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- ItemSomente MetadadadosThe flagellar attachment zone of Trypanosoma cruzi epimastigote forms(Elsevier B.V., 2006-04-01) Rocha, G. M.; Brandao, B. A.; Mortara, R. A.; Attias, M.; Souza, W. de; Carvalho, TMU; Universidade Federal do Rio de Janeiro (UFRJ); Universidade Federal de São Paulo (UNIFESP)The flagellar attachment zone (FAZ) is an adhesion region of Trypanosoma cruzi epimastigote forms where the flagellum emerges from the flagellar pocket and remains attached to the cell body. This region shows a junctional complex which is formed by a linear series of apposed macular structures that are separated by amorphous material and clusters of intramembranous particles. Two protein groups appear to be important in the FAZ region: a membrane glycoprotein of 72 kDa and several high molecular weight proteins. To gain a better understanding of the FAZ region, we compared wild-type Y strain T cruzi epimastigotes with a mutant cell in which the 72-kDa surface glycoprotein (Gp72), involved in cell body-flagellum adhesion, had been deleted by target gene replacement. Using immunofluorescence confocal microscopy and electron microscopy techniques to analyze the FAZ region the results suggest that, in the absence of Gp72, other proteins involved in the formation of FAZ remain concentrated in the flagellar pocket region. the analysis of a 3-D reconstruction model of wild-type epimastigotes showed that the endoplasmic reticulum and mitochondrion are in intimate association with FAZ, in contrast to the null mutant cells where the endoplasmic reticulum was not visualized. (c) 2005 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosImmunolocalization of Leishmania (Viannia) braziliensis membrane antigens recognized by mAbs SST-2, SST-3, and SST-4(Cambridge Univ Press, 2003-11-01) Silveira, TGV; Takahashi, H. K.; Straus, A. H.; Universidade Federal de São Paulo (UNIFESP)The immunolocalization of Leishmania (Viannia) braziliensis stage-specific antigens recognized by mAbs was analysed by transmission electron microscopy. the antigen recognized by mAb SST-2 was present at the surface of promastigotes, including the flagellum and flagellar pocket. the reactivity of SST-2 with isolates of different serodemes showed a pronounced microheterogencity in terms of the number of reactive bands within the low molecular weight range from 24 to 33 kDa. the 180 kDa glycoprotein recognized by mAb SST-3 was present only in the flagellar membrane. SST-3 also recognized multiple discrete bands from 160 to 200 kDa, as observed in several serodemes. in contrast, mAb SST-4, which recognizes a 98 kDa antigen, showed weak labelling on the promastigote surface by transmission electron microscopy and indirect immunofluorescence. Based on Western blotting, indirect immunofluorescence, and solid-phase radioimmunoassay, the antigens recognized by mAbs SST-2, SST-3 and SST-4 were present in all L. (V.) braziliensis analysed, from 7 different serodemes.
- ItemSomente MetadadadosIn vivo and in vitro phosphorylation and subcellular localization of trypanosomatid cytoskeletal giant proteins(Wiley-Blackwell, 2000-09-01) Baqui, MMA; Milder, R.; Mortara, Renato Arruda [UNIFESP]; Pudles, J.; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Promastigote forms of Phytomonas serpens, Leptomonas samueli, and Leishmania tarentolae express cytoskeletal giant proteins with apparent molecular masses of 3,500 kDa (Ps 3500), 2,500 kDa (Ls 2500), and 1,200 kDa (Lt 1200). respectively. Polyclonal antibodies to it 1200 and to Ps 3500 specifically recognize similar polypeptides of the same genera of parasite. in addition to reacting with giant polypeptides of the Leptomonas species, anti-is 2500 also cross reacts with Ps 3500, and with a 500-kDa polypeptide of Leishmania. Confocal immunofluorescence and immunogold electron microscopy showed major differences in topological distribution of these three proteins, though they partially share a common localization at the anterior end of the cell body skeleton. Furthermore, Ps 3500. Ls 2500, and it 1200 are in vivo phosphorylated at serine and threonine residues, whereas, in vitro phosphorylation of cytoskeletal fractions reveal that only Ps 3500 and Ls 2500 are phosphorylated. Heat treatment (100 degrees C) of high salt cytoskeletal extracts demonstrates that Ps 3500 and Ls 2500 remain stable in solution, whereas it 1200 is denatured. Kinase assays with immunocomplexes of heat-treated giant proteins show that only Ps 3500 and Ls 2500 are phosphorylated. These results demonstrate the existence of a novel class of megadalton phosphoproteins in promastigote forms of trypanosomatids that appear to be genera specific with distinct cytoskeletal functions. in addition, there is also evidence that Ps 3500 and is 2500, in contrast to it 1200, seem to be autophosphorylating serine and threonine protein kinases, suggesting that they might play regulatory roles in the cytoskeletal organization. (C) 2000 Wiley-Liss, Inc.
