論文 - 田村　悦臣

Methotrexate significantly induces apoptosis by inhibiting STAT3 activation in NPM-ALK-positive ALCL cells.

Uchihara Y, Komori R, Tago K, Tamura H, Funakoshi-Tago M

Biochemical pharmacology （Biochemical Pharmacology）  170   113666 2019年12月

研究論文（学術雑誌）,  ISSN  0006-2952

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© 2019 Elsevier Inc. Anaplastic large cell lymphoma (ALCL) is associated with a characteristic chromosomal translocation that generates the oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Methotrexate is a commonly used chemotherapeutic drug in the treatment of multiple cancers due to its inhibition of dihydrofolate reductase (DHFR), which suppresses the synthesis of DNA. In the present study, we found that low-dose methotrexate significantly induced apoptosis in transformed Ba/F3 cells expressing NPM-ALK by inhibiting the activation of signal transducer and activator of transcription factor 3 (STAT3), a critical downstream molecule of NPM-ALK. Although methotrexate prevented the phosphorylation of STAT3, it did not affect the activity of NPM-ALK. A co-treatment with folinic acid prevented the methotrexate-induced inhibition of STAT3 activation and induction of apoptosis, suggesting that methotrexate exerts its cytotoxic effects by depleting tetrahydrofolate (THF) in transformed cells by NPM-ALK. Furthermore, methotrexate induced the down-regulation of the anti-apoptotic protein, MCL-1, DNA damage, and the activation of a p53 tumor suppressor, leading to apoptosis through the inhibition of STAT3. Methotrexate significantly induced apoptosis in ALK inhibitor-resistant cells expressing the NPM-ALK mutant harboring the point mutation, G262R, and in ALCL patient-derived NPM-ALK-positive Ki-JK cells. Collectively, these results demonstrate the potential therapeutic application of methotrexate, which inhibits the activation of STAT3, to NPM-ALK-positive ALCL.

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Tyrosine-phosphorylated SOCS3 negatively regulates cellular transformation mediated by the myeloproliferative neoplasm-associated JAK2 V617F mutant

Funakoshi-Tago M., Tsuruya R., Ueda F., Ishihara A., Kasahara T., Tamura H., Tago K.

Cytokine （Cytokine）  123   154753 2019年11月

研究論文（学術雑誌）, 共著,  ISSN  10434666

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© 2019 Elsevier Ltd In the majority of myeloproliferative neoplasms (MPNs) patients, a point mutation, V617F has been found in Janus kinase 2 (JAK2) gene, and this JAK2 mutant provoked aberrant signaling pathway. In the current study, we found that suppressor of cytokine signaling proteins 3 (SOCS3) possessed the tumor suppressive activity against the JAK2 V617F mutant-provoked cellular transformation. The knockdown of SOCS3 increased the expression level of the JAK2 V617F mutant, which enhanced the activation of signaling mediators, including signal transducer and activator of transcription 3 and 5 (STAT3, STAT5) and extracellular signal-regulated kinase (ERK), and also increased of the proliferation rate and tumorigenesis activity of Ba/F3 cells expressing the JAK2 V617F mutant and erythropoietin receptor (EpoR). In contrast, the enforced expression of SOCS3 significantly inhibited the JAK2 V617F mutant-induced activation of downstream signaling molecules, cell proliferation, and tumorigenesis by down-regulating the expression level of the JAK2 V617F mutant. SOCS3 interacted with the JAK2V617F mutant through its SH2 domain and was phosphorylated at Tyr-204 and Tyr-221 in its SOCS box by the JAK2V617F mutant. SOCS3 mutants carrying a mutation in the SH2 domain (R71E) and a substitution at Tyr-221 (Y221F) failed to exert inhibitory effects on JAK2V617F mutant-induced cellular transformation and tumorigenesis. Collectively, these results imply that SOCS3 plays a negative role in the JAK2 V617F mutant-induced oncogenic signaling pathway through its SH2 domain and the phosphorylation of Tyr-221 in its SOCS box.

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3D in vivo imaging of the keratin filament network in the mouse stratum granulosum reveals profilaggrin-dependent regulation of keratin bundling.

Usui K, Kadono N, Furuichi Y, Shiraga K, Saitou T, Kawasaki H, Toyooka K, Tamura H, Kubo A, Amagai M, Matsui T

Journal of dermatological science （Journal of Dermatological Science）  94 （ 3 ） 346 - 349 2019年06月

研究論文（学術雑誌）, 共著,  ISSN  0923-1811

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N-Acetyl cysteine prevents activities of STAT3 inhibitors, Stattic and BP-1-102 independently of its antioxidant properties.

