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Mario Steindel - One of the best experts on this subject based on the ideXlab platform.
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Characterization of D-glucose transport in Trypanosoma rangeli.
Parasitology, 2006Co-Authors: Luiz Claudio Miletti, Leonardo B Koerich, Mario Steindel, L K Pacheco, B U StambukAbstract:Like in other trypanosomatids D-glucose is a crucial source of energy to Trypanosoma rangeli, a non-pathogenic parasite that in Central and South America infects triatomine vectors and different mammalian species, including humans. In several trypanosome species, D-glucose transporters were already described and cloned. In this study, we characterized the D-glucose transport activity present in 2 life-stage forms of T. rangeli (Epimastigotes and trypomastigotes) using D-[U-14C]glucose as substrate. Our results indicate that T. rangeli transports D-glucose with high affinity in both Epimastigote (Km 30 microM) and trypomastigotes (Km 80 microM) life-forms. Both transport activities were inhibited by Cytochalasin B and Phloretin, indicating that probably D-glucose uptake in T. rangeli is mediated by facilitated diffusion of the sugar. Significant differences were observed between Epimastigotes and trypomastigotes in relation to their affinity for D-glucose analogues, and the predicted amino acid sequence of a putative D-glucose transporter from T. rangeli (TrHT1) showed a larger identity with the T. cruzi D-glucose transporter encoded by the TcrHT1 gene than with other transporters already characterized in trypanosomatids.
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Penetration of the salivary glands of Rhodnius domesticus Neiva & Pinto, 1923 (Hemiptera: Reduviidae) by Trypanosoma rangeli Tejera, 1920 (Protozoa: Kinetoplastida)
Parasitology Research, 2005Co-Authors: Rosane M. S. Meirelles, Maurilio J Soares, Andrea Henriques-pons, Mario SteindelAbstract:Penetration of the heteroxenous protozoan Trypanosoma rangeli into the salivary glands of its invertebrate host Rhodnius domesticus has been investigated here using different approaches. Electron microscopy showed that Epimastigotes coming from the insect hemocoel cross the basal lamina that surrounds the salivary glands and penetrate through the gland cells cytoplasm. After reaching the gland lumen, Epimastigote forms remain adhered to the gland cell microvilli by their flagella, while metacyclic trypomastigotes are found swimming free in the saliva. Analysis by flow cytometry, western blotting and hemolytic activity allowed to demonstrate the presence in T. rangeli of a hemolytic molecule with antigenic cross-reactivity with murine perforin, which could be used by the parasites to reach the salivary gland lumen. This molecule, which we named as rangelysin, has 120 kDa molecular weight, is able to induce hemolysis only in acidic pH, and is produced by both trypomastigote and Epimastigote forms.
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penetration of the salivary glands of rhodnius domesticus neiva pinto 1923 hemiptera reduviidae by trypanosoma rangeli tejera 1920 protozoa kinetoplastida
Parasitology Research, 2005Co-Authors: Rosane M. S. Meirelles, Maurilio J Soares, Andrea Henriquespons, Mario SteindelAbstract:Penetration of the heteroxenous protozoan Trypanosoma rangeli into the salivary glands of its invertebrate host Rhodnius domesticus has been investigated here using different approaches. Electron microscopy showed that Epimastigotes coming from the insect hemocoel cross the basal lamina that surrounds the salivary glands and penetrate through the gland cells cytoplasm. After reaching the gland lumen, Epimastigote forms remain adhered to the gland cell microvilli by their flagella, while metacyclic trypomastigotes are found swimming free in the saliva. Analysis by flow cytometry, western blotting and hemolytic activity allowed to demonstrate the presence in T. rangeli of a hemolytic molecule with antigenic cross-reactivity with murine perforin, which could be used by the parasites to reach the salivary gland lumen. This molecule, which we named as rangelysin, has 120 kDa molecular weight, is able to induce hemolysis only in acidic pH, and is produced by both trypomastigote and Epimastigote forms.
Rosane M. S. Meirelles - One of the best experts on this subject based on the ideXlab platform.
