The Experts below are selected from a list of 69 Experts worldwide ranked by ideXlab platform
Niels Haugaard - One of the best experts on this subject based on the ideXlab platform.
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Adverse effects of hyperbaric oxygen on [^3H]Uridine incorporation and Uridine Kinase activity in B104 rat neuroblastoma cells
Molecular and Cellular Biochemistry, 1990Co-Authors: Gerard J. Gendimenico, Niels HaugaardAbstract:The effects of hyperbaric oxygen on uracil nucleotide metabolism in B104 rat neuroblastoma cells were investigated. Cells exposed to 10 atm O_2 for 4 h incorporated markedly less [^3H]Uridine into the acid-soluble fraction and RNA compared to cells kept in ambient air. The acid-soluble fraction of the oxygen-treated cells contained less total [^3H]Uridine phosphates ([^3H]UMP + [^3H]UDP + [^3H]UTP) than air-treated cells. Uridine Kinase activity, assayed in cytosolic extracts from cells exposed to 10 atm O_2 for 4 h, was decreased by 46% compared to the air controls. The reduced enzyme activity which appears to account for the depressed [^3H]Uridine incorporation, may contribute to the lethal effects of oxygen in these cells.
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Stimulation of the phosphorylation of Uridine in skeletal muscle by insulin and vanadate
Molecular and Cellular Biochemistry, 1990Co-Authors: Niels Haugaard, Aliza Torbati, Thomas Smithgall, Gary WildeyAbstract:The action of insulin and sodium vanadate on the phosphorylation of Uridine by skeletal muscle was studied in vitro . Insulin significantly increased the incorporation of ^3H-Uridine into uracil nucleotides by pieces of rat diaphragm incubated for 15 min in a phosphate-buffered medium. This action of the hormone was exceptionally consistent when MgATP was added to the incubation medium. In experiments in which pieces of psoas muscle were incubated in TRIS buffer in the presence and absence of insulin, the hormone caused a significant activation of Uridine Kinase measured in cytosolic extracts of the incubated tissue. In experiments with rat diaphragm similar to those with insulin, the vanadate ion caused a significant increase in phosphorylation of Uridine. The results of these experiments provide preliminary support for the proposal that uracil nucleotide metabolism is regulated by insulin and that insulin activates Uridine Kinase, the limiting enzyme in the synthesis of uracil nucleotides from Uridine by the salvage pathway.
Laszlo Szekely - One of the best experts on this subject based on the ideXlab platform.
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epstein barr virus encoded nuclear protein ebna 3 binds a novel human Uridine Kinase uracil phosphoribosyltransferase
BMC Cell Biology, 2002Co-Authors: Elena Kashuba, V I Kashuba, Tatjana Sandalova, George Klein, Laszlo SzekelyAbstract:Epstein-Barr virus (EBV) infects resting B-lymphocytes and transforms them into immortal proliferating lymphoblastoid cell lines (LCLs) in vitro. The transformed immunoblasts may grow up as immunoblastic lymphomas in immuno-suppressed hosts. In order to identify cellular protein targets that may be involved in Epstein-Barr virus mediated B-cell transformation, human LCL cDNA library was screened with one of the transformation associated nuclear antigens, EBNA-3 (also called EBNA-3A), using the yeast two-hybrid system. A clone encoding a fragment of a novel human protein was isolated (clone 538). The interaction was confirmed using in vitro binding assays. A full-length cDNA clone (F538) was isolated. Sequence alignment with known proteins and 3D structure predictions suggest that F538 is a novel human Uridine Kinase/uracil phosphoribosyltransferase. The GFP-F538 fluorescent fusion protein showed a preferentially cytoplasmic distribution but translocated to the nucleus upon co-expression of EBNA-3. A naturally occurring splice variant of F538, that lacks the C-terminal uracil phosphoribosyltransferase part but maintain Uridine Kinase domain, did not translocate to the nucleus in the presence of EBNA3. Antibody that was raised against the bacterially produced GST-538 protein showed cytoplasmic staining in EBV negative Burkitt lymphomas but gave a predominantly nuclear staining in EBV positive LCL-s and stable transfected cells expressing EBNA-3. We suggest that EBNA-3 by direct protein-potein interaction induces the nuclear accumulation of a novel enzyme, that is part of the ribonucleotide salvage pathway. Increased intranuclear levels of UK/UPRT may contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation.