Uchihara Y, Ohe T, Mashino T, Kidokoro T, Tago K, Tamura H, Funakoshi-Tago M

Pharmacological reports : PR （Pharmacological Reports）  71 （ 6 ） 1067 - 1078 2019年05月

研究論文（学術雑誌）,  ISSN  1734-1140

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© 2019 Institute of Pharmacology, Polish Academy of Sciences Background: Inhibitors for signal transducer and activator of transcription 3 (STAT3), Stattic, BP-1-102, and LLL12 significantly induce apoptosis in transformed Ba/F3 cells expressing an oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) that induces the activation of STAT3. We found that the antioxidant reagent, N-acetyl cysteine (NAC) prevented the abilities of Stattic and BP-1-102, but not LLL12 to induce apoptosis in transformed cells expressing NPM-ALK, providing a novel problem in use of STAT3 inhibitors. We herein investigated the mechanisms how NAC prevented the effects of Sttatic and BP-1-102. Methods: Ba/F3 cells expressing NPM-ALK and SUDHL-1 cells were treated with antioxidants such as NAC, Trolox or edaravone in combination with STAT3 inhibitors. Phosphorylation of STAT3, cell proliferation rate, cell viability, cell cycle, internucleosomal DNA fragmentation and the intracellular accumulation of reactive oxygen species (ROS) was investigated. The binding of STAT3 inhibitors and NAC was analyzed by LC–MS. Results: NAC but not Trolox and edaravone diminished the abilities of Stattic and BP-1-102 to induce apoptosis in cells expressing NPM-ALK. The ROS levels in cells expressing NPM-ALK were not markedly affected by the treatments with Stattic and BP-1-102 in combination with NAC, suggesting that NAC inhibited the activity of Stattic and BP-1-102 independent of its antioxidant activity. LC–MS analysis revealed that NAC directly bound to Stattic and BP-1-102. Furthermore, these NAC adducts exhibited no cytotoxicity, and failed to affect the activity of STAT3. Conclusions: NAC antagonizes the activities of Stattic and BP-1-102, which inhibit STAT3 activation by interacting with cysteine residues in STAT3.

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Coffee brew intake can prevent the reduction of lens glutathione and ascorbic acid levels in HFD-fed animals.

Nakazawa Y, Ishimori N, Oguchi J, Nagai N, Kimura M, Funakoshi-Tago M, Tamura H

Experimental and therapeutic medicine 17 （ 2 ） 1420 - 1425 2019年02月

研究論文（学術雑誌）,  ISSN  1792-0981

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Chemoenzymatic synthesis of hydroxytyrosol monoesters and their suppression effect on nitric oxide production stimulated by lipopolysaccharides

田村 悦臣

Bioscience, Biotechnology and Biochemistry （Bioscience, Biotechnology and Biochemistry）  83 （ 2 ） 185 - 191 2018年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09168451

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© 2018 Japan Society for Bioscience, Biotechnology, and Agrochemistry. Fatty acid monoesters of hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol] were synthesized in two steps from tyrosol (4-hydroxyphenylethanol) by successive Candida antarctica lipase B-catalyzed chemoselective acylation on the primary aliphatic hydroxy group over phenolic hydroxy group in tyrosol, and 2-iodoxybenzoic acid (IBX)-mediated hydroxylation adjacent to the remaining free phenolic hydroxy group. Examination of their suppression effects on nitric oxide production stimulated by lipopolysaccharides in RAW264.7 cells showed that hydroxytyrosol butyrate exhibited the highest inhibition (IC 50 7.0 μM) among the tested compounds.

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Hydroxytyrosol butyrate inhibits 6-OHDA-induced apoptosis through activation of the Nrf2/HO-1 axis in SH-SY5Y cells

Funakohi-Tago M., Sakata T., Fujiwara S., Sakakura A., Sugai T., Tago K., Tamura H.

European Journal of Pharmacology （European Journal of Pharmacology）  834   246 - 256 2018年09月

研究論文（学術雑誌）, 共著,  ISSN  00142999

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© 2018 Elsevier B.V. Hydroxytyrosol (HT) is a polyphenol contained in olives and exhibits antioxidant activity. We herein investigated the effects of HT and its derivatives, hydroxytyrosol acetate (HT-A) and hydroxytyrosol butyrate (HT-B), on the protection of neuronal cells against apoptosis induced by the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA). The pretreatment of SH-SY5Y cells with HT-B, but not HT or HT-A significantly reduced the 6-OHDA-induced generation of reactive oxygen species, activation of caspase-3, and subsequent cell death. HT-B also induced the protein expression of the transcription factor, NF-E2-related factor-2 (Nrf2) and its transcriptional activation, resulting in the up-regulated expression of heme oxygenase-1 (HO-1), which conferred neuroprotection against 6-OHDA-induced oxidative damage. Furthermore, three cysteine residues, Cys151, Cys273, and Cys288 in Kelch-like ECH-associated protein 1 (Keap1) were necessary for the HT-B-induced activation of Nrf2. Collectively, the present results demonstrated that HT-B, harboring higher fat solubility than HT and HT-A, effectively elicited adaptive responses to oxidative stress by activating the Nrf2/HO-1 axis in neuronal cells.

