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Ching C. Wang - One of the best experts on this subject based on the ideXlab platform.
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steady state kinetics of the hypoxanthine guanine xanthine phosphoribosyltransferase from Tritrichomonas Foetus the role of threonine 47
Biochemistry, 1998Co-Authors: Narsimha R Munagala, Marian S Chin, Ching C. WangAbstract:Tritrichomonas Foetus, an anaerobic flagellated protozoan, causes urogenital trichomoniasis in cattle. Hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRTase), an essential enzyme in T. Foetus required for salvaging exogenous purine bases, has been regarded as a promising target for anti-tritrichomonial chemotherapy. The steady-state kinetic analyses of synthesis and pyrophosphorolysis of IMP, GMP, and XMP and product inhibition studies have been used to elucidate the reaction mechanisms. Double-reciprocal plots of initial velocities versus the varying concentrations of one substrate at a fixed concentration of the other show intersecting lines indicating a sequential mechanism for both the forward and the reverse reactions. In terms of the kcat/Km ratios, hypoxanthine is the most effective substrate whereas guanine and xanthine are converted equally well into their corresponding nucleotides. The minimum kinetic model from the data in product inhibition studies is an ordered bi−bi mechanism, ...
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crystal structure of the hypoxanthine guanine xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas Foetus
Biochemistry, 1996Co-Authors: John R Somoza, Ching C. Wang, Marian S Chin, Pamela J Focia, Robert J FletterickAbstract:The crystal structure of the hypoxanthine−guanine−xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas Foetus has been determined and refined against X-ray data to 1.9 A resolution. T. Foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short “hinge” region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5° relative to the other. T. Foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. Foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there a...
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crystal structure of the hypoxanthine guanine xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas Foetus
Biochemistry, 1996Co-Authors: John R Somoza, Ching C. Wang, Marian S Chin, Pamela J Focia, Robert J FletterickAbstract:The crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas Foetus has been determined and refined against X-ray data to 1.9 A resolution. T. Foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short "hinge" region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5 degrees relative to the other. T. Foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. Foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there are substantial differences between the active sites of these two homologs. These differences suggest that it will be possible to find compounds that selectively inhibit the parasite enzyme.
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Identification of the IMP binding site in the IMP dehydrogenase from Tritrichomonas Foetus.
Biochemistry, 1995Co-Authors: Jorge A. Huete-pérez, Frank G. Whitby, Ching C. WangAbstract:The IMP dehydrogenase from Tritrichomonas Foetus has been identified as a potential target for antitritrichomonial chemotherapy. The gene encoding this enzyme was expressed in transformed Escherichia coli, and the recombinant protein was purified to homogeneity with an average yield of 3 mg of protein per liter of bacterial culture. Kinetic characterizations verified that the recombinant enzyme is in the authentic native state. 6-Cl-IMP, an irreversible inhibitor of the enzyme, was found to protect cysteine residue 319 of the enzyme against carboxymethylation by iodoacetamide. Radiolabeled IMP was covalently bound to the enzyme during the enzyme-catalyzed reaction via the formation of a specific adduct with cysteine residue 319. It is thus postulated that the conversion of IMP to XMP catalyzed by the IMP dehydrogenase from T. Foetus is mediated by a nucleophilic attack of cysteine-319 in the enzyme protein to IMP at, most likely, its 2-position to facilitate a hydride transfer to NAD, resulting in the formation of a covalent intermediate between substrate and enzyme.
Marlene Benchimol - One of the best experts on this subject based on the ideXlab platform.
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with Trichomonas vaginalis and Tritrichomonas Foetus
2016Co-Authors: Ricardo Chaves Vilela, Marlene BenchimolAbstract:Trichomonas vaginalis and Tritrichomonas Foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. Foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine ovi-duct epithelial cells were exposed to either T. vaginalis or T. Foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. Foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. Foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells. Key words: T. vaginalis- T. Foetus- cytokines- host-cells- IL-10 Human and bovine trichomoniases are sexually transmitted diseases caused by the parasites Trichomo-nas vaginalis and Tritrichomonas Foetus, respectively. These parasites are capable of causing severe vaginal
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cytotoxic effects exerted by Tritrichomonas Foetus pseudocysts
Protist, 2012Co-Authors: Antonio Pereiraneves, Ligia Ferreira Nascimento, Marlene BenchimolAbstract:The protozoan parasite Tritrichomonas Foetus displays a pear-shaped form and a pseudocyst stage. However, little is known about the biology of the pseudocyst. The aim of this work was to assess whether pseudocysts exert cytotoxic effects during their interaction with MDCK cells (an epithelial kidney canine cell line) and compare their behavior to that of the pear-shaped parasites. Pseudocysts and pear-shaped parasites from both cultured and freshly isolated T. Foetus were used. Electron microscopy revealed that the epithelial cells exhibited more signs of injury, such as depletion of microvilli, retraction from neighboring cells and swollen mitochondria with loss of electron density in the matrix, when the pseudocysts were used in interaction experiments. In addition, during the co-incubation with MDCK cells, pseudocysts exhibited a more intense amoeboid transformation than that found in pear-shaped parasites. The MTT viability assay demonstrated that the pseudocysts were more cytotoxic when in contact with host cells as compared to the flagellated pear-shaped parasites. The JC-1 viability assay revealed that pseudocysts induced a higher loss of mitochondrial membrane potential compared to pear-shaped parasites. Pseudocysts undergoing a budding process were observed after 2.5 h of co-incubation with MDCK cells. Our results suggest that the T. Foetus pseudocyst might be a more aggressive form.