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Penetration of the salivary glands of Rhodnius domesticus Neiva & Pinto, 1923 (Hemiptera: Reduviidae) by Trypanosoma rangeli Tejera, 1920 (Protozoa: Kinetoplastida)
Parasitology Research, 2005Co-Authors: Rosane M. S. Meirelles, Maurilio J Soares, Andrea Henriques-pons, Mario SteindelAbstract:Penetration of the heteroxenous protozoan Trypanosoma rangeli into the salivary glands of its invertebrate host Rhodnius domesticus has been investigated here using different approaches. Electron microscopy showed that Epimastigotes coming from the insect hemocoel cross the basal lamina that surrounds the salivary glands and penetrate through the gland cells cytoplasm. After reaching the gland lumen, Epimastigote forms remain adhered to the gland cell microvilli by their flagella, while metacyclic trypomastigotes are found swimming free in the saliva. Analysis by flow cytometry, western blotting and hemolytic activity allowed to demonstrate the presence in T. rangeli of a hemolytic molecule with antigenic cross-reactivity with murine perforin, which could be used by the parasites to reach the salivary gland lumen. This molecule, which we named as rangelysin, has 120 kDa molecular weight, is able to induce hemolysis only in acidic pH, and is produced by both trypomastigote and Epimastigote forms.
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penetration of the salivary glands of rhodnius domesticus neiva pinto 1923 hemiptera reduviidae by trypanosoma rangeli tejera 1920 protozoa kinetoplastida
Parasitology Research, 2005Co-Authors: Rosane M. S. Meirelles, Maurilio J Soares, Andrea Henriquespons, Mario SteindelAbstract:Penetration of the heteroxenous protozoan Trypanosoma rangeli into the salivary glands of its invertebrate host Rhodnius domesticus has been investigated here using different approaches. Electron microscopy showed that Epimastigotes coming from the insect hemocoel cross the basal lamina that surrounds the salivary glands and penetrate through the gland cells cytoplasm. After reaching the gland lumen, Epimastigote forms remain adhered to the gland cell microvilli by their flagella, while metacyclic trypomastigotes are found swimming free in the saliva. Analysis by flow cytometry, western blotting and hemolytic activity allowed to demonstrate the presence in T. rangeli of a hemolytic molecule with antigenic cross-reactivity with murine perforin, which could be used by the parasites to reach the salivary gland lumen. This molecule, which we named as rangelysin, has 120 kDa molecular weight, is able to induce hemolysis only in acidic pH, and is produced by both trypomastigote and Epimastigote forms.
Jose Roberto Meyerfernandes - One of the best experts on this subject based on the ideXlab platform.
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modulation of trypanosoma rangeli ecto phosphatase activity by hydrogen peroxide
Free Radical Biology and Medicine, 2009Co-Authors: Daniela Cosentinogomes, Thais Russoabrahao, Andre L Fonsecadesouza, Clara Rodrigues Ferreira, Antonio Galina, Jose Roberto MeyerfernandesAbstract:Abstract As a protozoan parasite of hematophagous insects, Trypanosoma rangeli Epimastigotes are exposed to reactive oxygen species during development in hosts. In this work, we investigated the role of H2O2 as a modulator of the ecto-phosphatase activity present in living T. rangeli. We observed that H2O2 inhibits ecto-phosphatase activities in the short and long Epimastigote forms of T. rangeli. Ecto-phosphatase activity found in the short form was more sensitive than that found in the long form. Moreover, H2O2 inhibited ecto-phosphatase activity of the short form in a dose-dependent manner and this inhibition was reversible after H2O2 removal. This effect was not observed for T. rangeli ecto-ATPase, another ecto-enzyme present on the external surface of T. rangeli. Cysteine, β-mercaptoethanol, and reduced glutathione were able to revert the enzyme inhibition promoted by H2O2. Catalase and glutathione peroxidase stimulated this ecto-phosphatase activity, whereas superoxide dismutase was not able to modulate this activity. The ecto-phosphatase activity was also activated by FCCP and inhibited by oligomycin. It seems that H2O2 plays a fundamental role in the regulation of cellular processes of these organisms. We showed, for the first time, that these parasites can produce H2O2, and it is able to regulate ecto-phosphatase activity.
