhibition of ALDH2 by Aldi, contributed for the MOA sciatic nerve accumulation in the of operated mice, 28 days afterwards. Together, our data indicates that Alda-1 presents analgesic effect in neuropathie pain models. This effect seems to be due the inhibition of circulating aldehydes and accumulation at the injury site. Thereby, ALDH2 could be a new therapeutic for the neuropathie pain control.

O controle da dor neuropática é um desafio e uma necessidade clínica não atendida. A aldeído-desidrogenase 2 (ALDH2) é uma enzima mitocondrial responsável pelo metabolismo de aldeídos reativos, incluindo o acetaldeído, 4-hidroxinonenal (4-HNE) e malondialdeído (MOA). A geração endógena de aldeídos reativos ocorre sob estresse oxidative e peroxidação lipídica. Nosso grupo demonstrou que aldeídos reativos estão diretamente relacionados com a gênese da dor aguda inflamatória, uma vez que a Alda-1, uma pequena molécula que ativa seletivamente a ALDH2 induz analgesia por reduzir os níveis desses aldeídos no tecido inflamado. No entanto, o papel dessa enzima na dor neuropática é desconhecido. Portanto, o objetivo deste projeto foi investigar se a ativação sustentada da ALDH2 seria capaz de controlar a dor neuropática em camundongos. Para tanto, utilizamos duas estratégias: (a) perda de função da ALDH2, por meio da utilização de camundongos transgênicos portadores de uma mutação que inativa a ALDH2 (ALDH2*2) ou inibição farmacológica da ALDH2 (Aldi) e (b) ganho de função da ALDH2, por meio da administração de Alda-1. Animais selvagens C57/BL foram utilizados como controle. A neuropatia foi induzida pela constrição crônica do nervo isquiático (CCI). O limiar nociceptive foi determinado, antes e por 28 dias após a cirurgia, utilizando o método de filamentos de von Frey. Após a avaliação nociceptiva, amostras de tecidos envolvidos nas vias nociceptivas como a medula espinal, gânglio da raiz dorsal (DRG), nervo isquiático e sangue foram obtidas para detecção de adutos de 4-HNE por Western blot ou ensaio de ELISA competitivo. O MOA foi quantificado por detecção de substâncias reativas ao ácido tiobarbitúrico (TBARS). Nossos resultados mostraram que CCI diminuiu o limiar nociceptive (hiperalgesia), tanto nos camundongos selvagens quanto ALDH2*2, por todo o período de observação, ou seja, 28 dias). A Alda-1 bloqueia a hiperalgesia induzida pela CCI nos animais selvagens por 14 dias. Esse efeito não é mais detectado no 21º e 28° dias. Com relação aos animais ALDH2*2, a Alda-1 induz analgesia por 14 dias. Este efeito analgésico é parcial no 21° e 28° dias. Os ensaios bioquímicos demonstraram que a 12 CCI não alterou os níveis de adutos de 4-HNE com proteínas da medula espinhal, nervo isquiático ou DRG. lnteressantemente, detectamos aumento nos níveis deste aldeído no soro de animais, 7 dias após a CCI. A elevação sistêmica de 4-HNE é acompanhada de aumento de MOA no nervo isquiático. A elevação desses aldeídos é controlada pela ativação sustentada da ALDH2 pela Alda-1. Não detectamos alteração significante nos níveis desses aldeídos em animais ALDH2*2. Por outro lado, a inibição da ALDH2 pela Aldi favoreceu o acúmulo de MOA no nervo isquiático de animais operados, 28 dias após a CCI. Em conjunto, nossos resultados indicam que a Alda-1 apresenta efeito analgésico em modelo de neuropatia. Esse efeito parece ser decorrente da inibição de aldeídos circulantes e do seu acúmulo no local da lesão. Assim, a ALDH2 pode ser um novo alvo terapêutico para o controle da dor neuropática.