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Roasted Coffee Reduces β-Amyloid Production by Increasing Proteasomal β-Secretase Degradation in Human Neuroblastoma SH-SY5Y Cells

田村 悦臣

Molecular Nutrition and Food Research （Molecular Nutrition and Food Research）  62 （ 21 ） e1800238 2018年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  16134125

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© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Scope: Epidemiological studies have shown that coffee consumption may be associated with a lower risk of developing several neurological disorders, including Alzheimer's disease (AD). Caffeine is a prominent candidate component underlying the preventive effects of coffee; however, the contribution of other constituents is unclear. To clarify this issue, the effect of roasting coffee beans on β-secretase (BACE1) expression in human neuroblastoma SH-SY5Y cells is investigated. Methods and results: Coffee (2%) reduces Aβ accumulation in culture medium to 80% of control levels after 24 h. Accordingly, BACE1 expression is decreased to 70% of control levels at 12 h. Experiments using cycloheximide and MG132, a proteasome inhibitor, reveal that coffee enhanced BACE1 degradation through activation of proteasomal activity. Furthermore, coffee activates cAMP-dependent protein kinase, and consequently, phosphorylation of a serine residue of proteasome 26S subunit, non-ATPase 11 (PSMD11). Pyrocatechol, a strong antioxidant known as catechol or 1,2-dihydroxybenzene, produced from chlorogenic acid during roasting, also reduces BACE1 expression by activation of proteasomal activity. Furthermore, pyrocatechol reduces Aβ production in SH-SY5Y cells. Conclusion: The data suggest that the roasting process may be crucial for the protective effects of coffee consumption in AD.

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Effect of hesperetin derivatives on the development of selenite-induced cataracts in rats

Nakazawa Y., Pauze M., Fukuyama K., Nagai N., Funakoshi-Tago M., Sugai T., Tamura H.

Molecular Medicine Reports （Molecular Medicine Reports）  18 （ 1 ） 1043 - 1050 2018年07月

研究論文（学術雑誌）, 共著,  ISSN  17912997

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Copyright © Spandidos Publications 2017. All rights reserved. Cataracts are a major cause of blindness worldwide. As anti-cataract pharmaceutical therapies require long-term treatment, identifying anti-cataract compounds that are ubiquitous in the human diet, have no adverse effects and are affordable, is of paramount importance. The present study focused on hesperetin and its derived compounds, hesperetin stearic acid ester (Hes-S) and hesperetin oleic acid ester (Hes-O), in order to investigate their therapeutic potential to treat cataracts in a selenite animal model. Thirteen-day-old Sprague Dawley rats were divided into 12 groups. Animals in groups 1 and 7 were subcutaneously injected with vehicle, those in groups 2 and 8 were administered hesperetin, those in groups 3 and 9 received stearic acid, those in groups 4 and 10 were injected with oleic acid, those in groups 5 and 11 were administered Hes-S, and those in groups 6 and 12 received Hes-O (10 nmol/kg body weight on days 0, 1 and 2). Animals in groups 7 to 12 were treated with sodium selenite (20 µmol/kg body weight given 4 h following the test compound treatment on day 0) to induce cataract. On day 6, rats had less severe central opacities and lower stage cataracts than rats in the selenite treatment-only control groups. The levels of glutathione (GSH) and ascorbic acid (AsA) in lenses with selenite-induced cataracts declined to one-third of that of controls, and the reduction in GSH and AsA levels was rescued following hesperetin, Hes-S or Hes-O treatment, with concentrations remaining to 70-80% of that of controls. However, there were no changes in the plasma levels of GSH and AsA following treatments. Administration of either hesperetin or hesperetin-derived compounds prevented the reduction of chaperone activity in the lens, and rats treated with Hes-S or Hes-O treatment had significantly greater chaperone activity than hesperetin-treated rats. Collectively, these results suggested that hesperetin and hesperetin-derived compounds may be novel drug compounds that have the potential to prevent or delay the onset of cataracts.