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a mg2 dependent ecto phosphatase activity on the external surface of trypanosoma rangeli modulated by exogenous inorganic phosphate
Acta Tropica, 2008Co-Authors: Andre L Fonsecadesouza, Claudia F Dick, Andre Luiz Araujo Dos Santos, Jose Roberto MeyerfernandesAbstract:Abstract In this work, we characterized a Mg2+-dependent ecto-phosphatase activity present in live Trypanosoma rangeli Epimastigotes. This enzyme showed capacity to hydrolyze the artificial substrate for phosphatases, p-nitrophenylphosphate (p-NPP). At saturating concentration of p-NPP, half-maximal p-NPP hydrolysis was obtained with 0.23 mM Mg2+. Ca2+ had no effect on the basal phosphatase activity, could not substitute Mg2+ as an activator and in contrast inhibited the p-NPP hydrolysis stimulated by Mg2+. The dependence on p-NPP concentration showed a normal Michaelis–Menten kinetics for this phosphatase activity with values of Vmax of 8.94 ± 0.36 nmol p-NP × h−1 × 10−7 cells and apparent Km of 1.04 ± 0.16 mM p-NPP. Mg2+-dependent ecto-phosphatase activity was stimulated by the alkaline pH range. Experiments using inhibitors, such as, sodium fluoride, sodium orthovanadate and ammonium molybdate, inhibited the Mg2+-dependent ecto-phosphatase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibited reversibly in 50% this activity. Okadaic acid and microcystin-LR, specific phosphoserine/threonine phosphatase inhibitors, inhibited significantly the Mg2+-dependent ecto-phosphatase activity. In addition, this phosphatase activity was able to recognize as substrates only o-phosphoserine and o-phosphothreonine, while o-phosphotyrosine was not a good substrate for this phosphatase. Epimastigote forms of T. rangeli exhibit a typical growth curve, achieving the stationary phase around fifth or sixth day and the Mg2+-dependent ecto-phosphatase activity decreased around 10-fold with the cell growth progression. Cells maintained at Pi-deprived medium (2 mM Pi) present Mg2+-dependent ecto-phosphatase activity approximately threefold higher than that maintained at Pi-supplemented medium (50 mM Pi).
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ecto phosphatase activity on the external surface of rhodnius prolixus salivary glands modulation by carbohydrates and trypanosoma rangeli
Acta Tropica, 2008Co-Authors: S A O Gomes, Andre Luiz Fonseca De Souza, Tina Kiffermoreira, Claudia F Dick, Andre Luiz Araujo Dos Santos, Jose Roberto MeyerfernandesAbstract:Abstract The salivary glands of insect's vectors are target organs to study the vectors–pathogens interactions. Rhodnius prolixus an important vector of Trypanosoma cruzi can also transmit Trypanosoma rangeli by bite. In the present study we have investigated ecto-phosphatase activity on the surface of R. prolixus salivary glands. Ecto-phosphatases are able to hydrolyze phosphorylated substrates in the extracellular medium. We characterized these ecto-enzyme activities on the salivary glands external surface and employed it to investigate R. prolixus – T. rangeli interaction. Salivary glands present a low level of hydrolytic activity (4.30 ± 0.35 nmol p -nitrophenol ( p -NP) × h −1 × gland pair −1 ). The salivary glands ecto-phosphatase activity was not affected by pH variation; and it was insensitive to alkaline inhibitor levamisole and inhibited approximately 50% by inorganic phosphate (Pi). MgCl 2 , CaCl 2 and SrCl 2 enhanced significantly the ecto-phosphatase activity detected on the surface of salivary glands. The ecto-phosphatase from salivary glands surface efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate release when phospho-tyrosine is used as a substrate. This ecto-phosphatase activity was inhibited by carbohydrates as d -galactose and d -mannose. Living short Epimastigotes of T. rangeli inhibited salivary glands ecto-phosphatase activity at 75%, while boiled parasites did not. Living long Epimastigote forms induced a lower, but significant inhibitory effect on the salivary glands phosphatase activity. Interestingly, boiled long Epimastigote forms did not loose the ability to modulate salivary glands phosphatase activity. Taken together, these data suggest a possible role for ecto-phosphatase on the R. prolixus salivary glands– T. rangeli interaction.
Maurilio J Soares - One of the best experts on this subject based on the ideXlab platform.
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transferrin uptake may occur through detergent resistant membrane domains at the cytopharynx of trypanosoma cruzi Epimastigote forms
Memorias Do Instituto Oswaldo Cruz, 2007Co-Authors: Jose R Correa, Georgia C. Atella, Camila Vargas, Maurilio J SoaresAbstract:Uptake of transferrin by Epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi Epimastigotes, being the main route for transferrin uptake.
