Browsing by Author "Campos, Alexandre"
Now showing 1 - 8 of 8
Results Per Page
Sort Options
- Analysis of Pelagia noctiluca proteome Reveals a Red Fluorescent Protein, a Zinc Metalloproteinase and a PeroxiredoxinPublication . Frazão, Bárbara; Campos, Alexandre; Osório, Hugo; Thomas, Benjamin; Leandro, Sérgio; Teixeira, Alexandre; Vasconcelos, Vitor; Antunes, AgostinhoPelagia noctiluca is the most venomous jellyfish in the Mediterranean Sea where it forms dense blooms. Although there is several published research on this species, until now none of the works has been focused on a complete protein profile of the all body constituents of this organism. Here, we have performed a detailed proteomics characterization of the major protein components expressed by P. noctiluca. With that aim, we have considered the study of jellyfish proteins involved in defense, body constituents and metabolism, and furthered explore the significance and potential application of such bioactive molecules. P. noctiluca body proteins were separated by1D SDS-PAGE and 2DE followed by characterization by nanoLC-MS/MS and MALDI-TOF/TOF techniques. Altogether, both methods revealed 68 different proteins, including a Zinc Metalloproteinase, a Red Fluorescent Protein (RFP) and a Peroxiredoxin. These three proteins were identified for the first time in P. noctiluca. Zinc Metalloproteinase was previously reported in the venom of other jellyfish species. Besides the proteins described above, the other 65 proteins found in P. noctiluca body content were identified and associated with its clinical significance. Among all the proteins identified in this work we highlight: Zinc metalloproteinase, which has a ShK toxin domain and therefore should be implicated in the sting toxicity of P. noctiluca.; the RFP which are a very important family of proteins due to its possible application as molecular markers; and last but not least the discovery of a Peroxiredoxin in this organism makes it a new natural resource of antioxidant and anti-UV radiation agents.
- Avaliação do efeito da microcistina-LR no crescimento, sistema antioxidante e indução de apoptose em Saccharomyces cerevisiaePublication . Valério, Elisabete; Vilares, Arminda; Campos, Alexandre; Pereira, Paulo; Vasconcelos, VitorNeste trabalho pretendeu-se usar a levedura Saccharomyces cerevisiae como modelo para compreender melhor os mecanismos de toxicidade induzidos pela microcistina-LR. Este é o organismo eucariótico mais simples e estudado que tem sido amplamente utilizado como modelo no estudo de mecanismos de toxicidade, devido à facilidade de acesso à informação sobre o seu genoma (totalmente sequenciado e anotado), proteoma e processos bioquímicos correspondentes.
- Avaliação do efeito da microcistina-LR no crescimento, sistema antioxidante e indução de apoptose em Saccharomyces cerevisiaePublication . Valério, Elisabete; Vilares, Arminda; Campos, Alexandre; Pereira, Paulo; Vasconcelos, VitorAs hepatotoxinas microcistinas são mundialmente frequentemente encontradas em corpos de água doce. Estas são conhecidas por causar hepatotoxicidade aguda em humanos / animais, promoção de tumores em animais e ao seu potencial carcinogénico. Em células de mamíferos, o mecanismo de toxicidade das microcistinas é atribuída a um processo que envolve várias vias, um deles relacionado com a inibição das fosfatases proteicas PP1 / PP2A e a produção de espécies reativas de oxigénio (ROS). No entanto, uma vez que ainda não estão completamente caracterizados os mecanismos moleculares de ação das microcistinas e os efeitos correspondentes, não é possível ainda fazer a identificação completa destes compostos tóxicos. Neste estudo foram avaliados os efeitos de várias concentrações de microcistina-LR no crescimento, níveis de ROS, resposta do sistema antioxidante e indução de apoptose da levedura Saccharomyces cerevisiae. Verificou-se que o crescimento microbiano não foi inibido na presença das várias concentrações de toxina. Contudo, após coloração das células com fluorocromos, verificou-se que a exposição das células à toxina induziu um aumento dos níveis intracelulares dos ROS. Este aumento provocou uma ativação do sistema antioxidante, especialmente na resposta da catalase. Além disso, observou-se uma inibição da SOD1, o que em conjunto com o tipo de ROS possivelmente presente, sugere que a espécie reativa de oxigénio maioritariamente induzida é peróxido de hidrogénio (H2O2). Observaram-se ainda sinais de apoptose após coloração das células com DAPI e após avaliação por citometria de fluxo, usando um kit de Anexina V-FITC. Os resultados obtidos neste estudo demonstram que a levedura Saccharomyces cerevisiae VL3 apresenta alguns dos principais efeitos tóxicos induzidos pela microcistina-LR em eucariotas superiores. Esta levedura, comprovou assim ser um simples e bom modelo eucariótico para estudar em mais detalhe os mecanismos moleculares de toxicidade induzidos pela microcistina-LR.