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Epstein-Barr virus encoded nuclear protein EBNA-3 binds a novel human Uridine Kinase/uracil phosphoribosyltransferase.
BMC Cell Biology, 2002Co-Authors: Elena Kashuba, V I Kashuba, Tatjana Sandalova, George Klein, Laszlo SzekelyAbstract:Epstein-Barr virus (EBV) infects resting B-lymphocytes and transforms them into immortal proliferating lymphoblastoid cell lines (LCLs) in vitro. The transformed immunoblasts may grow up as immunoblastic lymphomas in immuno-suppressed hosts. In order to identify cellular protein targets that may be involved in Epstein-Barr virus mediated B-cell transformation, human LCL cDNA library was screened with one of the transformation associated nuclear antigens, EBNA-3 (also called EBNA-3A), using the yeast two-hybrid system. A clone encoding a fragment of a novel human protein was isolated (clone 538). The interaction was confirmed using in vitro binding assays. A full-length cDNA clone (F538) was isolated. Sequence alignment with known proteins and 3D structure predictions suggest that F538 is a novel human Uridine Kinase/uracil phosphoribosyltransferase. The GFP-F538 fluorescent fusion protein showed a preferentially cytoplasmic distribution but translocated to the nucleus upon co-expression of EBNA-3. A naturally occurring splice variant of F538, that lacks the C-terminal uracil phosphoribosyltransferase part but maintain Uridine Kinase domain, did not translocate to the nucleus in the presence of EBNA3. Antibody that was raised against the bacterially produced GST-538 protein showed cytoplasmic staining in EBV negative Burkitt lymphomas but gave a predominantly nuclear staining in EBV positive LCL-s and stable transfected cells expressing EBNA-3. We suggest that EBNA-3 by direct protein-potein interaction induces the nuclear accumulation of a novel enzyme, that is part of the ribonucleotide salvage pathway. Increased intranuclear levels of UK/UPRT may contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation.
Thomas W Traut - One of the best experts on this subject based on the ideXlab platform.
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nifedipine and nimodipine competitively inhibit Uridine Kinase and orotidine phosphate decarboxylase theoretical relevance to poor outcome in stroke
Neurorehabilitation and Neural Repair, 2000Co-Authors: Thomas Najarian, Thomas W TrautAbstract:: Nifedipine and nimodipine, dihydropyridine calcium channel blockers, are commonly used as antihypertensive and antianginal agents in patients at risk for stroke. At least one stroke trial suggests that patients receiving calcium channel blockers at the time of an acute stroke have worse outcomes than those receiving other or no antihypertensive medications. We hypothesize that the poor outcome may not be related to blood pressure changes but instead may be mediated by competitive inhibition of important enzymes of pyrimidine synthesis whose products are needed to repair nerve cell membranes after an acute stroke. Both drugs acted as competitive inhibitors of the only enzymes that are known to synthesize the nucleotide Uridine-5'-phosphate: Uridine Kinase and orotidine-5'-phosphate decarboxylase. Nifedipine produced Ki values of 28 microM for Uridine Kinase and 105 microM for orotidine-5'-phosphate decarboxylase. Nimodipine produced Ki values of 20 microM for Uridine Kinase and 18 microM for orotidine-5'-phosphate decarboxylase. For Uridine Kinase, these inhibitors bound more tightly than the physiologic substrates Uridine or cytidine. For the decarboxylase, the inhibitors bound less tightly than the normal physiologic substrate orotidine-5'-phosphate. Additional experiments are needed to determine whether the concentrations of nifedipine or nimodipine, and of cytidine, Uridine, and orotidine-5'-phosphate in human brain, are such that this inhibition would affect stroke outcome.