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A 5-hydroxyoxindole derivative attenuates LPS-induced inflammatory responses by activating the p38-Nrf2 signaling axis

田村 悦臣

Biochemical Pharmacology （Biochemical Pharmacology）  155   182 - 197 2018年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00062952

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© 2018 Elsevier Inc. 5-Hydroxyoxindole is a urinary metabolite of indole that exhibits antioxidant activity. In the present study, we found that a 5-hydroxyoxindole derivative (5-HI) significantly inhibited LPS-induced inflammatory effects in the murine macrophage cell line, RAW264.7. 5-HI induced the expression of the transcription factor, Nrf2, which is typically ubiquitinated by Keap1, an adaptor component of the ubiquitin E3 ligase complex, resulting in its proteasomal degradation. By utilizing Keap1−/− MEFs reconstituted with Keap1 mutants harboring substitutions in their major cysteine residues, we clarified the importance of Cys151 in Keap1 as a sensor for 5-HI in the induction of Nrf2 expression. Furthermore, 5-HI induced the activation of the MKK3/6-p38 pathway, which is required for the transcriptional activation of Nrf2. The knockdown of Nrf2 enhanced the LPS-induced expression of inflammatory mediators, including iNOS, NO, and CCL2, and effectively repressed the inhibitory effects of 5-HI on their expression. Although 5-HI and antioxidant N-acetyl cysteine (NAC) both reduced LPS-induced ROS generation, the treatment with NAC did not affect the LPS-induced expression of inflammatory mediators, suggesting that the anti-inflammatory activity of 5-HI mediated by Nrf2 is independent of redox control. Furthermore, when injected into mice with 5-HI, the expression of Nrf2 was significantly increased, and the LPS-induced mRNA expression of CXCL1, CCL2, TNFα and IL-6 were remarkably inhibited in the kidneys, liver, and lungs, and the production of these cytokines in serum was effectively reduced. Collectively, these results suggest that 5-HI has potential in the treatment of inflammatory diseases through the activation of Nrf2.

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Taxodione induces apoptosis in BCR-ABL-positive cells through ROS generation

田村 悦臣

Biochemical Pharmacology （Biochemical Pharmacology）  154   357 - 372 2018年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00062952

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© 2018 Elsevier Inc. Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) are hematopoietic malignancies caused by the constitutive activation of BCR-ABL tyrosine kinase. Although direct BCR-ABL inhibitors, such as imatinib, were initially successful in the treatment of leukemia, many patients developed drug resistance over time due to the gatekeeper mutation of BCR-ABL T315I. In the present study, we found that taxodione, a quinone methide diterpene isolated from Taxodium distichum, significantly induced apoptosis in human myelogenous leukemia-derived K562 cells, which were transformed by BCR-ABL. Taxodione reduced the activities of mitochondrial respiratory chain (MRC) complexes III and V, which appeared to induce the production of reactive oxygen species (ROS). N-acetylcysteine (NAC), an antioxidant agent, canceled taxodione-induced ROS production, reductions in MRC activities, particularly complex V, and apoptotic cell death. Furthermore, in K562 cells treated with taxodione, BCR-ABL and its major signaling molecules, such as STAT5 and Akt were sequestered in mitochondrial fraction, and their localization changes decrease their abilities to stimulate cell proliferation, suggesting that these actions seem to be a mechanism how taxodione functions as an anti-tumor drug. Strikingly, NAC canceled these taxodione-caused anti-cancer effects. Taxodione induced apoptosis in transformed Ba/F3 cells induced not only by BCR-ABL, but also T315I-mutated BCR-ABL through the generation of ROS. Collectively, the present results suggest that in the treatment of leukemia, taxodione has potential as a compound with high efficacy to overcome BCR-ABL T315I mutation-mediated resistance in leukemia cells.

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Effect of hesperetin derivatives on the development of selenite‑induced cataracts in rats.

田村 悦臣

Molecular Medicine Reports 18   1043 - 1050 2018年03月

研究論文（学術雑誌）, 共著, 査読有り

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A major component of vitamin E, α-tocopherol inhibits the anti-tumor activity of crizotinib against cells transformed by EML4-ALK

田村 悦臣

European Journal of Pharmacology （European Journal of Pharmacology）  825   1 - 9 2018年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00142999

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© 2018 Elsevier B.V. Crizotinib is an inhibitor of anaplastic lymphoma kinase (ALK) and is of significant therapeutic benefit to patients with non-small cell lung cancer (NSCLC) harboring the EML4-ALK fusion gene. In the present study, we demonstrated that α-tocopherol, a major component of vitamin E, attenuated the effects of crizotinib independently of its anti-oxidant properties. α-Tocopherol significantly inhibited crizotinib-induced apoptosis in cells transformed by EML4-ALK. It also effectively attenuated the crizotinib-induced inhibition of EML4-ALK and its downstream molecules, STAT3 and ERK, and suppressed the inhibitory effects of crizotinib on EML4-ALK-mediated transformation in the focus formation assay. On the other hand, other members of the vitamin E family, namely, β-tocopherol, γ-tocopherol, δ-tocopherol, and α-tocotrienol, and a water-soluble analog of vitamin E, Trolox had no effects on the anti-tumor activity of crizotinib in cells transformed by EML4-ALK. Collectively, these results revealed the risk of the anti-tumor activity of crizotinib being attenuated when it is administrated in combination with vitamin E supplements containing α-tocopherol as a major component.

