Differential expression of PEPCK isoforms is correlated to Aedes aegypti oogenesis and embryogenesis.

The mosquito Aedes aegypti undertakes a shift in carbohydrate metabolism during embryogenesis, including an increase in the activity of phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, at critical steps of embryo development. All eukaryotes studied to date present two PEPCK isoforms, namely PEPCK-M (mitochondrial) and PEPCK-C (cytosolic). In A. aegypti, however, these proteins are so far uncharacterized. In the present work we describe two A. aegypti PEPCK isoforms by sequence alignment, protein modeling, and transcription analysis in different tissues, as well as PEPCK enzymatic activity assays in mitochondrial and cytoplasmic compartments during oogenesis and embryogenesis. First, we characterized the protein sequences compared to other organisms, and identified conserved sites and key amino acids. We also performed structure modeling for AePEPCK(M) and AePEPCK(C), identifying highly conserved structural sites, as well as a signal peptide in AePEPCK(M) localized in a very hydrophobic region. Moreover, after blood meal and during mosquito oogenesis and embryogenesis, both PEPCKs isoforms showed different transcriptional profiles, suggesting that mRNA for the cytosolic form is transmitted maternally, whereas the mitochondrial form is synthesized by the zygote. Collectively, these results improve our understanding of mosquito physiology and may yield putative targets for developing new methods for A. aegypti control.

Avaliação in silico da interação entre o receptor GABAA e metalocompostos derivados de benzodiazepínicos / Evaluation of the interaction between GABAA receptor and organometallics derived from benzodiazepines in silico

O receptor do ácido γ-aminobutírico do tipo A (GABAA) é o receptor de ação rápida mais amplamente distribuído no sistema nervoso central (SNC) dos mamíferos. Os receptores GABAA são canais iônicos pentaméricos transmembranares e apresentam alta heterogeneidadede entre suas subunidades. Quando estes receptores são ativados pelo neurotransmissor GABA, permitem a passagem de íons cloreto para dentro dos neurônios, resultando em uma hiperpolarização destas células, as tornando menos reativas a neurotransmissores excitatórios. Os receptores GABAA são alvos de vários grupos farmacológicos com propriedades anestésicas e sedativas. A ativação destes receptores pode ser modulada por diferentes grupos de compostos, incluindo os benzodiazepínicos (BZDs), que se tornaram o grupo farmacológico prescrito mais consumido no mundo, sendo indicados no tratamento de ansiedade, insônia, relaxamento muscular e epilepsia. Apesar de serem indicados no tratamento em diversas manifestações clínicas, podem apresentar efeitos adversos, como comprometimento da memória, síndrome de descontinuação, além da sua ineficiência no tratamento em alguns casos de epilepsia. Nesse contexto, é importante identificar e desenvolver novos compostos que apresentem as mesmas características e eficiência dos BZDs clássicos, porém minimizando seus efeitos adversos.

Uma abordagem interessante é a síntese de compostos de coordenação, a partir da associação entre compostos orgânicos com elementos metálicos a partir dos BZDs clássicos, como o diazepam. Neste trabalho, foi analisada a interação do receptor GABAA com cinco compostos de coordenação derivados do diazepam com íon paládio ([(DZP)PdOAcPPh3], [(DZP)PdClPPh3], [(DZP)PdClPy], ([(DZP)PdCl]2 e [(DZP)PdOAc]2) em um modelo heteropentamérico da principal combinação de isoformas do receptor GABAA (α1ß2γ2) construído pela técnica de modelagem comparativa. Com o intuito de predizer a pose dos ligantes, foram realizadas simulações de docking molecular entre os compostos de coordenação e o diazepam com o receptor GABAA, utilizando o software AutoDock. Nossos resultados indicam que os compostos de coordenação apresentaram energia livre de ligação estimada mais baixa que o ligante diazepam, com destaque para o composto o [(DZP)PdOAc]2. Foram identificados quatro resíduos que aparentemente contribuem para a interação proteína-ligante: His101(α1), Ser204(α1), Tyr58(γ2) e Phe77(γ2). O metal paládio incluso nos compostos de coordenação apresentou um papel de estabilidade estrutural, conferindo um maior número de interações com o receptor GABAA no sítio dos benzodiazepínicos. (AU)

Detection and genotyping of Trypanosoma cruzi from açai products commercialized in Rio de Janeiro and Pará, Brazil.

BACKGROUND: Several cases of food-borne acute Chagas disease (ACD) have been reported in the Brazilian Amazon so far. Up to 2004, the occurrence of ACD by oral transmission, associated with food consumption, was rare. Recent cases of ACD in Brazil have been attributed to the consumption of juice from the açai palm containing reservoir animals or insect vectors waste, infected with Trypanosoma cruzi. This study aimed to determine the T. cruzi contamination rate and to genotype the parasite in food samples prepared from açai, which are commercialized in Rio de Janeiro and the Pará States in Brazil. METHODS: The amplificability of DNA extracted from açai samples, and T. cruzi and Triatominae detection were performed by conventional PCR. Molecular characterization was done by multilocus PCR analysis, to determine the parasite discrete type units (DTUs) based on the size of PCR products in agarose gels, using the intergenic region of the spliced leader (SL), 24 Sα rDNA and nuclear fragment A10 as targets. RESULTS: From the 140 samples of açai-based products analyzed, T. cruzi DNA was detected in 14 samples (10%); triatomine DNA was detected in one of these 14 samples. The parasite genotyping demonstrated that food samples containing açai showed a mixture of T. cruzi DTUs with TcIII, TcV and TcI prevailing. CONCLUSIONS: In this study, the molecular detection and identification of T. cruzi from açai-based manufactured food samples, was performed for the first time. Although parasite DNA is a marker of possible contamination during food manufacturing, our findings do not indicate that açai is a source of Chagas disease via oral transmission per se, as live parasites were not investigated. Nevertheless, a molecular approach could be a powerful tool in the epidemiological investigation of outbreaks, supporting previous evidence that açai-based food can be contaminated with T. cruzi. Furthermore, both food quality control and assessment of good manufacturing practices involving açai-based products can be improved, assuring the safety of açai products.