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Uridine Kinase altered enzyme with decreased affinities for Uridine and ctp
Archives of Biochemistry and Biophysics, 1998Co-Authors: Patricia A Ropp, Thomas W TrautAbstract:Abstract Uridine Kinase is the rate-limiting enzyme in the salvage pathway for Uridine or cytidine of mammalian cells. Alignment of the Uridine Kinase sequence with other nucleoside and nucleotide Kinases supports a common ancestor for all of these. Three polypeptide segments for the ATP site and three polypeptide segments for the acceptor nucleoside site have been identified. We report here the characterization of an altered form of the enzyme with a single amino acid change, Q146R, within or near the Uridine-binding site. This single amino acid change leads to a 160-fold increase in K m for Uridine ( K m = 6.5 mM) and a decrease in k cat by more than 99%. This variant has normal affinity for ATP ( K m = 130 μM), but shows substrate inhibition at ATP concentrations >3 mM. Mouse Uridine Kinase is normally an active tetramer that will dissociate to inactive monomers in response to CTP. In contrast, the altered protein is monomeric, but will associate to dimers and then to tetramers with increasing ATP. The Q146R enzyme has a 100-fold loss in affinity for the allosteric inhibitor CTP; this supports a model for CTP inhibition being caused by CTP binding backward at the catalytic site, as a bisubstrate analog.
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cloning and expression of a cdna encoding Uridine Kinase from mouse brain
Archives of Biochemistry and Biophysics, 1996Co-Authors: Patricia A Ropp, Thomas W TrautAbstract:Abstract Uridine Kinase is the rate-limiting enzyme in the pyrimidine salvage pathway of all mammalian cells. A cDNA for Uridine Kinase from mouse brain has been isolated, sequenced, and characterized. This is the first report of a complete nucleotide sequence for mammalian Uridine Kinase. The isolated cDNA is only 95% complete, missing the first 17 codons. The correct 5′-terminus sequence was obtained from high-stringency screening of a mouse liver genomic DNA library. The translated cDNA sequence encodes a protein of 277 amino acids (Mr31,068). A truncated form of the cDNA was expressed inEscherichia coli.The expressed protein displayed Uridine Kinase activity and readily formed a tetramer, the most active form of the wild-type enzyme. Analysis of the amino acid sequence identified the three ATP-binding site consensus motifs. The predicted secondary structure for Uridine Kinase and the sequence comparison with three Kinases having known crystal structures are consistent with Uridine Kinase having an α/β core structure of the nucleotide-binding fold found in many Kinases. We have also isolated and cloned a nonfunctional, processed pseudogene from mouse genomic DNA. This pseudogene sequence is 94% identical with the coding DNA.
Elena Kashuba - One of the best experts on this subject based on the ideXlab platform.
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epstein barr virus encoded nuclear protein ebna 3 binds a novel human Uridine Kinase uracil phosphoribosyltransferase
BMC Cell Biology, 2002Co-Authors: Elena Kashuba, V I Kashuba, Tatjana Sandalova, George Klein, Laszlo SzekelyAbstract:Epstein-Barr virus (EBV) infects resting B-lymphocytes and transforms them into immortal proliferating lymphoblastoid cell lines (LCLs) in vitro. The transformed immunoblasts may grow up as immunoblastic lymphomas in immuno-suppressed hosts. In order to identify cellular protein targets that may be involved in Epstein-Barr virus mediated B-cell transformation, human LCL cDNA library was screened with one of the transformation associated nuclear antigens, EBNA-3 (also called EBNA-3A), using the yeast two-hybrid system. A clone encoding a fragment of a novel human protein was isolated (clone 538). The interaction was confirmed using in vitro binding assays. A full-length cDNA clone (F538) was isolated. Sequence alignment with known proteins and 3D structure predictions suggest that F538 is a novel human Uridine Kinase/uracil phosphoribosyltransferase. The GFP-F538 fluorescent fusion protein showed a preferentially cytoplasmic distribution but translocated to the nucleus upon co-expression of EBNA-3. A naturally occurring splice variant of F538, that lacks the C-terminal uracil phosphoribosyltransferase part but maintain Uridine Kinase domain, did not translocate to the nucleus in the presence of EBNA3. Antibody that was raised against the bacterially produced GST-538 protein showed cytoplasmic staining in EBV negative Burkitt lymphomas but gave a predominantly nuclear staining in EBV positive LCL-s and stable transfected cells expressing EBNA-3. We suggest that EBNA-3 by direct protein-potein interaction induces the nuclear accumulation of a novel enzyme, that is part of the ribonucleotide salvage pathway. Increased intranuclear levels of UK/UPRT may contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation.
