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Miura T.,Hokkaido Pharmaceutical University School of Pharmacy
Chemico-Biological Interactions | Year: 2015

To investigate the mechanisms of cardiotoxicity induced by adriamycin (ADM), the enzymatic activities of ADM-Fe3+, including the peroxidase and lipoxygenase (LOX) activity, and participation of active oxygen species in the damage to biological components were examined. ADM-Fe3+, but not ADM, steadily oxidized tetramethyl-p-phenylenediamine in the presence of peroxides, indicating that ADM-Fe3+ acts as a peroxidase. However, the activity of ADM-Fe3+ as peroxidase was very low compared with that of heme peroxidase, but was similar to that of LOX, which has a known peroxidase activity. Conversely, the activity of ADM-Fe3+ as a LOX was also very low compared with that of LOX itself. However, the lipid hydroperoxides (LOOH) produced by ADM-Fe3+ were the substrate for ADM-Fe3+ as a peroxidase. These findings indicate that lipid peroxidation cooperates with the peroxidase activity of ADM-Fe3+. Hydroxyl radicals (HO) were generated when ADM-Fe3+ was incubated with H2O2, but not with LOOH. Alcohol dehydrogenase was inactivated by LOOH. Conversely, DNA was mainly damaged by ADM-Fe3+ with H2O2. A small amount of DNA remained at the starting point on agarose gels during incubation with ADM-Fe3+ with LOOH and ADM-Fe3+ with H2O2. It seems that HO and compound I-like species participate in the strand breaks and the aggregation of DNA, respectively. ©2015 Published by Elsevier Ireland Ltd.


Oshiumi H.,Hokkaido University | Miyashita M.,Hokkaido University | Miyashita M.,Hokkaido Pharmaceutical University School of Pharmacy | Matsumoto M.,Hokkaido University | Seya T.,Hokkaido University
PLoS Pathogens | Year: 2013

The innate immune system is essential for controlling viral infections, but several viruses have evolved strategies to escape innate immunity. RIG-I is a cytoplasmic viral RNA sensor that triggers the signal to induce type I interferon production in response to viral infection. RIG-I activation is regulated by the K63-linked polyubiquitin chain mediated by Riplet and TRIM25 ubiquitin ligases. TRIM25 is required for RIG-I oligomerization and interaction with the IPS-1 adaptor molecule. A knockout study revealed that Riplet was essential for RIG-I activation. However the molecular mechanism underlying RIG-I activation by Riplet remains unclear, and the functional differences between Riplet and TRIM25 are also unknown. A genetic study and a pull-down assay indicated that Riplet was dispensable for RIG-I RNA binding activity but required for TRIM25 to activate RIG-I. Mutational analysis demonstrated that Lys-788 within the RIG-I repressor domain was critical for Riplet-mediated K63-linked polyubiquitination and that Riplet was required for the release of RIG-I autorepression of its N-terminal CARDs, which leads to the association of RIG-I with TRIM25 ubiquitin ligase and TBK1 protein kinase. Our data indicate that Riplet is a prerequisite for TRIM25 to activate RIG-I signaling. We investigated the biological importance of this mechanism in human cells and found that hepatitis C virus (HCV) abrogated this mechanism. Interestingly, HCV NS3-4A proteases targeted the Riplet protein and abrogated endogenous RIG-I polyubiquitination and association with TRIM25 and TBK1, emphasizing the biological importance of this mechanism in human antiviral innate immunity. In conclusion, our results establish that Riplet-mediated K63-linked polyubiquitination released RIG-I RD autorepression, which allowed the access of positive factors to the RIG-I protein. © 2013 Oshiumi et al.


Terayama N.,Kogakuin University | Yasui E.,Kogakuin University | Mizukami M.,Hokkaido Pharmaceutical University School of Pharmacy | Miyashita M.,Kogakuin University | Nagumo S.,Kogakuin University
Organic Letters | Year: 2014

The first total synthesis of 14-membered macrolide sekothrixide and the originally proposed structure are reported. Seven contiguous asymmetric centers in the side chain were constructed using ring-openings of several kinds of epoxide. Assembly of the left segment and right segment was performed on the basis of the RCM reaction to generate 14-membered lactones having an E-trisubstituted olefin. These synthetic results led to a revision of C4, C6, and C8 stereochemistry in the structure of natural sekothrixide. © 2014 American Chemical Society.


Miura T.,Hokkaido Pharmaceutical University School of Pharmacy
Journal of Biochemistry | Year: 2012

During the oxidation of NADH by horseradish peroxidase (HRP-Fe 3+), superoxide (O- 2) is produced, and HRP-Fe3+ is converted to compound III. Superoxide dismutase inhibited both the generation of O- 2 and the formation of compound III. In contrast, catalase inhibited only the generation of O- 2. Under anaerobic conditions, the formation of compound III did not occur in the presence of NADH, thus indicating that compound III is produced via formation of a ternary complex consisting of HRP-Fe3+, NADH and oxygen. The generation of hydroxyl radicals was dependent upon O - 2 and H2O2 produced by HRP-Fe 3+-NADH. The reaction of compound III with H2O2 caused the formation of compound II without generation of hydroxyl radicals. Only HRP-Fe3+-NADH (but not K+O- 2 and xanthine oxidase-hypoxanthine) was able to induce the conversion of metmyoglobin to oxymyoglobin, thus suggesting the participation of a ternary complex made up of HRP-Fe2+..O2 ..NAD . (but not free O- 2 or H2O 2) in the conversion of metmyoglobin to oxymyoglobin. It appears that a cyclic pathway is formed between HRP-Fe3+, compound III and compound II in the presence of NADH under aerobic conditions, and a ternary complex plays the central roles in the generation of O- 2 and hydroxyl radicals. © 2012 The Authors.


Miura T.,Hokkaido Pharmaceutical University School of Pharmacy
Journal of Biochemistry | Year: 2015

In this study, bleomycin-Fe3+ steadily oxidized tetramethylbenzidine (TMB) in the presence of peroxides. However, the ability of bleomycin-Fe3+ to function as a peroxidase was extremely low compared with that of other peroxidases. A characteristic property of bleomycin-Fe3+ different from that observed for other peroxidases is its ability to oxidize TMB at the similar rate at both a pH 5 and 8 in the presence of lipid hydroperoxide (LOOH). In the present experiments, hydroxyl radicals (HO•) were generated only when bleomycin-Fe3+ was incubated with H2O2 at a pH of 5. No generation of HO• was observed during the incubation of bleomycin-Fe3+ with LOOH. Meanwhile, bleomycin-Fe3+ induced the formation of LOOH from linoleic acid and alcohol dehydrogenase was inactivated by bleomycin-Fe3+ with peroxides. Thiobarbituric acid reactive substances were formed from DNA by bleomycin-Fe3+ with H2O2, and strand breaks were caused by bleomycin-Fe3+ with LOOH. The oxidative substrates for bleomycin-Fe3+ blocked the damage to biological components induced by bleomycin-Fe3+. These results suggest that compound I-like species contribute to the process of damage to biological components induced by bleomycin-Fe3+. © 2014 The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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