Multielemental analyses of Lonomia obliqua (Lepidoptera, Saturniidae) caterpillar using EDXRF and INAA techniques

In this study, multielemental analysis of Lonomia obliqua (Lepidoptera, Saturniidae) caterpillar was performed using energy dispersive X-ray fluorescence and instrumental neutron activation analysis techniques. This caterpillar is poisonous and has the ability to cause fatal hemorrhagic effects in humans after contact. The need of this study is related to morphological changes (mainly size and color) observed in some caterpillars used for preparation of antilonomic serum (antivenom). The samples were classified as healthy (caterpillars of control) and unhealthy (caterpillars visibly modified). The XRF measurements were performed in an energy dispersive X-ray fluorescence spectrometer and the instrumental neutron activation analysis using the IEA-R1 nuclear reactor at IPEN. The results show significant differences for several elements (mainly, P, Cl, Ca, Ti, Mn, Fe, Ni, and Zn) in unhealthy caterpillars that can affect the development of this species as well as the quality and yield of the antivenom. Furthermore, its elemental characterization contributes for the understanding the potential pharmacological (procoagulant and antithrombotic) in the prevention of life-threatening blood clots

Proteomic informed by transcriptomic for salivary glands components of the camel tick Hyalomma dromedarii

Background The hard tick Hyalomma dromedarii is one of the most injurious ectoparasites affecting camels and apparently best adapted to deserts. As long-term blood feeders, ticks are threatened by host defense system compounds that can cause them to be rejected and, ultimately, to die. However, their saliva contains a cocktail of bioactive molecules that enables them to succeed in taking their blood meal. A recent sialotranscriptomic study uncovered the complexity of the salivary composition of the tick H. dromedarii and provided a database for a proteomic analysis. We carried out a proteomic-informed by transcriptomic (PIT) to identify proteins in salivary glands of both genders of this tick species. Results We reported the array of 1111 proteins identified in the salivary glands of H. dromedarii ticks. Only 24% of the proteins were shared by both genders, and concur with the previously described sialotranscriptome complexity. The comparative analysis of the salivary glands of both genders did not reveal any great differences in the number or class of proteins expressed their enzymatic composition or functional classification. Indeed, few proteins in the entire proteome matched those predicted from the transcriptome while others corresponded to other proteins of other tick species. Conclusion This investigation represents the first proteomic study of H. dromedarii salivary glands. Our results shed light on the differences between the composition of H. dromedarii male and female salivary glands, thus enabling us to better understand the gender-specific strategy to feed successfully.

Crotalus durissus ruruima snake venom and a Phospholipase A2 isolated from this venom elicit macrophages to form lipid droplets and synthesize inflammatory lipid mediators

Viper snake Crotalus durissus ruruima (Cdr) is a subspecies found in northern area of Brazil. Among the snakes of Crotalus genus subspecies, the venom of Cdr presents highest level of crotoxin, which is the major component of Crotalus snake venoms, formed by two subunits (crotapotin and a phospholipase A2 named CBr) and presents potent neurotoxic activity. Curiously, the venom of C. d. ruruima (CdrV) is better neutralized by antibothropic than by anticrotalic serum, strongly suggesting that this venom has similarities with venom of Bothrops genus snakes with regard to the ability to induce inflammation. Macrophages are cells with a central role in inflammatory and immunological responses. Upon inflammatory stimuli, these cells exhibit increased numbers of lipid droplets, which are key organelles in the synthesis and release of inflammatory mediators. However, the effects of CdrV and CBr in macrophage functions are unknown. We herein investigated the ability of CdrV and CBr to activate macrophages with focus on the formation of lipid droplets (LDs), synthesis of lipid mediators, and mechanisms involved in these effects. The involvement of LDs in PGE2 biosynthesis was also assessed. Stimulation of murine macrophages with CdrV and CBr induced an increased number of LDs and release of prostanoids (PGE2, PGD2, and TXB2). Neither CdrV nor CBr induced the expression of COX-1 and COX-2 by macrophages. LDs induced by both CdrV and CBr are associated to PLIN2 recruitment and expression and were shown to be dependent on COX-1, but not COX-2 activity. Moreover, PGE2 colocalized to CdrV- and CBr-induced LDs, revealing the role of these organelles as sites for the synthesis of prostanoids. These results evidence, for the first time, the ability of a whole snake venom to induce formation of LDs and the potential role of these organelles for the production of inflammatory mediators during envenomation by Crotalus snakes.