- Effects of microcystin-LR on Saccharomyces cerevisiae growth, oxidative stress and apoptosisPublication . Valério, Elisabete; Vilares, Arminda; Campos, Alexandre; Pereira, Paulo; Vasconcelos, VitorMicrocystins (MC) are cyanotoxins occurring globally, known for causing acute hepatotoxicity in humans/animals, tumor promotion in animals and potential carcinogenicity. The mechanism of MC toxicity is considered a multi-pathway process involving the inhibition of protein phosphatases PP1/PP2A and the production of reactive oxygen species (ROS). However, their mechanism of action is not fully characterized, thus hampering the complete hazard identification. In this study, we evaluated the effect of several microcystin-LR concentrations on the growth, ROS levels, antioxidant system response and apoptosis induction on Saccharomyces cerevisiae. Our results showed that the growth of S. cerevisiae was not inhibited when compared to control cells. However, the staining of cells with DHR123 and DHE revealed an intracellular increase of the ROS levels. This ROS increase resulted in an augment of catalase activity and inhibition of SOD. All these facts suggest that hydrogen peroxide was the main ROS induced by MCLR. Signs of apoptosis were also detected in the cells exposed to toxin. Our results show that S. cerevisiae VL3 displays MCLR toxicity effects known to occur in higher eukaryotes and confirmed that it can be a simple and good model to help further in the elucidation of MCLR molecular mechanisms of toxicity.
- New Insights in Saccharomyces cerevisiae Response to the Cyanotoxin Microcystin-LR, Revealed by Proteomics and Gene ExpressionPublication . Valério, Elisabete; Barreiros, Sara; Rodrigues, Sara; Turkina, Maria V.; Vasconcelos, Vitor M.; Campos, AlexandreMicrocystins (MCs) are hepatotoxins produced by some cyanobacteria. They are cyclic peptides that inhibit the serine/threonine protein phosphatases (PPs) PP1 and PP2A, especially PP2A. The inhibition of PP2A triggers a series of molecular events, which are responsible for most MC cytotoxic and genotoxic effects on animal cells. It is also known that MCs induce oxidative stress in cells due to the production of reactive oxygen species (ROS). However, a complete characterization of the toxic effects of MCs is still not accomplished. This study aimed to clarify additional molecular mechanisms involved in MC-LR toxicity, using Saccharomyces cerevisiae as eukaryotic model organism. First, a shotgun proteomic analysis of S. cerevisiae VL3 cells response to 1 nM, 10 nM, 100 nM, and 1 µM MC-LR was undertaken and compared to the control (cells not exposed to MC-LR). This analysis revealed a high number of proteins differentially expressed related with gene translation and DNA replication stress; oxidative stress; cell cycle regulation and carbohydrate metabolism. Inference of genotoxic effects of S. cerevisiae VL3 cells exposed to different concentrations of MC-LR were evaluated by analyzing the expression of genes Apn1, Apn2, Rad27, Ntg1, and Ntg2 (from the Base Excision Repair (BER) DNA repair system) using the Real-Time RT-qPCR technique. These genes displayed alterations after exposure to MC-LR, particularly the Apn1/Apn2/Rad27, pointing out effects of MC-LR in the Base Excision Repair system (BER). Overall, this study supports the role of oxidative stress and DNA replication stress as important molecular mechanisms of MC-LR toxicity. Moreover, this study showed that even at low-concentration, MC-LR can induce significant changes in the yeast proteome and in gene expression.
- New insights on the mode of action of microcystins in animal cells - a reviewPublication . Valério, Elisabete; Vasconcelos, Vitor; Campos, AlexandreMicrocystins (MCs) are the most commonly occurring hepatotoxins produced by cyanobacteria. The inhibition of PP2A is widely assumed as the principal mechanism of toxicity of MCs, however recently it has been found that MC modulates PP2A activity not only by direct inhibition of its activity, but also by regulating its expression. Nevertheless the mechanisms of toxicity of MCs seem to be more complex to interpret than expected. The induction of some cellular-molecular mechanisms appears to be biphasic in time and concentration of MC and in most cases related with the intracellular ROS generation. These intracellular ROS levels cause oxidative stress which leads to changes in several markers of MC-LR-induced oxidative stress ultimately resulting in apoptosis or cell damage and also genotoxicity. MCs can also induce severe changes in the cytoskeleton elements: microfilaments, intermediate filaments and microtubules, which results in changes in the cytoskeleton architecture and cell viability. There are also indications that there are second messengers involved in MC-LR mediated cytotoxicity and apoptosis. Different congeners of these toxins induce different degrees of responses in the cell, assumed to be related with the capacity of toxin internalization, affinity towards PP1 and PP2A, and the ability to cause oxidative stress. MCs have also been implicated in neurotoxicity and in damages in reproductive organs. The regulation of transcription factors and proto-oncogenes by MC is the mode of action of MCs tumor promotion. This review summarizes mainly the findings from the last five years about the molecular mechanisms behind MC toxicity in animal cells.