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Penetration of the salivary glands of Rhodnius domesticus Neiva & Pinto, 1923 (Hemiptera: Reduviidae) by Trypanosoma rangeli Tejera, 1920 (Protozoa: Kinetoplastida)
Parasitology Research, 2005Co-Authors: Rosane M. S. Meirelles, Maurilio J Soares, Andrea Henriques-pons, Mario SteindelAbstract:Penetration of the heteroxenous protozoan Trypanosoma rangeli into the salivary glands of its invertebrate host Rhodnius domesticus has been investigated here using different approaches. Electron microscopy showed that Epimastigotes coming from the insect hemocoel cross the basal lamina that surrounds the salivary glands and penetrate through the gland cells cytoplasm. After reaching the gland lumen, Epimastigote forms remain adhered to the gland cell microvilli by their flagella, while metacyclic trypomastigotes are found swimming free in the saliva. Analysis by flow cytometry, western blotting and hemolytic activity allowed to demonstrate the presence in T. rangeli of a hemolytic molecule with antigenic cross-reactivity with murine perforin, which could be used by the parasites to reach the salivary gland lumen. This molecule, which we named as rangelysin, has 120 kDa molecular weight, is able to induce hemolysis only in acidic pH, and is produced by both trypomastigote and Epimastigote forms.
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penetration of the salivary glands of rhodnius domesticus neiva pinto 1923 hemiptera reduviidae by trypanosoma rangeli tejera 1920 protozoa kinetoplastida
Parasitology Research, 2005Co-Authors: Rosane M. S. Meirelles, Maurilio J Soares, Andrea Henriquespons, Mario SteindelAbstract:Penetration of the heteroxenous protozoan Trypanosoma rangeli into the salivary glands of its invertebrate host Rhodnius domesticus has been investigated here using different approaches. Electron microscopy showed that Epimastigotes coming from the insect hemocoel cross the basal lamina that surrounds the salivary glands and penetrate through the gland cells cytoplasm. After reaching the gland lumen, Epimastigote forms remain adhered to the gland cell microvilli by their flagella, while metacyclic trypomastigotes are found swimming free in the saliva. Analysis by flow cytometry, western blotting and hemolytic activity allowed to demonstrate the presence in T. rangeli of a hemolytic molecule with antigenic cross-reactivity with murine perforin, which could be used by the parasites to reach the salivary gland lumen. This molecule, which we named as rangelysin, has 120 kDa molecular weight, is able to induce hemolysis only in acidic pH, and is produced by both trypomastigote and Epimastigote forms.
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reservosome an endocytic compartment in Epimastigote forms of the protozoan trypanosoma cruzi kinetoplastida trypanosomatidae correlation between endocytosis of nutrients and cell differentiation
Parasitology, 2004Co-Authors: Regina Celia Bressan Queiroz De Figueiredo, Daniela Santoro Rosa, Yara M Gomes, M Nakasawa, Maurilio J SoaresAbstract:Reservosomes are large membrane-bound organelles found at the posterior end of Epimastigote forms of Trypanosoma cruzi , but absent in amastigotes and trypomastigotes. We have transferred bloodstream trypomastigotes to LIT medium supplemented with gold-labelled transferrin in order to analyse, at the ultrastructural level, the occurrence of reservosomes and endocytosis during the trypomastigote to Epimastigote differentiation. After 24 h, the trypomastigotes differentiated into amastigotes, which adhered to each other forming large clusters. Electron-dense vesicles were detected close to the Golgi complex in cells with intermediary characteristics between amastigotes and Epimastigotes, but typical reservosomes at the posterior cell tip were still absent. Transferrin–gold complexes were observed only bound to the surface of clustered cells. After 72 h, Epimastigotes were observed being released from the clusters and free-swimming Epimastigotes appeared, containing electron-dense vesicles at their posterior region. Typical reservosomes, labelled with transferrin–gold, were observed only in free-swimming Epimastigotes. When fully differentiated Epimastigotes were incubated with transferrin–gold complexes and then processed for the immunocytochemical detection of cysteine proteinase, all reservosomes were positive for the enzyme, but co-localization of both markers did not occur in all organelles. Our data demonstrate that in T. cruzi Epimastigotes endocytosis is strongly related to reservosome biogenesis during the trypomastigote to Epimastigote differentiation process.