A secreted phospholipase A2 induces formation of smooth muscle foam cells which transdifferentiate to macrophage-like state

Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-gama and -ß/d. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.

Inflammation induced by platelet-activating viperid snake venoms: perspectives on thromboinflammation

Envenomation by viperid snakes is characterized by systemic thrombotic syndrome and prominent local inflammation. To date, the mechanisms underlying inflammation and blood coagulation induced by Viperidae venoms have been viewed as distinct processes. However, studies on the mechanisms involved in these processes have revealed several factors and signaling molecules that simultaneously act in both the innate immune and hemostatic systems, suggesting an overlap between both systems during viper envenomation. Moreover, distinct classes of venom toxins involved in these effects have also been identified. However, the interplay between inflammation and hemostatic alterations, referred as to thromboinflammation, has never been addressed in the investigation of viper envenomation. Considering that platelets are important targets of viper snake venoms and are critical for the process of thromboinflammation, in this review, we summarize the inflammatory effects and mechanisms induced by viper snake venoms, particularly from the Bothrops genus, which strongly activate platelet functions and highlight selected venom components (metalloproteases and C-type lectins) that both stimulate platelet functions and exhibit pro-inflammatory activities, thus providing insights into the possible role(s) of thromboinflammation in viper envenomation.

Experimental antivenom against serine proteases from the Bothrops jararaca venom obtained in mice, and its comparison with the antibothropic serum from the Butantan Institute

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly made up of proteolytic enzymes. Although the antibothropic serum produced by the Butantan Institute is remarkable in saving lives, studies show that some symptoms observed in cases of envenoming are not efficiently neutralized. Moreover, our group has shown that the commercial antivenom does not fully neutralize in vitro some serine proteases present in the Bothrops jararaca venom. Therefore, this study focuses on a new method in the production of specific immunoglobulins capable of neutralizing the activities of these enzymes in vitro. For this, a pool of serine proteases that was not inhibited by the commercial antivenom, made up of four enzymes (KN-BJ2, BjSP, HS112 and BPA) from the B. jararaca venom was obtained through two chromatographic steps (DEAE-HPLC and C8-RP-HPLC). The identities of these proteases were confirmed by SDS-PAGE, followed by tryptic digestion and mass spectrometry analysis. This pool was inoculated into BALB/c and C57BL/6 mice, using SBA-15 as adjuvant, and the produced IgGs were purified by affinity chromatography. The sera were characterized by ELISA, avidity and proteolytic neutralization assays. Both animal models responded to the immunization, producing higher IgGs titers when compared to the commercial antivenom. The experimental serum from BALB/c mice presented a better hydrolysis inhibition of the selective fluorescent substrate for serine proteases (~80%) when compared to C57BL/6 (~25%) and the commercial antivenom (<1%) at the dose of 500:1 (weight of antivenom:weight of venom). These results show that a different immunization method using isolated serine proteases improves the toxins neutralizing efficacy and could lead to a better end product to be used as a supplemental medicine to the currently used immunotherapy.

Purification and biochemical characterization of TsMS 3 and TsMS 4: neuropeptide-degrading metallopeptidases in the tityus serrulatus venom

Although omics studies have indicated presence of proteases on the Tityus serrulatus venom (TsV), little is known about the function of these molecules. The TsV contains metalloproteases that cleave a series of human neuropeptides, including the dynorphin A (1-13) and the members of neuropeptide Y family. Aiming to isolate the proteases responsible for this activity, the metalloserrulase 3 and 4 (TsMS 3 and TsMS 4) were purified after two chromatographic steps and identified by mass spectrometry analysis. The biochemical parameters (pH, temperature and cation effects) were determined for both proteases, and the catalytic parameters (Km, kcat, cleavage sites) of TsMS 4 over fluorescent substrate were obtained. The metalloserrulases have a high preference for cleaving neuropeptides but presented different primary specificities. For example, the Leu-enkephalin released from dynorphin A (1-13) hydrolysis was exclusively performed by TsMS 3. Neutralization assays using Butantan Institute antivenoms show that both metalloserrulases were well blocked. Although TsMS 3 and TsMS 4 were previously described through cDNA library studies using the venom gland, this is the first time that both these toxins were purified. Thus, this study represents a step further in understanding the mechanism of scorpion venom metalloproteases, which may act as possible neuropeptidases in the envenomation process.

