| 1. |
- Clausen, Anders Ranegaard, et al.
(författare)
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A multisubstrate deoxyribonucleoside kinase from plants.
- 2008
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Ingår i: Nucleic acids symposium series. - : Oxford University Press (OUP). - 0261-3166 .- 1746-8272. ; :52, s. 489-490
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Tidskriftsartikel (refereegranskat)abstract
- Deoxyribonucleoside kinases catalyze the rate limiting step during the salvage of deoxyribonucleosides and convert them into the corresponding monophosphate compounds. We have identified and characterized a unique multisubstrate deoxyribonucleoside kinase from plants. The phylogenetic relationship and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases. The broad substrate specificity makes this enzyme an interesting candidate to be evaluated as a suicide gene in anti-cancer therapy.
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| 2. |
- Clausen, Anders Ranegaard, et al.
(författare)
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Plants salvage deoxyribonucleosides in mitochondria.
- 2014
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 33:4-6, s. 291-295
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Tidskriftsartikel (refereegranskat)abstract
- Deoxyribonucleoside kinases phosphorylate deoxyribonucleosides into the corresponding 5'-monophosphate deoxyribonucleosides to supply the cell with nucleic acid precursors. In mitochondrial fractions of the model plant Arabidopsis thaliana, we detected deoxyadenosine and thymidine kinase activities, while the cytosol fraction contained six-fold lower activity and chloroplasts contained no measurable activities. In addition, a mitochondrial fraction isolated from the potato Solanum tuberosum contained thymidine kinase and deoxyadenosine kinase activities. We conclude that an active salvage of deoxyribonucleosides in plants takes place in their mitochondria. In general, the observed localization of the plant dNK activities in the mitochondrion suggests that plants have a different organization of the deoxyribonucleoside salvage compared to mammals.
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| 3. |
- Clausen, Anders Ranegaard, et al.
(författare)
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Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana.
- 2012
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X. ; 279:20, s. 3889-3897
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Tidskriftsartikel (refereegranskat)abstract
- Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized in mammals but in contrast little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding mono-phosphate compounds with a surprising preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1-like genes (AtTK1a and AtTK1b) and deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, but AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. Our results point out a crucial role for salvage of thymidine during early plant development. © 2012 The Authors Journal compilation © 2012 FEBS.
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| 4. |
- Khan, Zahidul, et al.
(författare)
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Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.
- 2010
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Ingår i: Neuro-Oncology. - : Oxford University Press (OUP). - 1523-5866 .- 1522-8517. ; 12, s. 549-558
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Tidskriftsartikel (refereegranskat)abstract
- The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen for central nervous system (CNS) tumors, several obstacles have been encountered such as inefficient gene transfer to the tumor cells, limited prodrug penetration into the CNS, and inefficient enzymatic activity of the suicide gene. We report here the cloning and successful application of a novel thymidine kinase 1 (TK1) from the tomato plant, with favorable characteristics in vitro and in vivo. This enzyme (toTK1) is highly specific for the nucleoside analog prodrug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. An important feature of toTK1 is that it efficiently phosphorylates its substrate AZT not only to AZT monophosphate, but also to AZT diphosphate, with excellent kinetics. The efficiency of the toTK1/AZT system was confirmed when toTK1-transduced human glioblastoma (GBM) cells displayed a 500-fold increased sensitivity to AZT compared with wild-type cells. In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumor growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls. The novel toTK1/AZT suicide gene therapy system in combination with stem cell-mediated gene delivery promises new treatment of malignant gliomas.
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| 5. |
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| 6. |
- Munch-Petersen, Birgitte, et al.
(författare)
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Jure Piskur (1960-2014)
- 2015
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Ingår i: Journal of Genetics and Genomics. - : Elsevier BV. - 1673-8527. ; 42:5, s. 275-277
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 7. |
- Mutahir, Zeeshan, et al.
(författare)
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Characterization of oligomeric and kinetic properties of tomato thymidine kinase 1.
- 2011
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 30:12, s. 1223-1226
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Tidskriftsartikel (refereegranskat)abstract
- The gene encoding thymidine kinase 1 from tomato (toTK1) has in combination with azidothymidine (AZT) recently been proposed as a powerful suicide gene for anticancer gene therapy. The toTK1/AZT combination has been demonstrated to have several advantages for the treatment of glioblastomas because AZT can easily penetrate the blood-brain barrier and toTK1 can efficiently phosphorylate AZT and also AZT-monophosphate. In a pursuit to further understand the properties of toTK1, we examined the oligomerization properties of recombinant toTK1 and its effect on enzyme kinetics. Previously, it has been shown that human TK1 is a dimer in the absence of ATP and a tetramer if preincubated with ATP. However, we show here that ATP preincubation did not result in a structural shift from dimer to tetramer in toTK1. For human TK1 pretreated with ATP, the K(m) value decreased 20-fold, but toTK1's K(m) value did not show a dependence on the presence or absence of ATP. Furthermore, toTK1 was always found in a highly active form.
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| 8. |
- Mutahir, Zeeshan, et al.