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DIFFERENTIATION OF TRYPANOSOMA CRUZI EpimastigoteS: METACYCLOGENESIS AND ADHESION TO SUBSTRATE ARE TRIGGERED BY NUTRITIONAL STRESS
Journal of Parasitology, 2000Co-Authors: Regina Celia Bressan Queiroz De Figueiredo, Daniela Santoro Rosa, Maurilio J SoaresAbstract:Differentiation of Trypanosoma cruzi Epimastigotes to metacyclic trypomastigotes occurs in the insect rectum, after adhesion of the Epimastigotes to the intestinal wall. We investigated the effect of the nutritional stress on the metacyclogenesis process in vitro by incubating Epimastigotes in the chemically defined TAU3AAG medium supplemented with different nutrients. Addition of fetal bovine serum induced Epimastigote growth but inhibited metacyclogenesis. In this medium, few parasites attached to the substrate. Ultrastructural analysis demonstrated reservosomes at the posterior end of the Epimastigotes. Incubation of the cells in TAU3AAG medium containing gold-labeled transferrin resulted in high endocytosis of the marker by both adhered and free-swimming Epimastigotes. No intracellular gold particles could be detected in trypomastigotes. Addition of transferrin–gold complexes to adhered Epimastigotes cultivated for 4 days in TAU3AAG medium resulted in decrease of both metacyclogenesis and adhesion to ...
Samuel Goldenberg - One of the best experts on this subject based on the ideXlab platform.
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Further characterization ofTrypanosoma cruzi GP57/51 as the major antigen expressed by differentiating Epimastigotes
Parasitology Research, 1991Co-Authors: Myrna C. Bonaldo, Julio Scharfstein, Ana Cristina M. Murta, Samuel GoldenbergAbstract:Study of Trypanosoma cruzi metacyclogenesis under chemically defined conditions showed that the expression of a group of acidic polypeptides with a molecular weight of 45–50 kDa is markedly increased on adhesion of Epimastigotes to the culture vessels. Immunochemical analysis revealed that these developmentally regulated polypeptides are structurally related to, and possibly homologous with, a major T. cruzi antigen, namely, GP57/51, a glycoprotein that has recently been characterized as a cysteine proteinase. The differentiation of Epimastigotes into metacyclic trypomastigotes is accompanied by a 2- to 5-fold reduction in the concentration of GP57/51 antigen as determined by radioimmunoassays using monoclonal antibodies. Two-dimensional polyacrylamide gel electrophoretic analysis of metabolically labelled parasites indicated that this antigen is synthesized as a precursor with a molecular weight of 60 kDa, which is then processed to a level of 45–50 kDa via the formation of at least one intermediate processing product. The observation that expression of GP57/51-related products increases during Epimastigote differentiation suggests an improtant role for this proteinase during the life cycle of T. cruzi .
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Further characterization of Trypanosoma cruzi GP57/51 as the major antigen expressed by differentiating Epimastigotes.
Parasitology Research, 1991Co-Authors: Myrna C. Bonaldo, Julio Scharfstein, Ana Cristina M. Murta, Samuel GoldenbergAbstract:Study ofTrypanosoma cruzi metacyclogenesis under chemically defined conditions showed that the expression of a group of acidic polypeptides with a molecular weight of 45–50 kDa is markedly increased on adhesion of Epimastigotes to the culture vessels. Immunochemical analysis revealed that these developmentally regulated polypeptides are structurally related to, and possibly homologous with, a majorT. cruzi antigen, namely, GP57/51, a glycoprotein that has recently been characterized as a cysteine proteinase. The differentiation of Epimastigotes into metacyclic trypomastigotes is accompanied by a 2- to 5-fold reduction in the concentration of GP57/51 antigen as determined by radioimmunoassays using monoclonal antibodies. Two-dimensional polyacrylamide gel electrophoretic analysis of metabolically labelled parasites indicated that this antigen is synthesized as a precursor with a molecular weight of 60 kDa, which is then processed to a level of 45–50 kDa via the formation of at least one intermediate processing product. The observation that expression of GP57/51-related products increases during Epimastigote differentiation suggests an improtant role for this proteinase during the life cycle ofT. cruzi.