Preclinical evaluation of Amblyomin-X, a Kunitz-type protease inhibitor with antitumor activity

Amblyomin-X, a Kunitz-type protease inhibitor, is a recombinant protein that selectively induces apoptosis in tumor cells and promotes tumor reduction in vivo in melanoma animal models. Furthermore, Amblyomin-X was able to drastically reduce lung metastasis in a mice orthotopic kidney tumor model. Due to its antitumor activity, Amblyomin-X potential to become a new drug is currently under investigation, therefore the aim of the present study was to perform preclinical assays to evaluate Amblyomin-X toxicity in healthy mice. Exploratory toxicity assays have shown that treatment with 512?mg/kg of Amblyomin-X lead to animal mortality, therefore two groups of treatment were evaluated in the present work: in the acute toxicity assay, animals were injected once with doses ranging from 4 to 256?mg/kg of Amblyomin-X, while in the subacute toxicity assay, animals were injected with 0.25, 0.57 and 1?mg/kg of Amblyomin-X daily, during 28 days. Following this treatment regimens, Amblyomin-X did not cause any mortality; moreover, toxicity signs were discrete, reversible and observed only at the higher doses, thus establishing a safety profile for administration in mice, which can be further used to determine the dose translation of this novel drug candidate for treatment in other species.

Identificação e caracterização de novos criptídeos obtidos pela hidrólise da hemoglobina e suas atividades sobre a enzima conversora de angiotensina (EC 3.4.15.1) e a renina (EC 3.4.23.15) / Identification and characterization of new cryptides obtained through the hydrolysys of hemoglobin and its activities on the angiotensin converting enzyme (EC 3.4.15.1) and renin (EC 3.4.23.15)

Cryptids are peptides with bioactive function that are hidden in other proteins, having similar or even different function from these mother proteins. The objective of this work is to analyze the generation of these cryptides from the hydrolysis of hemoglobin, which already has active peptides described as hemocidin, hemorphine and hemopressin, and to analyze their interaction with the enzymes ACE (Angiotensin Converting Enzyme) and Renin that are part of the system Renin- Angiotensin which is fundamental for cardio circulatory regulation. Hydrolysis of the hemoglobin was performed by trypsin, and the material was analyzed in SDS-Page and fractionated in High Pressure Liquid Chromatography. The mass of the peptides generated in LC-MS and in silico was determined. Preliminary tests were performed with ACE and the fractions, with results above 60% inhibition. The tests were redone in a fluorimetric assay again showing results above 60% inhibition in 4 fractions. The fluorimetric test was also used with the enzyme renin, with results around 20% inhibition. These same fractions were tested for MTT in fibroblast cell culture and no toxicity or proliferation was observed.

Os criptídeos são peptídeos com função bioativa que se encontram ocultos em outras proteínas, tendo função semelhante ou até distinta dessas proteínas-mãe. O objetivo deste trabalho é analisar a geração destes criptídeos a partir da hidrólise de hemoglobina, que já possui peptídeos ativos descritos como a hemocidina, hemorfina e hemopressina, e analisar a interação deles com as enzimas ECA (Enzima Conversora de Angiotensina) e Renina que fazem parte do sistema Renina- Angiotensina que é fundamental na regulação cardiocirculatória. Para isso foi realizada uma hidrólise da hemoglobina por tripsina, sendo esse material analisado em SDS-PAGE e fracionado em Cromatografia Líquida de Alta Pressão. Foi determinada a massa dos peptídeos gerados em LC-MS e in silico. Foram realizados testes preliminares com a ECA e as frações, tendo resultados acima de 60% de inibição. Os testes foram refeitos em ensaio fluorimétrico apresentando novamente resultados acima de 60% de inibição diante 4 frações. O teste fluorimétrico foi usado também com a enzima renina, apresentando resultados em torno de 20% de inibição. Essas mesmas frações foram testadas por MTT em cultura celular de fibroblastos e não foi notada toxicidade ou proliferação.

