Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.(AU)

A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.(AU)

The effects of qualitative and quantitative protein malnutrition on electrolyte homeokinesis in serum of Wistar rats

Background: Proteins having structural and various regulatory functions are essential components of all unicellular and multicellular organisms. The only source of proteins and their building blocks, amino acids, for human and many animals are the proteins and amino acids in their foods. Although qualitative and quantitative protein malnutrition are common problems in animals and men, the impacts of dietary proteins on serum electrolytes are still controversial. Materials, Methods & Results: Adult male Wistar rats were randomly divided into three groups of 6 or 7 animals serving as controls, and quantitative or qualitative protein malnutrition groups. Animals were held in metabolic cages individually in a conventional room with 12:12 h day/night cycle, 29°C temperature and 50-70% relative humidity. After a 10-day acclimation period controls (n = 7) were given rat chow diet consisting of 24% protein, while other groups received an almost N-free diet (quantitative malnutrition) or a diet containing 20% gelatin as protein source (qualitative malnutrition) for 35 days. Food and tap water were given animals ad libitum during acclimation period and throughout the experiment. At the end of experiment, blood samples were collected and Na+, K+ and Cl- concentrations in serum were determined. Data were analyzed by ANOVA, ANCOVA and Pearson's correlation test. Dietary interventions had significant effects on mean body weights of animals (P = 0.000), but not on their food consumptions. However, in the last week controls consumed significantly more food than both malnourished groups (P = 0.001). If row data were used for statistical evaluation, it was seen that qualitative and quantitative protein malnutrition affected only the serum K+, and Cl- concentrations significantly (P = 0.003, P = 0.000). Controls had higher K+ concentrations than those of gelatin-given group (P = 0.002) and N-free group had higher Cl- concentrations than control and gelatin-given groups (P = 0.001). However, if body-weight corrected electrolyte values were considered, compared to controls a two-fold increase in mean concentrations of all three electrolytes in sera of both malnourished groups were seen (P = 0.000). There were certain negative or positive correlations between different variables interested. Discussion: The findings of this study revealed that, on the basis of the row data, the dietary protein inanition increase serum Cl - concentration, while gelatin in the diet decrease the K+ concentration in sera of rats. Because of the body weights of malnourished animals decreased while controls continued to gain weight throughout the experiment, a substantial difference in body weights of malnourished animals and controls occurred. And, because of physiological variables are reflections of body weights, it was necessary to eliminate the possible effects of the body weights of animals. If the effect of the body weights of animals eliminated, a two-fold increase of all three electrolytes studied was seen. It was concluded that dietary proteins exert signifi cant control on the homeokinesis of serum electrolytes. If physiopathological conditions are questioned, standardization for comparison of the results from control and experimental groups in a study as well as from different studies is indispensible