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Administration of antioxidant compounds affects the lens chaperone activity and prevents the onset of cataracts.

田村 悦臣

Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie （Biomedicine and Pharmacotherapy）  95   137 - 143 2017年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0753-3322

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© 2017 Elsevier Masson SAS To prevent lens opacification and cataract formation, the lens contains α-crystallin, which has been shown to function as a molecular chaperone that maintains the correct folding of other proteins. Oxidative stress is known to be an important factor in the initiation and progression of a cataract. So far, several antioxidant compounds have been reported to prevent cataracts in vivo and in vitro. This stress also triggers α-crystallin modifications and alters its chaperone activity. However, few studies have examined the relationship between the consumption of antioxidant compounds and lens chaperone activity. To elucidate the effect of antioxidants on lens chaperone activity, antioxidants were administered to a selenite-induced cataract model of rats. The chaperone activity in lens water-soluble fraction was measured using aldehyde dehydrogenase. All antioxidant treatment groups, except decaffeinated coffee treatment, had less severe central opacities and lower stage cataracts than control groups. The chaperone activity was weaker in lens of selenite cataract rats, but antioxidant compounds and coffee treatment can prevent the chaperone activity decreasing, but not decaffeinated coffee. These results suggested that the treatment with antioxidant compounds could prevent cataract formation by the maintenance of the chaperone activity in water-soluble lens proteins. Thus, this study describes the development of an anticataract drug target for lens chaperone activity.

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Occludin as a functional marker of vascular endothelial cells on tube-forming activity

田村 悦臣

Journal of Cellular Physiology （Journal of Cellular Physiology）  233 （ 2 ） 1700 - 1711 2017年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00219541

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© 2017 Wiley Periodicals, Inc. Cell therapy using endothelial progenitor cells (EPCs) is a promising strategy for the treatment of ischemic diseases. Two types of EPCs have been identified: early EPCs and late EPCs. Late EPCs are able to form tube structure by themselves, and have a high proliferative ability. The functional marker(s) of late EPCs, which relate to their therapeutic potential, have not been fully elucidated. Here we compared the gene expression profiles of several human cord blood derived late EPC lines which exhibit different tube formation activity, and we observed that the expression of occludin (OCLN) in these lines correlated with the tube formation ability, suggesting that OCLN is a candidate functional marker of late EPCs. When OCLN was knocked down by transfecting siRNA, the tube formation on Matrigel, the S phase + G 2 /M phase in the cell cycle, and the spheroid-based sprouting of late EPCs were markedly reduced, suggesting the critical role of OCLN in tube formation, sprouting, and proliferation. These results indicated that OCLN plays a novel role in neovascularization and angiogenesis.

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Alpha-tocopherol attenuates the anti-tumor activity of crizotinib against cells transformed by NPM-ALK.

田村 悦臣

PloS one （PLoS ONE）  12 （ 8 ） e0183003 2017年08月

研究論文（学術雑誌）, 共著, 査読有り

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© 2017 Uchihara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Anaplastic large cell lymphomas (ALCL) are mainly characterized by harboring the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). The ALK inhibitor, crizotinib specifically induced apoptosis in Ba/F3 cells expressing NPM-ALK by inhibiting the activation of NPM-ALK and its downstream molecule, signal transducer and activator of transcription factor 3 (STAT3). We found that α-tocopherol, a major component of vitamin E, attenuated the effects of crizotinib independently of its anti-oxidant properties. Although α-tocopherol suppressed the inhibitory effects of crizotinib on the signaling axis including NPM-ALK and STAT3, it had no influence on the intake of crizotinib into cells. Crizotinib also directly inhibited the kinase activity of NPM-ALK; however, this inhibitory effect was not altered by the co-treatment with α-tocopherol. Whereas the nuclear localization of NPM-ALK was disappeared by the treatment with crizotinib, the co-treatment with α-tocopherol swept the effect of crizotinib and caused the localization of NPM-ALK in nucleus. The administration of α-tocopherol attenuated the anti-tumor activity of crizotinib against NPM-ALK-provoked tumorigenesis in vivo. Furthermore, the α-tocopherol-induced inhibition of crizotinib-caused apoptosis was also observed in NPM-ALK-positive cells derived from ALCL patients, namely, SUDHL-1 and Ki-JK. Collectively, these results not only revealed the novel mechanism underlying crizotinib-induced apoptosis in NPM-ALK-positive cells, but also suggest that the anti-tumor effects of crizotinib are attenuated when it is taken in combination with vitamin E.

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Coffee reduces KRAS expression in Caco-2 human colon carcinoma cells via regulation of miRNAs.