Caracterizando o perfil enzimático de extratos de tentáculos da água-viva do Atlântico Sul Olindias sambaquiensis (Hydrozoa) / Characterising the enzymatic profile of tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Hydrozoa)

Jellyfishes are amongst the most familiar of venomous marine animals. Jellyfish encounters with humans are common, although subsequent envenomation has varying toxicity ranging from usually mild symptoms to sometimes lethal consequences. Jellyfish venoms are of biotechnological importance, with toxins displaying antimicrobial, analgesic and anti-tumor activities. Furthermore, proteolytic enzymes have also been described. Recently, the proteomic profile of putative toxins isolated from nematocysts of the hydromedusae Olindias sambaquiensis was reported, which included protease and phospholipase enzymes. Here we present the results of a study to experimentally confirm enzymatic activity in extract of O. sambaquiensis tentacles. Specimens of O. sambaquiensis were collected at the coast of São Sebastião, SP, Brazil. A procedure to generate tentacle extract was standardised, yielding high quantities of proteins of different molecular masses. The extract was assayed for the presence of metalloproteases, serine proteases and phospholipase A2 activities using substrates that cover a wide range of each class of these enzymes and also specific substrates for metalloproteases ADAMs and MMPs. The extract showed activity towards all substrates confirming previous proteomic evidence for the presence of these enzymes. In addition, proteolytic activity was also detected by zymography assays on casein and gelatin. However, when comparing these activities to snake venoms, which are rich in proteases and phospholipases, the metalloprotease activity detected was lower than the high levels observed in Bothrops jararaca venom, but levels of serine protease and phospholipase A2 enzyme activity was comparable to the one observed in venoms of Bothrops snakes, known for its strong anti-coagulant and myotoxic activities due to serine proteases and phospholipases A2 molecules, respectively. These data validate the functional properties of proteins characterised previously by proteomics and provide a better understanding of the toxin arsenal of this underexplored venomous animal, indicating the role of active serine proteases and phospholipases A2 in the action of the venom.

As águas-vivas ou medusas estão entre os animais peçonhentos marinhos mais populares. Acidentes envolvendo medusas são comuns e sua toxicidade para humanos pode variar de sintomas leves até envenenamentos fatais em alguns casos. Os venenos de medusas são de importância biotecnológica, apresentando toxinas com atividade antimicrobiana, analgésica e antitumoral. Além disso, enzimas proteolíticas também já foram descritas. Recentemente o perfil proteômico de toxinas putativas dos nematocistos isolados da hidromedusa Olindias sambaquiensis foi caracterizado, identificando a presença de proteases e fosfolipases. Nesse trabalho nós apresentamos os resultados de um estudo que confirma a presença de atividade enzimática no extrato de tentáculos de O. sambaquiensis. Os espécimes foram coletados na costa de São Sebastião, SP, Brasil. O procedimento para obtenção do extrato de tentáculos foi padronizado, rendendo grande quantidade de proteínas de diferentes massas moleculares. O extrato foi avaliado quanto a presença de atividade de metaloproteases, serino proteases e fosfolipases A2, utilizando substratos genéricos para essas enzimas e também substratos específicos para as metaloproteases do tipo ADAMs e MMPs. O extrato apresentou atividade sobre todos os substratos, confirmando evidências proteômicas anteriores. Além disso, a atividade proteolítica também foi detectada por ensaios de zimografia em caseína e gelatina. Quando comparamos essas atividades com venenos de serpentes, que são abundantes em proteases e fosfolipases, a atividade de metaloproteases obtida foi bem menor que os altos índices demonstrados pelo veneno de Bothrops jararaca, entretanto, as atividades observadas para serino proteases e fosfolipases A2 foram comparáveis à atividade observada em venenos de serpentes Bothrops, conhecidas pela sua forte atividade anticoagulante e miotóxica devido a presença de serino proteases e fosfolipases A2, respectivamente. Esses resultados validam as propriedades funcionais das proteínas caracterizadas previamente pelo proteoma e fornecem um melhor entendimento do arsenal tóxico desse animal peçonhento pouco explorado, indicando o papel das serino proteases e fosfolipases A2 na ação do veneno.