Atividade moluscicida e intera??es moleculares da nitazoxanida com Limnoperna fortunei.

Programa de P?s-Gradua??o em Ci?ncias Biol?gicas. N?cleo de Pesquisas em Ci?ncias Biol?gicas, Pr?-Reitoria de Pesquisa de P?s Gradua??o, Universidade Federal de Ouro Preto.O Limnoperna fortunei ? respons?vel por impactos econ?micos, principalmente no setor hidrel?trico respons?vel pela maior parte da produ??o de energia el?trica no Brasil. Atualmente o cloro ? o agente mais empregado no combate ?s infesta??es pelo L. fortunei, entretanto, sua utiliza??o ? limitada pelas ag?ncias ambientais devido ao potencial t?xico. A subst?ncia 2-acetolyloxy-N-(5-nitro-2-thiazolyl)-benzamide (NTZ) induziu mortalidade de 80 a 60% em concentra??es de 5 a 1 ppm respectivamente. Nessas concentra??es, a droga possui baixa toxicidade em organismos superiores aer?bicos. Estudos em peixes demonstraram baixo potencial toxicol?gico, indicando que pode ser usada com seguran?a na dose 1 ppm. Visando alcan?ar melhor desempenho da droga e utiliza-la em menores concentra??es, a am?nia foi associada a 1 ppm para ampliar o efeito moluscicida do NTZ. Nossos resultados mostram uma potencializa??o do efeito e uma mortalidade de 80% com um dose 10 vezes menor associada a 1 ppm de ammonia. A droga pode ser facilmente removida da ?gua de descarte logo ap?s o tratamento de tubula??es, se recolhida em reservat?rio contendo 1% de suspens?o de crav?o ativado sob agita??o. Essa suspens?o remove pelo menos 99% de pulsos de concentra??es de 5 ppm em 15 segundos de exposi??o. Al?m disso, a capacidade m?xima de reten??o do carv?o atinge 10% do seu peso. Esse estudo traz como perspectiva imediata a cria??o de um sistema de fluxo cont?nuo de remo??o do NTZ da ?gua de rejeito e a possibilidade dispensa-la em ?guas fluviais sem causar danos ambientais. O NTZ foi imobilizado em Sepharose, com o objetivo de isolar e identificar as prote?nas que interagem de forma espec?fica ao NTZ. Empregando-se a espectrometria de massas foram identificadas algumas prote?nas ligantes que foram analisadas como poss?veis alvos de a??o. A arginina quinase, considerada alvo de drogas antiparasit?rias e enzima fundamental para a manuten??o do organismo em anaerobiose, foi identificada na fra??o retida. Os resultados de ensaios enzim?ticos demonstram que o NTZ inibe arginina quinase em concentra??es molares de 10-4M. Devido a import?ncia dessa enzima e a possiblidade de ac?mulo da droga durante a filtra??o de ?gua realizada por esses organismos consideramos a possibilidade que esse mecanismo possa explicar o efeito t?xico do NTZ sobre o L. fortunei. Al?m disso, a intera??o entre a droga e prote?nas envolvidas na regula??o da apoptose tamb?m poderiam contribuir para essa atividade t?xica.The Limnoperna fortunei is responsible for economic impacts, mainly in the hydroelectric sector responsible for most of the Brazil electricity production. Chlorine is currently the most widely used agent for controlling L. fortunei infestations, however, its use is limited by environmental agencies due to toxic potential. The substance 2-acetolyloxy-N- (5-nitro-2-thiazolyl) -benzamide (NTZ) induced mortality from 80 to 60% in concentrations of 5 to 1 ppm respectively. At these concentrations, the drug has low toxicity in upper aerobic organisms. Studies in fish have shown low toxicological potential, indicating that it can safely be used at the dose of 1 ppm. In order to achieve better performance of the drug and to use it at lower concentrations, ammonia was associated with 1 ppm to increase the molluscicidal effect of NTZ. Our results show potentiation of the effect and a mortality of 80% with a 10-fold lower dose associated with 1 ppm of ammonia. The drug can be easily removed from the wastewater soon after treatment of pipes if collected in a reservoir containing 1% activated charcoal suspension under agitation. This suspension removes at least 99% of pulses at concentrations of 5 ppm in 15 seconds of exposure. In addition, the maximum retention capacity of coal reaches 10% of its weight. This study has as an immediate perspective the creation of a continuous flow system for the removal of NTZ from waste water and the possibility to dispense it in river waters without causing environmental damages. NTZ was immobilized on Sepharose, in order to isolate and identify proteins that specifically interact with NTZ. Using mass spectrometry some ligand proteins were identified and analyzed as possible targets for action. Arginine kinase, considered a target of antiparasitic drugs and an enzyme essential for the maintenance of the organism in anaerobiosis, was identified in the retained fraction. Results from enzymatic assays demonstrate that NTZ inhibits arginine kinase at molar concentrations of 10-4M. Due to the importance of this enzyme and the possibility of accumulation of the drug during the filtration of water performed by these organisms, we consider the possibility that this mechanism can explain the toxic effect of NTZ on the L. fortunei. In addition, the interaction between the drug and proteins involved in the regulation of apoptosis could also contribute to this toxic activity

Exposure of cultured fibroblasts to the peptide PR-11 for the identification of induced proteome alterations and discovery of novel potential ligands.

The PR-11 peptide corresponds to the N-terminal and active region of the endogenously synthesized PR-39 molecule, of porcine origin. It is known to possess various biological effects including antimicrobial properties, angiogenic and anti-inflammatory activities. Apart from its reported activity as a proteasome inhibitor, a more comprehensive understanding of its function, at the molecular level, is still lacking. In this study, we used a label-free shotgun strategy to evaluate the proteomic alterations caused by exposure of cultured fibroblasts to the peptide PR-11. This approach revealed that more than half of the identified moleculeswere related to signalling, transcription and translation. Proteins directly associated to regulation of angiogenesis and interaction with the hypoxia-inducible factor 1-? (HIF-1?) were significantly altered. In addition, at least three differentially expressed molecules of the NF-?B pathway were detected, suggesting an anti-inflammatory property of PR-11. At last, we demonstrated novel potential ligands of PR-11, through its immobilization for affinity chromatography. Among the elutedmolecules, gC1qR, a known complement receptor, appearedmarkedly enriched. This provided preliminary evidence of a PR-11 ligand possibly involved in the internalization of this peptide. Altogether, our findings contributed to a better understanding of the cellular pathways affected by PR-39 derived molecules