Navegando por Palavras-chave "immunolocalization"

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    Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme
    (Elsevier B.V., 2008-06-01) Silva, Edson Roberto da; Laranjeira da Silva, Maria Fernanda; Fischer, Hannes; Mortara, Renato A. [UNIFESP]; Mayer, Mario Gustavo; Framesqui, Karine; Silber, Ariel Mariano; Floeter-Winter, Lucile Maria; Universidade de São Paulo (USP); Univ Luterana Brasil; Universidade Federal de São Paulo (UNIFESP); Inst Butantan Serv Genet
    Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. in Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. in the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. for the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. the interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. the determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.
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    Human antibody responses of patients living in endemic areas for schistosomiasis to the tegumental protein Sm29 identified through genomic studies
    (Blackwell Publishing, 2006-06-01) Cardoso, F. C.; Pacifico, RNA; Mortara, Renato Arruda [UNIFESP]; Oliveira, S. C.; Universidade Federal de Minas Gerais (UFMG); Universidade Federal de São Paulo (UNIFESP)
    Surface proteins of schistosomes are exposed to host tissues and thus present as potential candidate molecules for the development of new intervention strategies. Herein, we have identified a new tegumental protein of Schistosoma mansoni, termed Sm29. in silico analysis revealed a signal peptide, three glycosylation sites and a transmembrane region on Sm29 amino acid sequence. Sm29 transcription in mammalian developmental stages cDNA libraries of S. mansoni was verified by PCR using specific primers for Sm29 nucleotide sequence and it revealed the presence of transcripts in schistosomula and adult worm stages of the parasite. Sm29 (40-169) fragment was produced in Escherichia coli and purified by affinity chromatography to be used in the immunological assays. Confocal microscopy confirmed bioinformatic studies, revealing that Sm29 is a membrane-bound protein localized on the tegument of S. mansoni adult worm. ELISA was performed using rSm29 protein to investigate the antibody isotype profile to Sm29 in sera of patients living in endemic areas for schistosomiasis. IgG1 and IgG3 subclass antibodies to rSm29 were predominant in sera of individuals naturally resistant to infection and resistant to re-infection whereas low levels of IgM, IgA or IgE were measured. Since, IgG1 and IgG3 are involved in parasite killing and in protective immunity the findings reported here suggest the use of Sm29 as a potential candidate vaccine against schistosomiasis.
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    Molecular characterization and immunolocalization of Schistosoma mansoni ATP-diphosphohydrolase
    (Elsevier B.V., 2003-08-08) DeMarco, R.; Kowaltowski, A. T.; Mortara, R. A.; Verjovski-Almeida, S.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
    Schistosoma mansoni, a human parasite that constitutes a major health problem in developing countries, escapes from host defenses and survives in the human bloodstream. Here, we report the cloning of a S. mansoni ATP-diphosphohydrolase ortholog (SmATPDase1). Southern blots indicated that in S. mansoni it is a single-copy gene. RT-PCR revealed that SmATPDase1 is expressed in five stages of the parasite life cycle, namely cercaria, schistosomula, adults, eggs, and miracidia. Using confocal microscopy, SmATPDase1 protein was immunolocalized on the external surface in all stages, except eggs, being conspicuously present in adults. ATPDase, which is present on the outer surface of endothelial cells lining human blood vessels, has been implicated in thromboregulation by promoting ADP hydrolysis and inhibition of platelet aggregation. the presence of an ATPDase ortholog on the surface of S. mansoni suggests that the enzyme might play a role in the escape from host defenses that would involve platelet activation. (C) 2003 Elsevier Inc. All rights reserved.
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    Schistosonia mansoni: Expression of Fes-like tyrosine kinase SmFes in the tegument and terebratorium suggests its involvement in host penetration
    (Elsevier B.V., 2007-07-01) Bahia, Diana; Mortara, Renato A.; Kusel, John R.; Andrade, Luiza F.; Ludolf, Fernanda; Kuser, Paula R.; Avelar, Livia; Trolet, Jacques; Dissous, Colette; Pierce, Raymond J.; Oliveira, Guilherme; Universidade Federal de São Paulo (UNIFESP); Fiocruz MS; Univ Glasgow; Programa Pos Grad & Pequisa; Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA); INSERM; Univ Lille 2; Inst Pasteur
    Protein Tyrosine Kinases (PTKs) are important molecules in intra- and inter-cellular communication, playing a major role in signal transduction processes. We have previously identified and characterized the molecular structure of a new PTK in Schistosoma mansoni, SmFes. SmFes exhibits the characteristic features of Fes/Fps protein tyrosine kinase subfamily of which it is the first member described in helminths. Herein, we show that genes orthologous to SmFes are also present in other Schistosoma species and the transcript is detected in Schistosoma japonicum. the SmFes protein was detected at all the main life-cycle stages and was most abundant in cercariae and newly-transformed schistosomula. However, no protein was detected in schistosomula maintained in vitro for 7 days. By immunolocalization assays we showed that SmFes is particularly concentrated at the terebratorium of miracidia and tegument of cercaria and schistosomula skin-stage. These findings suggest that SmFes may play a role in signal transduction pathways involved in larval transformation after penetration into intermediate and definitive hosts. (c) 2007 Elsevier Inc. All rights reserved.
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    The Ultrastructural Study of Tumorigenic Cells Using Nanobiomarkers
    (Mary Ann Liebert Inc, 2010-06-01) Pavon, Lorena Favaro; Marti, Luciana Cavalheiro; Sibov, Tatiana Tais; Malheiros, Suzana M. F. [UNIFESP]; Oliveira, Daniela Mara; Guilhen, Daiane Donna; Camargo-Mathias, Maria Izabel; Amaro Junior, Edson; Gamarra, Lionel Fernel; SBIBHAE; IIEPAE; Universidade Federal de São Paulo (UNIFESP); Univ Fed Sao Joao Del Rei; UNESP; Universidade de São Paulo (USP)
    Despite recent advances, patients with malignant brain tumors still have a poor prognosis. Glioblastoma (WHO grade 4 astrocytoma), the most malignant brain tumor, represents 50% of all astrocytomas, with a median survival rate of <1 year. It is, therefore, extremely important to search for new diagnostic and therapeutic approaches for patients with glioblastoma. This study describes the application of superparamagnetic nano-particles of iron oxide, as well as monoclonal antibodies, of immunophenotypic significance, conjoined to quantum dots for the ultrastructural assessment of glioblastoma cells. for this proposal, an immunophenotypic study by flow cytometry was carried out, followed by transmission electron microscopy analysis. the process of tumor cell labeling using nanoparticles can successfully contribute to the identification of tumorigenic cells and consequently for better understanding of glioblastoma genesis and recurrence. in addition, this method may help further studies in tumor imaging, diagnosis, and prognostic markers detection.