ABSTRACT
Os tripanossomatídeos são organismos caracterizados pelo controle póstranscricional da expressão gênica, principalmente em nível de tradução. Na tradução em eucariotos, tem grande destaque o complexo eIF4F, sendo um de seus principais componentes o fator de iniciação da tradução eIF4E. Já foram descritos em tripanossomatídeos seis homólogos para o eIF4E, nomeados EIF4E1 a 6. Em um estudo com Leishmania amazonensis, focado no EIF4E3, percebeu-se que seu perfil de expressão se alterava rapidamente numa curva de crescimento, com este apresentando ao menos duas bandas. As mudanças observadas sugeriam modificações pós-traducionais do tipo fosforilação, algo posteriormente confirmado. Analisando-se a sequência do EIF4E3 de Leishmania, foi possível identificar a presença de possíveis sítios de fosforilação e de ligação a parceiros funcionais como homólogos do eIF4G, outro componente do complexo eIF4F, e da proteína de ligação á cauda poli-A (PABP). No presente estudo foi analisado o perfil de expressão e a capacidade de ligação a parceiros funcionais do EIF4E3 de Leishmania superexpresso em células transfectadas e no qual foram introduzidas mutações em motivos específicos. Os resultados mostraram um perfil de expressão de ao menos três bandas para o EIF4E3 de L. amazonensis e duas para L. infantum, com o sítio S75, presente apenas na primeira, sendo o responsável por esta diferença...
Subject(s)
Humans , Animals , Gene Expression , Leishmania infantum/genetics , Leishmania infantum/metabolism , Leishmania mexicana/genetics , Leishmania mexicana/metabolism , Poly(A)-Binding Proteins , Protein Binding , Protein Biosynthesis , Protozoan Proteins , Sequence Homology, Nucleic AcidABSTRACT
The role of glycosphingolipids (GSLs) present in amastigote forms of Leishmania (Leishmania) amazonensis during infection of macrophages was analyzed, with particular emphasis on GSLs presenting the terminal Galpß1-3Galpa disaccharide. Macrophage invasion by L. (L.) amazonensis amastigotes was reduced by 37 percent when the disaccharide Galpß1-3Galp (1 mM) was added to the culture medium. The putative macrophage receptor/lectin for ß-Gal-globotriaosylceramide (Galpß1-3Galpa1-4Galpß1-4Glc pß1-1Cer) and other structurally related GSLs from L. (L.) amazonensis amastigotes were analyzed by micelles and parasite binding assay to peritoneal macrophage proteins fractionated by SDS-PAGE under nonreducing conditions. Micelles containing purified amastigote GSLs or a suspention of L. (L.) amazonensis amastigotes fixed with 2 percent formaldehyde were incubated with nitrocellulose membrane containing the macrophage proteins transferred by Western blotting. Binding of micelles containing purified GSLs from amastigote forms or fixed L. (L.) amazonensis amastigotes to nitrocellulose membrane was probed using monoclonal antibody ST-3, which recognizes the glycoepitope Galpß1-3Galpa1-R present either in the micelle preparation or on the amastigote surface. Macrophage protein with molecular mass ~30 kDa bound the amastigote GSL and appeared to be a doublet on electrophoresis. The specificity of this interaction was confirmed using fixed L. (L.) chagasi amastigotes, which do not express GSLs such as ß-Galp-globotriaosylceramides, and which do not bind to 30-kDa protein.
Subject(s)
Animals , Cricetinae , Mice , Antibodies, Monoclonal/immunology , Glycosphingolipids/metabolism , Leishmania mexicana/metabolism , Macrophages/parasitology , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Glycosphingolipids/immunology , Leishmania mexicana/immunology , Leishmania mexicana/pathogenicity , Mice, Inbred BALB C , Macrophages/immunologyABSTRACT
Activated macrophages simultaneously synthesize nitric oxide and superoxide anion which can react with each other producing peroxynitrite. Consequently, it has been difficult to assess the precise contribution of each of the formed reactive oxygen- and nitrogenderived species to the microbicidal activities of macrophages, particularly in vivo. To explore this problem, we are examining the formation and potential roles of nitrogen-derived intermediates in Leishmania amazonensis murine infection. Thus far, our results have demonstrated that peroxynitrite is a potent leishmanicidal agent in vitro and that both nitric oxide and peroxynitrite are formed during infection of susceptible BALB/c mouse strain. Nitric oxide was detected as the nitrosyl-hemoglobin complex by electron paramagnetic resonance analysis of blood drawn from mice at different times of infection, and it was shown to increase with the evolution of the disease. These results will be discussed in the context of the dual physiological role of nitric oxide either as a signaling molecule or as a deleterious agent.
Subject(s)
Animals , Mice , In Vitro Techniques , Leishmania mexicana/metabolism , Leishmaniasis/metabolism , Nitrites/metabolism , Nitric Oxide/metabolism , Peroxides/metabolism , Anions/metabolism , Electron Spin Resonance Spectroscopy , Reactive Oxygen Species/metabolism , Free Radicals , Hemoglobins/biosynthesis , Leishmania major/drug effects , Leishmania major/immunology , Leishmania major/metabolism , Leishmania mexicana/drug effects , Leishmania mexicana/immunology , Leishmaniasis/immunology , Macrophage Activation , Mice, Inbred BALB C , Nitrites/pharmacology , Nitrogen/physiology , Nitrogen/metabolism , Oxidants/metabolism , Nitric Oxide/pharmacology , Nitric Oxide/physiology , Nitric Oxide/chemical synthesis , Peroxides/pharmacology , Superoxides/metabolism , Tyrosine/biosynthesisABSTRACT
Inhibiçäo do crescimento de um subespécie de Leishmania por exometabólitos de outra subespécie, um fenômeno ainda näo notificado, é sugerido em nossas recentes observaçöes em experimentos de clonagem celular com Leishmania mexicana mexicana e Leishmania mexicana amazonensis. Os clones foram identificados usando a técnica de análise de esquizodemas. O fenômeno observado é claramente relevante em estudos de isolamento parasitário, metabolismo, imunidade cruzada e quimioterapia