田村 悦臣

Oncology letters （Oncology Letters）  14 （ 1 ） 1109 - 1114 2017年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1792-1074

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© 2016, Spandidos Publications. All rights reserved. Previous epidemiological studies have demonstrated that moderate coffee consumption is associated with a lower risk of certain types of cancer, particularly colon cancer. To elucidate the molecular basis for this protective action, the effect of coffee on Caco-2 human colon carcinoma cells was investigated. Low concentrations of coffee (<5%) inhibited proliferation of Caco-2 cells without affecting cell viability. Coffee also reduced KRAS proto-oncogene, GTPase (KRAS) gene expression in a dose-dependent manner; however, caffeine, caffeic acid and chlorogenic acid, three major constituents of coffee, did not exhibit this effect. Increasing the duration of coffee bean roasting increased the reduction in KRAS expression, suggesting that the active constituents responsible for this effect emerged during the roasting process. MicroRNA (miR) analysis revealed that coffee induced the expression of miR-30c and miR-96, both of which target the KRAS gene. The results of the present study suggested that daily coffee consumption may reduce KRAS activity, thereby preventing the malignant growth of colon carcinoma cells.

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Coffee extract inhibits adipogenesis in 3T3-L1 preadipocyes by interrupting insulin signaling through the downregulation of IRS1.

田村 悦臣

PloS one （PLoS ONE）  12 （ 3 ） e0173264 2017年03月

研究論文（学術雑誌）, 共著, 査読有り

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© 2017 Maki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Although epidemiological data have indicated that a strong negative association exists between coffee consumption and the prevalence of obesity-associated diseases, the molecular mechanisms by which coffee intake prevents obesity-associated diseases has not yet been elucidated. In this study, we found that coffee intake significantly suppressed high-fat diet (HFD)-induced metabolic alternations such as increases in body weight and the accumulation of adipose tissue, and up-regulation of glucose, free fatty acid, total cholesterol and insulin levels in the blood. We also found that coffee extract significantly inhibited adipogenesis in 3T3-L1 preadipocytes. In the early phase of adipogenesis, 3T3-L1 cells treated with coffee extract displayed the retardation of cell cycle entry into the G2/M phase called as mitotic clonal expansion (MCE). Coffee extract also inhibited the activation of CCAAT/enhancer-binding protein β (C/EBPβ) by preventing its phosphorylation by ERK. Furthermore, the coffee extract suppressed the adipogenesis-related events such as MCE and C/EBPβ activation through the down-regulation of insulin receptor substrate 1 (IRS1). The stability of the IRS1 protein was markedly decreased by the treatment with coffee extract due to proteasomal degradation. These results have revealed an anti-adipogenic function for coffee intake and identified IRS1 as a novel target for coffee extract in adipogenesis.

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Data on the effect of hypomyelinating leukodystrophy 6 (HLD6)-associated mutations on the TUBB4A properties.

田村 悦臣

Data in brief 11   284 - 289 2017年02月

研究論文（学術雑誌）, 共著, 査読有り

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Roasting Enhances the Anti-Cataract Effect of Coffee Beans: Ameliorating Selenite-Induced Cataracts in Rats.

田村 悦臣

Current eye research （Current Eye Research）  42 （ 6 ） 864 - 870 2017年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0271-3683

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© 2017 Taylor & Francis. Purpose: Coffee is a widely consumed beverage. While recent studies have linked its intake to a reduced risk of cataracts, caffeine is believed to be the key factor for its effect. To know how roasting beans affects the effect of coffee on cataract formation, we investigated the impact roasting using a selenite-induced cataract rat model. Materials and Methods: Sprague Dawley rats were given a single injection of sodium selenite, which induced formation of nuclear cataracts by day 6, with or without coffee intake (100% coffee, 0.2 mL/day) for following 3 days. Results: The concentrations of glutathione (GSH) and ascorbic acid (AsA) in selenite-induced cataract lenses declined to half that of controls. However, 3 days of coffee intake ameliorated the reduction of GSH and AsA so that concentrations remained at 70–80% that of controls. Roasting enhanced the preventive effect of coffee by further reducing cataract formation and ameliorating selenite-induced reduction of antioxidants. High-performance liquid chromatography analysis revealed degradation of chlorogenic acid and generation of pyrocatechol during the coffee roasting process. We discovered that pyrocatechol, at doses equivalent to that found in dark-roasted coffee, was equally effective as caffeine at reducing cataract formation and ameliorating the reduction of antioxidants. Conclusion: Our results indicate that pyrocatechol, generated during the roasting process, acts as an antioxidant together with caffeine to prevent cataract formation.

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Phosphorylated CIS suppresses the Epo or JAK2 V617F mutant-triggered cell proliferation through binding to EpoR.

