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Peter Reichard - One of the best experts on this subject based on the ideXlab platform.
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effects of mutational loss of nucleoside kinases on deoxyadenosine 5 phosphate deoxyadenosine substrate cycle in cultured cem and v79 cells
Journal of Biological Chemistry, 1994Co-Authors: Vera Bianchi, Paola Ferraro, Stefania Borella, Paolo Bonvini, Peter ReichardAbstract:Abstract The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.
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effects of mutational loss of nucleoside kinases on deoxyadenosine 5 phosphate deoxyadenosine substrate cycle in cultured cem and v79 cells
Journal of Biological Chemistry, 1994Co-Authors: Vera Bianchi, Paola Ferraro, Stefania Borella, Paolo Bonvini, Peter ReichardAbstract:Abstract The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.
Vera Bianchi - One of the best experts on this subject based on the ideXlab platform.
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effects of mutational loss of nucleoside kinases on deoxyadenosine 5 phosphate deoxyadenosine substrate cycle in cultured cem and v79 cells
Journal of Biological Chemistry, 1994Co-Authors: Vera Bianchi, Paola Ferraro, Stefania Borella, Paolo Bonvini, Peter ReichardAbstract:Abstract The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.
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effects of mutational loss of nucleoside kinases on deoxyadenosine 5 phosphate deoxyadenosine substrate cycle in cultured cem and v79 cells
Journal of Biological Chemistry, 1994Co-Authors: Vera Bianchi, Paola Ferraro, Stefania Borella, Paolo Bonvini, Peter ReichardAbstract:Abstract The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.
Caterina Garone - One of the best experts on this subject based on the ideXlab platform.
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deoxycytidine and deoxythymidine treatment for thymidine kinase 2 deficiency
Annals of Neurology, 2017Co-Authors: Carlos Lopezgomez, Caterina Garone, Beatriz Garciadiaz, Saba Tadesse, Rebecca J Levy, Maria J Sanchezquintero, Marti Juanolafalgarona, Emanuele BarcaAbstract:Objective Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2–/–) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. Methods To test these hypotheses, we assessed two therapies in Tk2–/– mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. Results We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2–/– animals compared to dCMP+dTMP. Interpretation Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641–652
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Research Article Deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency
2016Co-Authors: Caterina Garone, Valentina Emmanuele, Luis C Lopez, Saba Tadesse, Kurenai Tanji, Catarina M Quinzii, Beatriz Garcia-diaz, O Akman, Michio HiranoAbstract:Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 defi-ciency, we administered deoxycytidine and deoxythymidine mono-phosphates (dCMP+dTMP) to the Tk2 H126N (Tk2/) knock-in mouse model from postnatal day 4, when mutant mice are pheno-typically normal, but biochemically affected. Assessment of 13-day-old Tk2/ mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2/200dCMP/dTMP) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater pheno-typic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency
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deoxypyrimidine monophosphate bypass therapy for thymidine kinase 2 deficiency
Embo Molecular Medicine, 2014Co-Authors: Caterina Garone, Beatriz Garciadiaz, Valentina Emmanuele, Luis C Lopez, Saba Tadesse, Hasan O Akman, Kurenai Tanji, Catarina M QuinziiAbstract:Autosomal recessive mutations in the thymidine kinase 2 gene (TK2) cause mitochondrial DNA depletion, multiple deletions, or both due to loss of TK2 enzyme activity and ensuing unbalanced deoxynucleotide triphosphate (dNTP) pools. To bypass Tk2 deficiency, we administered deoxycytidine and deoxythymidine monophosphates (dCMP+dTMP) to the Tk2 H126N (Tk2 � /� ) knock-in mouse model from postnatal day 4, when mutant mice are phenotypically normal, but biochemically affected. Assessment of 13-day-old Tk2 � /� mice treated with dCMP+dTMP 200 mg/kg/day each (Tk2 � /� 200dCMP/dTMP ) demonstrated that in mutant animals, the compounds raise dTTP concentrations, increase levels of mtDNA, ameliorate defects of mitochondrial respiratory chain enzymes, and significantly prolong their lifespan (34 days with treatment versus 13 days untreated). A second trial of dCMP+dTMP each at 400 mg/kg/day showed even greater phenotypic and biochemical improvements. In conclusion, dCMP/dTMP supplementation is the first effective pharmacologic treatment for Tk2 deficiency.
Paolo Bonvini - One of the best experts on this subject based on the ideXlab platform.
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effects of mutational loss of nucleoside kinases on deoxyadenosine 5 phosphate deoxyadenosine substrate cycle in cultured cem and v79 cells
Journal of Biological Chemistry, 1994Co-Authors: Vera Bianchi, Paola Ferraro, Stefania Borella, Paolo Bonvini, Peter ReichardAbstract:Abstract The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.
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effects of mutational loss of nucleoside kinases on deoxyadenosine 5 phosphate deoxyadenosine substrate cycle in cultured cem and v79 cells
Journal of Biological Chemistry, 1994Co-Authors: Vera Bianchi, Paola Ferraro, Stefania Borella, Paolo Bonvini, Peter ReichardAbstract:Abstract The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.
Stefania Borella - One of the best experts on this subject based on the ideXlab platform.
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effects of mutational loss of nucleoside kinases on deoxyadenosine 5 phosphate deoxyadenosine substrate cycle in cultured cem and v79 cells
Journal of Biological Chemistry, 1994Co-Authors: Vera Bianchi, Paola Ferraro, Stefania Borella, Paolo Bonvini, Peter ReichardAbstract:Abstract The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.
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effects of mutational loss of nucleoside kinases on deoxyadenosine 5 phosphate deoxyadenosine substrate cycle in cultured cem and v79 cells
Journal of Biological Chemistry, 1994Co-Authors: Vera Bianchi, Paola Ferraro, Stefania Borella, Paolo Bonvini, Peter ReichardAbstract:Abstract The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP synthesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3% in CEM cells and to 10% in V79 cells. This strongly suggests the presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.