- ItemSomente MetadadadosMorphological events during the Trypanosoma cruzi cell cycle(Elsevier B.V., 2007-04-01) Elias, Maria Carolina; Cunha, Julia P. C. da; Faria, Flavio P. de; Mortara, Renato A.; Freymueller, Edna; Schenkman, Sergio; Universidade Federal de São Paulo (UNIFESP); Inst ButantanThe replication and segregation of organelles producing two identical daughter cells must be precisely controlled during the cell cycle progression of eukaryotes. in kinetoplastid flagellated protozoa, this includes the duplication of the single mitochondrion containing a network of DNA, known as the kinetoplast, and a flagellum that grows from a cytoplasmic basal body through the flagellar pocket compartment before emerging from the cell. Here, we show the morphological events and the timing of these events during the cell cycle of the epimastigote form of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease. DNA staining, flagellum labeling, bromodeoxyuridine incorporation, and ultra-thin serial sections show that nuclear replication takes 10% of the whole cell cycle time. in the middle of the G2 stage, the new flagellum emerges from the flagellar pocket and grows unattached to the cell body. While the new flagellum is still short, the kinetoplast segregates and mitosis occurs. the new flagellum reaches its final size during cytokinesis when a new cell body is formed. These precisely coordinated cell cycle events conserve the epimastigote morphology with a single nucleus, a single kinetoplast, and a single flagellum status of the interphasic cell. (C) 2006 Elsevier GmbH. All rights reserved.
- ItemSomente MetadadadosPhosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of GPI-anchored surface molecules of Trypanosoma cruzi triggers in vitro morphological reorganization of trypomastigotes(Soc Protozoologists, 2001-01-01) Mortara, R. A.; Minelli, LMS; Vandekerchove, F.; Nussenzweig, V; Ramalho-Pinto, F. J.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); NYU Med CtrTrypanosoma cruzi trypomastigotes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) in vitro are rapidly induced to differentiate into round forms. Using confocal microscopy, we were able to show that trypomastigotes treated with PI-PLC initiate the process of flagellum remodeling by 30 sec after contact with the enzyme and amastigote-like forms are detected as early as 10 min after PI-PLC treatment. Scanning and transmission electron microscopy indicate that trypomastigotes undergo a previously undescribed process of flagellum circularization and internalization. Analysis of the flagellar complex with monoclonal antibody 4D9 shows heterogeneous labeling among the parasites, suggesting a remodeling of these molecules. After PI-PLC treatment, parasites rapidly lose the surface marker Ssp-3 and 24 h post-treatment they begin to exhibit a circular nucleus and a rod-shaped kinetoplast. By flow cytometry analysis and confocal microscopy, the Ssp-4 amastigote-specific epitope can be detected on the parasite surface. This indicates that thr release of trypomastigote GPI-anchored molecules by exogenous PI-PLC in vitro can trigger morphological changes.
- ItemSomente MetadadadosThree-Dimensional Reconstruction of Trypanosoma cruzi Epimastigotes and Organelle Distribution Along the Cell Division Cycle(Wiley-Blackwell, 2011-07-01) Ramos, Thiago Cesar Prata [UNIFESP]; Freymüller-Haapalainen, Edna [UNIFESP]; Schenkman, Sergio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Trypanosoma cruzi is the protozoan that causes Chagas disease. It divides in the insect vector gut or in the cytosol of an infected mammalian cell. T cruzi has one mitochondrion, one Golgi complex, one flagellum, and one cytostome. Here, we provide three-dimensional (3D) models of this protozoan based on images obtained from serial sections on electron microscopy at different stages of the cell cycle. Ultrathin serial sections were obtained from Epon (TM) embedded parasites, photographed in a transmission electron microscope, and 3D models were generated using Reconstruct and Blender 3D modeling softwares. the localization and distribution of organelles was evaluated and attributed to specific morphological patterns and deduced by distribution of specific markers by immunofluorescence analysis. the new features found in the 3D reconstructions are (1) the electron-dense chromatin is interconnected leaving an internal space for a centrally located nucleolus; (2) the kinetoplast is accommodated within a separated branch of the tubular and single mitochondrion; (3) the disk shaped kinetoplast, which is the mitochondrial DNA, duplicates from the interior in G2 phase; (4) the mitochondrion faces the external membrane and shrinks to accommodate an enlarged number of cytosolic vesicles from G1 to G2; (5) the cytostome progress from the parasite surface toward the posterior end contouring the kinetoplast and nucleus and retracts during cell cycle. These new observations might help understanding how organelles are formed and distributed in early divergent eukaryotic cells and provides a useful method to understand the organelle distribution in small eukaryotic cells. (C) 2011 International Society for Advancement of Cytometry