田村 悦臣

Cellular signalling （Cellular Signalling）  31   41 - 57 2016年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0898-6568

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© 2016 Elsevier Inc. The JAK2 V617F mutant-mediated aberrant signaling pathway is a hallmark of myeloproliferative neoplasms (MPNs). Although cytokine-inducible Src homology 2 protein (CIS) and suppressors of cytokine signaling (SOCS) are negative regulators of the JAK-STAT pathway, the functional role of CIS/SOCS family members in the JAK2 V617F mutant-induced oncogenic signaling pathway has not yet been elucidated. In this study, we found that the expression of CIS and SOCS1 was induced through the activation of signal transducer and activator of transcription 5 (STAT5) in not only the cells stimulated with Epo or IL-3 but also the cells transformed by the JAK2 V617F mutant. Cell proliferation and tumor formation in nude mice induced by the JAK2 V617F mutant were significantly enhanced when the expression of CIS was silenced using an RNA interference technique, whereas the knockdown of SOCS1 had no effect. The enforced expression of CIS caused apoptotic cell death in the transformed by JAK2 V617F mutant and drastically inhibited the JAK2 V617F mutant-induced tumor formation. CIS interacted with phosphorylated EpoR at Y401, which was critical for the activation of STAT5 and ERK. Whereas the activation of STAT5 and ERK in the transformed cells by JAK2 V617F mutant was increased by the knockdown of CIS, the enforced expression of CIS reduced the activation of these molecules. Furthermore, these anti-tumor effects of CIS required the function of SH2 domain and its tyrosine phosphorylation at Y253. We herein elucidated the mechanism by which CIS functions as a novel type of tumor suppressor in JAK2 V617F mutant-induced tumorigenesis.

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Three Tyrosine Residues in the Erythropoietin Receptor Are Essential for Janus Kinase 2 V617F Mutant-induced Tumorigenesis.

田村 悦臣

The Journal of biological chemistry （Journal of Biological Chemistry）  292 （ 5 ） 1826 - 1846 2016年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021-9258

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© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. The erythropoietin receptor (EpoR) regulates development of blood cells, and its full activation normally requires the cytokine erythropoietin (Epo). In the case of myeloproliferative neoplasms (MPN), Epo-independent signaling through EpoR can be caused by a point mutation, V617F, in the EpoR-interacting tyrosine kinase Janus kinase 2 (JAK2). In cells expressing the JAK2 V617F mutant, eight tyrosine residues in the intracellular domain of EpoR are phosphorylated, but the functional role of these phosphorylations in oncogenic signaling is incompletely understood. Here, to evaluate the functional consequences of the phosphorylation of these tyrosine residues, we constructed an EpoR-8YF mutant in which we substituted all eight tyrosine residues with phenylalanine. Co-expression of EpoR-8YF with the JAK2 V617F mutant failed to induce cytokine-independent cell proliferation and tumorigenesis, indicating that JAK2-mediated EpoR phosphorylation is the reason for JAK2 V617F mutant-induced oncogenic signaling. An exhaustive mutational analysis of the eight EpoR tyrosine residues indicated that three of these residues, Tyr-343, Tyr-460, and Tyr-464, are required for the JAK2 V617F mutant to exhibit its oncogenic activity.We also showed that phosphorylation at these three residues was necessary for full activation of the transcription factor STAT5, which is a critical downstream factor of JAK2 V617F-induced oncogenic signaling. In contrast, Epo stimulation could moderately stimulate the proliferation of cells expressing wild type JAK2 and EpoR-8YF, suggesting that the requirement of the phosphorylation of these three tyrosine residues seems to be specific for the oncogenic proliferation provoked by V617F mutation. Collectively, these results have revealed that phosphorylation of Tyr-343, Tyr-460, and Tyr-464 in EpoR underlies JAK2 V617F mutant-induced tumorigenesis. We propose that the targeted disruption of this pathway has therapeutic utility for managing MPN.

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Roasted coffee induces aldo-keto reductase 1C3 (AKR1C3) expression in human prostate cancer LNCaP cells associated with Nrf2 activation.

田村 悦臣

Oncology letters    1 - 6 2016年11月

研究論文（学術雑誌）, 共著, 査読有り

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Inhibitory effects of flavonoids extracted from Nepalese propolis on the LPS signaling pathway.