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cytopathic effects of Tritrichomonas Foetus on bovine oviduct cells
Veterinary Parasitology, 2009Co-Authors: Victor Midlej, Ricardo Vilela, A B Dias, Marlene BenchimolAbstract:Tritrichomonas Foetus is an extracellular parasite of the reproductive tract in cattle. To investigate the cytopathic effects of T. Foetus in deeper parts of the reproductive tract, a bovine primary oviduct epithelial cell system (BOECs) was developed. Reproductive tracts were obtained from cows and the effect of co-incubating T. Foetus with BOECs was analyzed by scanning electron, transmission electron and fluorescence microscopy. Viability tests were performed using colorimetric methods, TUNEL (Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling), fluorescein diacetate, propidium iodide, JC-1 and annexin-V. The results demonstrate that: (1) the in vitro oviduct epithelium is useful for interaction experiments with T. Foetus; (2) T. Foetus adheres to the BOECs as single separate cells, and later on the cells aggregate as large clusters; (3) the posterior region of the cell initiates the process of adhesion and forms filopodia and digitopodia; (4) T. Foetus severely damages BOECs leaving imprints in the epithelial cells, wide intercellular spaces, and large lesions in the epithelium; and (5) T. Foetus provokes bovine oviduct cell death by apoptosis and secondary necrosis. Our observations indicate the possibility that T. Foetus can move through the reproductive tract to the oviduct and that infertility in cows can be mediated by an attack on the oviduct cells by T. Foetus.
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Tritrichomonas Foetus budding from multinucleated pseudocysts
Protist, 2009Co-Authors: Antonio Pereiraneves, Marlene BenchimolAbstract:Tritrichomonas Foetus is a flagellated protozoan parasite that causes trichomoniasis, a major sexually transmitted disease in cattle. T. Foetus presents a simple life cycle, exhibiting only the trophozoitic form. However, under unfavorable growth conditions, the trophozoites, which are polar and flagellated, can round up and internalize their flagella forming pseudocysts. In this form no cyst wall surrounds the cell and it also displays a distinct mitosis when compared with the trophozoite form. In pseudocyst mitosis, the cell proceeds with duplication of cytoskeletal and mastigont structures; nuclear division occurs but without the corresponding cytoplasm division. Thus, giant multinucleated cells which present many mastigont structures are formed (approximately 62% of the population). These polymastigont/multinucleated cells are maintained when the cells are under stress conditions. When environmental conditions become favorable, the flagella are externalized and new flagellated trophozoites one by one, gradually bud from the multinucleated cell. Thus, in order to better understand the pseudocyst mitosis, the polymastigont formation and the generation of new cells by this budding process, video microscopy and other complementary techniques, such as immunofluorescence and transmission electron microscopy were used.
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the effect of drugs on cell structure of Tritrichomonas Foetus
Parasitology Research, 2004Co-Authors: Rodrigo Madeiro Da Costa, Marlene BenchimolAbstract:The effects of the microtubule affecting drugs taxol, nocodazole and colchicine on the cell cycle and ultrastructure of Tritrichomonas Foetus, a protist parasite of cattle, were studied. Alterations in the cytoskeleton, motility and organellar ultrastructure were followed using anti-tubulin antibodies and fluorescence microscopy, scanning- and transmission-electron microscopy. Flow cytometry was also used to analyze the effect of the drugs on the cell cycle. T. Foetus was treated with 10 μM taxol, 15 μM nocodazole or 1.5 mM colchicine for 12 h. The first effect observed was pseudocyst formation and alterations in cell motility. The cell cycle was affected and the cells have blocked cytokinesis, but not karyokinesis. The behavior of Golgi, hydrogenosomes and vacuoles was analyzed. The following effects were seen following drug treatments: (1) cell motility was altered and flagella internalized; (2) microtubules of the pelta-axostyle complex were not depolymerized and the axostyle assumed a curved form; (3) hydrogenosomes were of abnormal size and shape; (4) cells became multinucleate; (5) the division process was blocked in cytokinesis; (6) autophagic vacuoles containing a large amount of microtubules were seen; (7) axoneme organization was altered; (8) zoids were formed; (9) signs of cell death, such as membrane blebbing, were observed.