- Proteomic and Real-Time PCR analyses of Saccharomyces cerevisiae VL3 exposed to microcystin-LR reveals a set of protein alterations transversal to several eukaryotic modelsPublication . Valério, Elisabete; Campos, Alexandre; Osório, Hugo; Vasconcelos, VitorSome of the most common toxins present in freshwater, in particular microcystins (MCs), are produced by cyanobacteria. These toxins have a negative impact on human health, being associated with episodes of acute hepatotoxicity and being considered potentially carcinogenic to humans. To date the exact mechanisms of MC-induced toxicity and tumor promotion were not completely elucidated. To get new insights underlying microcystin-LR (MCLR) molecular mechanisms of toxicity we have performed the proteomic profiling using two-dimensional electrophoresis and MALDI-TOF/TOF of Saccharomyces cerevisiae cells exposed for 4 h-1 nM and 1 μM of MCLR, and compared them to the control (cells not exposed to MCLR). We identified 14 differentially expressed proteins. The identified proteins are involved in metabolism, genotoxicity, cytotoxicity and stress response. Furthermore, we evaluated the relative expression of yeast's PP1 and PP2A genes and also of genes from the Base Excision Repair (BER) DNA-repair system, and observed that three out of the five genes analyzed displayed dose-dependent responses. Overall, the different proteins and genes affected are related to oxidative stress and apoptosis, thus reinforcing that it is probably the main mechanism of MCLR toxicity transversal to several organisms, especially at lower doses. Notwithstanding these MCLR responsive proteins could be object of further studies to evaluate their suitability as biomarkers of exposure to the toxin.
- Uso da levedura Saccharomyces cerevisiae para elucidar mecanismos de toxicidade da microcistina-LRPublication . Valério, Elisabete; Campos, Alexandre; Vilares, Arminda; Osório, Hugo; Pereira, Paulo; Vasconcelos, VitorAté à data, foram já efetuados diversos estudos com vários organismos e linhas celulares para desvendar os mecanismos moleculares de toxicidade da microcistina, uma das mais frequentes hepatotoxinas produzida por cianobactérias. Em células de mamíferos, o mecanismo de toxicidade da microcistina é atribuída a um processo que envolve várias vias, um deles relacionado com a inibição das fosfatases proteicas PP1 / PP2A e outro com a produção de espécies reativas de oxigénio (ROS). Contudo, existem ainda algumas lacunas na identificação destes mecanismos, que impede a total caracterização do modo de ação desta toxina. Por forma a contribuir para a elucidação dos mecanismos de toxicidade da variante química microcistina-LR (MCLR), neste estudo foram avaliados os efeitos de várias concentrações de MCLR nos níveis de ROS, resposta do sistema antioxidante, indução de apoptose, expressão diferencial de proteínas e alteração da expressão génica na levedura Saccharomyces cerevisiae. Verificou-se que após coloração das células com fluorocromos, a exposição das células à toxina induziu um aumento dos níveis intracelulares dos ROS. Este aumento provocou uma ativação do sistema antioxidante, especialmente na resposta da catalase. Além disso, observou-se uma inibição da superóxido dismutase citosólica, o que em conjunto com o tipo de espécies reativas de oxigénio passíveis de estarem presentes, sugere que a ROS maioritariamente induzida é peróxido de hidrogénio (H2O2). Observaram-se ainda sinais de apoptose após avaliação por citometria de fluxo, usando um kit de Anexina V-FITC. Da análise proteómica, verificou-se que 14 proteínas foram diferencialmente expressas nas células expostas a diferentes concentrações de MCLR, quando comparada com o controlo. A análise da expressão relativa dos genes homólogos das PP1/PP2A e de genes BER envolvidos na reparação de DNA revelou que alguns destes apresentaram respostas diferenciais, dependentes da concentração de toxina usada. Os resultados obtidos neste estudo demonstram que a levedura Saccharomyces cerevisiae VL3 apresenta alguns dos principais efeitos tóxicos induzidos pela microcistina-LR em eucariotas superiores e o seu uso revelou que existem proteínas e genes alterados pela exposição à MCLR que são transversais a vários modelos eucarióticos.