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Epstein-Barr virus encoded nuclear protein EBNA-3 binds a novel human Uridine Kinase/uracil phosphoribosyltransferase.
BMC Cell Biology, 2002Co-Authors: Elena Kashuba, V I Kashuba, Tatjana Sandalova, George Klein, Laszlo SzekelyAbstract:Epstein-Barr virus (EBV) infects resting B-lymphocytes and transforms them into immortal proliferating lymphoblastoid cell lines (LCLs) in vitro. The transformed immunoblasts may grow up as immunoblastic lymphomas in immuno-suppressed hosts. In order to identify cellular protein targets that may be involved in Epstein-Barr virus mediated B-cell transformation, human LCL cDNA library was screened with one of the transformation associated nuclear antigens, EBNA-3 (also called EBNA-3A), using the yeast two-hybrid system. A clone encoding a fragment of a novel human protein was isolated (clone 538). The interaction was confirmed using in vitro binding assays. A full-length cDNA clone (F538) was isolated. Sequence alignment with known proteins and 3D structure predictions suggest that F538 is a novel human Uridine Kinase/uracil phosphoribosyltransferase. The GFP-F538 fluorescent fusion protein showed a preferentially cytoplasmic distribution but translocated to the nucleus upon co-expression of EBNA-3. A naturally occurring splice variant of F538, that lacks the C-terminal uracil phosphoribosyltransferase part but maintain Uridine Kinase domain, did not translocate to the nucleus in the presence of EBNA3. Antibody that was raised against the bacterially produced GST-538 protein showed cytoplasmic staining in EBV negative Burkitt lymphomas but gave a predominantly nuclear staining in EBV positive LCL-s and stable transfected cells expressing EBNA-3. We suggest that EBNA-3 by direct protein-potein interaction induces the nuclear accumulation of a novel enzyme, that is part of the ribonucleotide salvage pathway. Increased intranuclear levels of UK/UPRT may contribute to the metabolic build-up that is needed for blast transformation and rapid proliferation.
Gary Wildey - One of the best experts on this subject based on the ideXlab platform.
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Stimulation of the phosphorylation of Uridine in skeletal muscle by insulin and vanadate
Molecular and Cellular Biochemistry, 1990Co-Authors: Niels Haugaard, Aliza Torbati, Thomas Smithgall, Gary WildeyAbstract:The action of insulin and sodium vanadate on the phosphorylation of Uridine by skeletal muscle was studied in vitro . Insulin significantly increased the incorporation of ^3H-Uridine into uracil nucleotides by pieces of rat diaphragm incubated for 15 min in a phosphate-buffered medium. This action of the hormone was exceptionally consistent when MgATP was added to the incubation medium. In experiments in which pieces of psoas muscle were incubated in TRIS buffer in the presence and absence of insulin, the hormone caused a significant activation of Uridine Kinase measured in cytosolic extracts of the incubated tissue. In experiments with rat diaphragm similar to those with insulin, the vanadate ion caused a significant increase in phosphorylation of Uridine. The results of these experiments provide preliminary support for the proposal that uracil nucleotide metabolism is regulated by insulin and that insulin activates Uridine Kinase, the limiting enzyme in the synthesis of uracil nucleotides from Uridine by the salvage pathway.
