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| 3. |
- Madeja, Zbigniew, et al.
(författare)
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The role of thioredoxin reductase activity in selenium-induced cytotoxicity
- 2005
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Ingår i: Biochemical Pharmacology. - : Elsevier. - 0006-2952 .- 1356-1839. ; 69:12, s. 1765-1772
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Tidskriftsartikel (refereegranskat)abstract
- The selenoprotein thioredoxin reductase is a key enzyme in selenium metabolism, reducing selenium compounds and thereby providing selenide to synthesis of all selenoproteins. We evaluated the importance of active TrxR1 in selenium-induced cytotoxicity using transfected TrxR1 over-expressing stable Human Embryo Kidney (HEK-293) cells and modulation of activity by pretreatment with low concentration of selenite. Treatment with sodium selenite induced cytotoxity in a dose-dependent manner in both TrxR1 over-expressing and control cells. However, TrxR1 over-expressing cells, which were preincubated for 72h with 0.1 microM selenite, were significantly more resistant to selenite cytotoxicity than control cells. To demonstrate the early effects of selenite on behaviour of HEK-293 cells, we also investigated the influence of this compound on cell motility. We observed inhibition of cell motility by 50 microM selenite immediately after administration. Moreover, TrxR1 over-expressing cells preincubated with a low concentration of selenite were more resistant to the inhibitory effect of 50 microM selenite than those not preincubated. It was also observed that the TrxR over-expressing cells showed higher TrxR1 activity than control cells and the preincubation of over-expressing cells with 0.1 microM selenite induced further significant increase in the activity of TrxR1. On the other hand, we demonstrated that TrxR1 over-expressing cells showed decreased glutathione peroxidase activity compared to control cells. These data strongly suggest that TrxR1 may be a crucial enzyme responsible for cell resistance against selenium cytotoxicity.
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- Nalvarte, Ivan, et al.
(författare)
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Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells : Effect on cell growth and differentiation
- 2004
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 279:52, s. 54510-54517
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Tidskriftsartikel (refereegranskat)abstract
- The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3'-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxR-overexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1- or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.
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| 8. |
- Nordman, Tomas
(författare)
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In vitro studies on the biosynthesis and reduction of ubiquinone
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis concerns the role of ubiquinone, the only endogenously synthesised lipid soluble antioxidant, in the cellular defence against peroxidation of proteins and lipids. The aims of the present investigations were to study the biosynthesis of ubiquinone in two different organelle fractions, i.e. microsomes and peroxisomes and to characterise the enzyme reactions of the three flavoenzymes, hpoamide dehydrogenase, glutathione reductase and thioredoxin reductase in the reduction of ubiquinone. A semipreparative HPLC method was established to rapidly isolate different polyprenols with high purity. The isolated compounds could be used for studies of different enzyme reactions in the mevalonate pathway, and as standards for quantitative HPLC-analysis. Compared to conventional chromatographic methods this new technique was much more rapid and polyprenols with higher purity was isolated. It was demonstrated that both peroxisomes and microsomes were involved in the biosynthesis of ubiquinone. Two enzymes involved in the synthesis of ubiquinone, trans-prenyltransferase and nonaprenyl-4-hydroxybenzoate (NPHB)-transferase, were investigated. The results clearly showed differences in the regulation of the synthesis of ubiquinone in those organelles. The specific activity of trans-prenyltransferase in peroxisomes was 30% of the total activity found in both organelles. The characteristics of the regeneration of ubiquinol by the flavoenzymes, hpoamide dehydrogenase, glutathione reductase and thioredoxin reductase were investigated. These enzymes belong to the same family of enzymes and are defined as homodimeric pyridine nucleotide-disulfide oxidoreductases. The reduction of ubiquinone by Hpoamide dehydrogenase and glutathione reductase was shown to be highly elevated by addition of zinc to the reaction mixture, whereas this reaction by thioredoxin reductase was inhibited by zinc. For Hpoamide dehydrogenase and glutathione reductase the pH optimum for the reaction was found at acidic pH, but at physiological pH for thioredoxin reductase. The reduction of ubiquinone by thioredoxin reductase was confirmed to be selenium dependent by use of full-length bovine and rat, E. coli (lacking selenocysteine), recombinant human (selenocysteine replaced by alanine), and truncated rat thioredoxin reductases, as well as with stable cell lines overexpressing thioredoxin reductase. Altogether, the novel biological findings in this thesis are that; ubiquinone is not only synthesised in microsomes but also to a high extent in peroxisomes; ubiquinone is efficiently reduced by glutathione reductase and thioredoxin reductase; the reduction of ubiquinone by thioredoxin reductase is entirely selenium dependent.
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| 9. |
- Nordman, Tomas, et al.
(författare)
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Regeneration of the antioxidant ubiquinol by lipoamide dehydrogenase, thioredoxin reductase and glutathione reductase
- 2003
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Ingår i: Biofactors. - : IOS Press. - 0951-6433 .- 1872-8081. ; 18:1-4, s. 45-50
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Tidskriftsartikel (refereegranskat)abstract
- Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.
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- Rissler, P., et al.
(författare)
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Adriamycin cytotoxicity may stimulate growth of hepatocellular tumours in an experimental model for adjuvant systemic chemotherapy in liver transplantation
- 2005
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Ingår i: Transplant International. - : Frontiers Media SA. - 0934-0874 .- 1432-2277. ; 18:8, s. 992-1000
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Tidskriftsartikel (refereegranskat)abstract
- Adjuvant treatment with adriamycin has been suggested to improve results after liver transplantation for hepatocellular cancer. Here we have applied an animal model for evaluation of treatment with adriamycin and/or cyclosporine A on liver tumour growth. Three chemically induced rat liver tumours with various degree of differentiation were transferred to the spleens of syngenic rats. Each recipient group was divided into four subgroups, treated with adriamycin and/or cyclosporine A or none of the drugs. When the tumour was well differentiated no proliferation was found in any of the subgroups. When the tumour exhibited a more pronounced dysplasia, adriamycin stimulated tumour growth. This effect was further increased by cyclosporine. In the animals transplanted with the most aggressive tumour, adriamycin inhibited tumour growth. When given together with cyclosporine this inhibition was counteracted. These data suggest that adriamycin, especially when given together with cyclosporine, may have a stimulatory effect on liver tumour cell growth.
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| 11. |
- Xia, Ling, et al.
(författare)
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Reduction of ubiquinone by lipoamide dehydrogenase : An antioxidant regenerating pathway
- 2001
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 268:5, s. 1486-1490
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Tidskriftsartikel (refereegranskat)abstract
- Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synthesized lipid-soluble antioxidant) to ubiquinol, the form in which it functions as an antioxidant. The reduction of ubiquinone was linear with time and exhibited turnover numbers of 5 and 1.2 min-1 in the presence and absence of zinc, respectively. The reaction was stimulated by zinc and cadmium but not by the other divalent ions tested. The zinc/cadmium-dependent stimulation of the reaction increased rapidly and linearly up to a concentration of 0.1 mM and was even further increased at 0.5 mM. At pH 6, the activity was three times higher than at physiological pH. Alteration of the NADPH : NADP+ ratio revealed that the reaction is inhibited by higher concentrations of the oxidized cofactors. FAD reduced ubiquinone in a dose-dependent manner at a considerably lower rate, suggesting that the reduction of ubiquinone by lipoamide dehydrogenase involves the FAD moiety of the enzyme.
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| 12. |
- Xia, Ling, et al.
(författare)
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The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress
- 2003
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 278:4, s. 2141-2146
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Tidskriftsartikel (refereegranskat)abstract
- The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.
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