(författare)
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Gene duplications and losses among vertebrate deoxyribonucleoside kinases of the non-TK1 Family
- 2016
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Ingår i: Nucleosides, Nucleotides and Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 35:10-12, s. 677-690
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Tidskriftsartikel (refereegranskat)abstract
- Deoxyribonucleoside kinases (dNKs) salvage deoxyribonucleosides (dNs) and catalyze the rate limiting step of this salvage pathway by converting dNs into corresponding monophosphate forms. These enzymes serve as an excellent model to study duplicated genes and their evolutionary history. So far, among vertebrates only four mammalian dNKs have been studied for their substrate specificity and kinetic properties. However, some vertebrates, such as fish, frogs, and birds, apparently possess a duplicated homolog of deoxycytidine kinase (dCK). In this study, we characterized a family of dCK/deoxyguanosine kinase (dGK)-like enzymes from a frog Xenopus laevis and a bird Gallus gallus. We showed that X. laevis has a duplicated dCK gene and a dGK gene, whereas G. gallus has a duplicated dCK gene but has lost the dGK gene. We cloned, expressed, purified, and subsequently determined the kinetic parameters of the dCK/dGK enzymes encoded by these genes. The two dCK enzymes in G. gallus have broader substrate specificity than their human or X. laevis counterparts. Additionally, the duplicated dCK enzyme in G. gallus might have become mitochondria. Based on our study we postulate that changing and adapting substrate specificities and subcellular localization are likely the drivers behind the evolution of vertebrate dNKs.
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| 9. |
- Mutahir, Zeeshan, et al.
(författare)
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Thymidine kinase 1 regulatory fine-tuning through tetramer formation.
- 2013
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X. ; 280:6, s. 1531-1541
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Tidskriftsartikel (refereegranskat)abstract
- Thymidine kinase 1 (TK1) provides a crucial precursor, thymidine monophosphate (dTMP), for nucleic acid synthesis, and the activity of TK1 increases up to 200-fold during the S-phase of cell division in humans. An important part of the regulatory check-points is the ATP and enzyme concentration-dependent transition of TK1 from a dimer with low catalytic efficiency to a tetramer with high catalytic efficiency. This regulatory fine-tuning serves as an additional control to provide the balanced pool of nucleic acid precursors in the cell. We sub-cloned and over-expressed ten different TK1s, originating from widely different organisms, and characterized their kinetic and oligomerization properties. While bacteria, plants and Dictyostelium only exhibited dimeric TK1, we found that all animals had a tetrameric TK1. However, a clear ATP dependent switch between dimer and tetramer was found only in higher vertebrates, and was especially pronounced in mammalian and bird TK1s. We suggest that the dimer form is the original one and the tetramer originated in the animal lineage after the split of Dictyostelium and the lineages leading to invertebrates and vertebrates. The efficient switching mechanism was likely settled first in the warm-blooded animals when they separated from the rest of vertebrates. © 2013 The Authors Journal compilation © 2013 FEBS.
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| 10. |
- Sandrini, Michael P. B., et al.
(författare)
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Nucleoside analogues are activated by bacterial deoxyribonucleoside kinases in a species-specific manner
- 2007
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 1460-2091 .- 0305-7453. ; 60:3, s. 510-520
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: To investigate the bactericidal activity of antiviral and anticancer nucleoside analogues against a variety of pathogenic bacteria and characterize the activating enzymes, deoxyribonucleoside kinases (dNKs). Methods: Several FDA-approved nucleoside analogue drugs were screened for their potential bactericidal activity against several clinical bacterial isolates and type strains. We identified and subcloned the genes coding for putative deoxyribonucleoside kinases in Escherichia coli, Pasteurella multocida, Salmonella enterica, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens and Listeria monocytogenes. These genes were tested for their ability to increase the susceptibility of a dNK-deficient E. coli strain to various analogues. We overexpressed, purified and characterized the substrate specificity and kinetic properties of the recombinant enzymes from S. enterica and B. cereus. Results: The tested Gram-negative bacteria were susceptible to 3 '-azido-3 '-deoxythymidine (AZT) in the concentration range 0.032-31.6 mu M except for a single E. coli isolate and two Pseudomonas aeruginosa isolates which were resistant to the tested AZT concentrations. Purified recombinant S. enterica thymidine kinase phosphorylated AZT efficiently with a K-m of 73.3 mM and k(cat)/K-m of 6.6 x 10(4) s(-1)M(-1) and is the activator of this drug in vivo. 2 ',2 '-Difluoro-2 '-deoxycytidine ( gemcitabine) was a potent antibiotic against Gram-positive bacteria in the concentration range between 0.001 and 1.0 mu M. The B. cereus deoxyadenosine kinase had a Km for gemcitabine of 33.5 mM and kcat/Km of 5.1 x 10(3) s(-1) M-1 and activates gemcitabine in vivo. S. enterica and B. cereus are now amongst the first bacteria with a completely characterized set of dNK enzymes. Conclusions: Bacterial dNKs efficiently activate nucleoside analogues in a species-specific manner. Therefore, nucleoside analogues have a potential to be employed as antibiotics in the fight against emerging multiresistant bacteria.
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