Robert J Fletterick - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the hypoxanthine guanine xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas Foetus
Biochemistry, 1996Co-Authors: John R Somoza, Ching C. Wang, Marian S Chin, Pamela J Focia, Robert J FletterickAbstract:The crystal structure of the hypoxanthine−guanine−xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas Foetus has been determined and refined against X-ray data to 1.9 A resolution. T. Foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short “hinge” region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5° relative to the other. T. Foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. Foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there a...
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crystal structure of the hypoxanthine guanine xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas Foetus
Biochemistry, 1996Co-Authors: John R Somoza, Ching C. Wang, Marian S Chin, Pamela J Focia, Robert J FletterickAbstract:The crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas Foetus has been determined and refined against X-ray data to 1.9 A resolution. T. Foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short "hinge" region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5 degrees relative to the other. T. Foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. Foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there are substantial differences between the active sites of these two homologs. These differences suggest that it will be possible to find compounds that selectively inhibit the parasite enzyme.
Marian S Chin - One of the best experts on this subject based on the ideXlab platform.
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steady state kinetics of the hypoxanthine guanine xanthine phosphoribosyltransferase from Tritrichomonas Foetus the role of threonine 47
Biochemistry, 1998Co-Authors: Narsimha R Munagala, Marian S Chin, Ching C. WangAbstract:Tritrichomonas Foetus, an anaerobic flagellated protozoan, causes urogenital trichomoniasis in cattle. Hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRTase), an essential enzyme in T. Foetus required for salvaging exogenous purine bases, has been regarded as a promising target for anti-tritrichomonial chemotherapy. The steady-state kinetic analyses of synthesis and pyrophosphorolysis of IMP, GMP, and XMP and product inhibition studies have been used to elucidate the reaction mechanisms. Double-reciprocal plots of initial velocities versus the varying concentrations of one substrate at a fixed concentration of the other show intersecting lines indicating a sequential mechanism for both the forward and the reverse reactions. In terms of the kcat/Km ratios, hypoxanthine is the most effective substrate whereas guanine and xanthine are converted equally well into their corresponding nucleotides. The minimum kinetic model from the data in product inhibition studies is an ordered bi−bi mechanism, ...
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crystal structure of the hypoxanthine guanine xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas Foetus
Biochemistry, 1996Co-Authors: John R Somoza, Ching C. Wang, Marian S Chin, Pamela J Focia, Robert J FletterickAbstract:The crystal structure of the hypoxanthine−guanine−xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas Foetus has been determined and refined against X-ray data to 1.9 A resolution. T. Foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short “hinge” region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5° relative to the other. T. Foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. Foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there a...
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crystal structure of the hypoxanthine guanine xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas Foetus
Biochemistry, 1996Co-Authors: John R Somoza, Ching C. Wang, Marian S Chin, Pamela J Focia, Robert J FletterickAbstract:The crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas Foetus has been determined and refined against X-ray data to 1.9 A resolution. T. Foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short "hinge" region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5 degrees relative to the other. T. Foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. Foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there are substantial differences between the active sites of these two homologs. These differences suggest that it will be possible to find compounds that selectively inhibit the parasite enzyme.
John R Somoza - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the hypoxanthine guanine xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas Foetus
Biochemistry, 1996Co-Authors: John R Somoza, Ching C. Wang, Marian S Chin, Pamela J Focia, Robert J FletterickAbstract:The crystal structure of the hypoxanthine−guanine−xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas Foetus has been determined and refined against X-ray data to 1.9 A resolution. T. Foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short “hinge” region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5° relative to the other. T. Foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. Foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there a...
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crystal structure of the hypoxanthine guanine xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas Foetus
Biochemistry, 1996Co-Authors: John R Somoza, Ching C. Wang, Marian S Chin, Pamela J Focia, Robert J FletterickAbstract:The crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas Foetus has been determined and refined against X-ray data to 1.9 A resolution. T. Foetus HGXPRTase crystallizes as an asymmetric dimer, with GMP bound to only one of the two molecules that form the asymmetric unit. Each molecule of HGXPRTase is formed by two lobes joined by a short "hinge" region, and the GMP binds in a cavity between the two lobes. A comparison of the two molecules in the asymmetric unit shows that the hinge region is flexible and that ligand binding affects the relative positions of the two lobes. The binding of GMP brings the two lobes closer together, rotating one lobe by about 5 degrees relative to the other. T. Foetus appears to depend on HGXPRTase for its supply of GMP, making this enzyme a target for antiparasite drug design. A comparison of the structures of T. Foetus HGXPRTase and human HGPRTase reveals that, while these enzymes retain a similar polypeptide fold, there are substantial differences between the active sites of these two homologs. These differences suggest that it will be possible to find compounds that selectively inhibit the parasite enzyme.