田村 悦臣

Int. Immunopharmacol. （International Immunopharmacology）  40   550 - 560 2016年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1567-5769

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© 2016 Elsevier B.V. Flavonoids, particularly those derived from plants, harbor biological effects such as anti-inflammation and the inhibition of cancer progression. In the present study, we investigated the effects of 10 kinds of flavonoids isolated from Nepalese propolis on the LPS signaling pathway in order to clarify their anti-inflammatory activities. Five types of flavonoids: isoliquiritigenin, chrysin, 3′,4′-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, and cearoin, markedly inhibited inflammatory responses including LPS-induced NO production by suppressing the expression of iNOS mRNA and LPS-induced mRNA expression of TNFα and CCL2. Their inhibitory effects on LPS-induced inflammatory responses correlated with the intensities of these flavonoids to suppress the LPS-induced activation of nuclear factor κB (NF-κB), an essential transcription factor for the mRNA expression of iNOS, TNFα, and CCL2. Among these flavonoids, 3′,4′-dihydroxy-4-methoxydalbergione and 4-methoxydalbergion markedly inhibited the LPS-induced activation of IKK, thereby abrogating the degradation of IκBα and nuclear localization of NF-κB. On the other hand, isoliquiritigenin, chrysin, and cearoin failed to inhibit these signaling steps, but suppressed the transcriptional activity of NF-κB, which caused their anti-inflammatory effects. The results of the present study revealed that these five kinds of flavonoids are the components of Nepalese propolis that exhibit anti-inflammatory activities with a different regulatory mechanism for the activation of NF-κB.

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The Extracellular C-loop Domain Plays an Important Role in the Cell Adhesion Function of Aquaporin 0.

田村 悦臣

Current eye research （Current Eye Research）  42 （ 4 ） 617 - 624 2016年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0271-3683

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© 2016 Taylor & Francis. Purpose: Although aquaporin 0 (AQP0) is a member of the AQP family, it has limited water permeability compared with other members. AQP0 may also have cell adhesion-related functions, but the evidence is still limited. Here, we studied the relationship of AQP0 to cell adhesion and determined the region required for cell adhesion.Methods: L-cell fibroblasts stably expressing AQP0 or AQP1 (L-AQP0 or L-AQP1) were established. One group of cells was stained with CellTracker Red and cultured into a confluent monolayer, whereas the other group was loaded with CellTracker Blue and seeded over the monolayer. To study cell adhesion, the percentages of lower and upper layer cells were measured using flow cytometry. To determine the region of AQP0 required for adhesion, activity was done by pull-down assay using glutathione S-transferase fusion proteins. To study the water permeability, Xenopus laevis oocyte expressing AQP0 wild-type or AQP0 mutated in C-loop was transferred to a hypotonic solution and photographed, and the diameter was measured to calculate the volume.Results: More cells adhered to the lower cells in the L-AQP0 homotypic pair than other pairs such as L-AQP1 homotypic or L-AQP0/L-AQP1 heterotypic pairs. Pull-down assays revealed that AQP0 could bind to itself via the C-loop extracellular domain. Furthermore, we determined that 109Pro and 110Pro in the C-loop were important for cell adhesion. However, mutation of the C-loop in AQP0 did not affect its water permeability.Conclusions: AQP0 is known to bind lipids in the opposing membrane. Our data suggest that this cell-to-cell adhesion occurs not only in the AQP0/liquids but also via AQP0/AQP0 interaction through the C-loop domain. Mutations in the C-loop amino acids did not affect the water permeability of AQP0 but did affect its cell adhesion function. These independent dual functions of AQP0 are important for lens transparency.

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A bis-malonic acid fullerene derivative significantly suppressed IL-33-induced IL-6 expression by inhibiting NF-κB activation.

田村 悦臣

International immunopharmacology （International Immunopharmacology）  40   254 - 264 2016年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1567-5769

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© 2016 IL-33 functions as a ligand for ST2L, which is mainly expressed in immune cells, including mast cells. IL-33 is a potent inducer of pro-inflammatory cytokines, such as IL-6, and has been implicated in the pathogenesis of allergic inflammatory diseases. Therefore, IL-33 has recently been attracting attention as a new target for the treatment of inflammatory diseases. In the present study, we demonstrated that a water-soluble bis-malonic acid fullerene derivative (C60-dicyclopropane-1,1,1′,1′-tetracarboxylic acid) markedly diminished the IL-33-induced expression of IL-6 in bone marrow-derived mast cells (BMMC). The bis-malonic acid fullerene derivative suppressed the canonical signaling steps required for NF-κB activation such as the degradation of IκBα and nuclear translocation of NF-κB by directly inhibiting the IL-33-induced IKK activation. Although p38 and JNK also contributed to IL-33-induced expression of IL-6, the bis-malonic acid fullerene derivative did not affect their activation. Furthermore, the bis-malonic acid fullerene derivative had no effect on the NF-κB activation pathway induced by TNFα and IL-1. These results suggest that the bis-malonic fullerene derivative has potential as a specific drug for the treatment of IL-33-induced inflammatory diseases by specifically inhibiting the NF-κB activation pathway.

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Effect of hesperetin on chaperone activity in selenite-induced cataract.

田村 悦臣

Open medicine (Warsaw, Poland) （Open Medicine (Poland)）  11 （ 1 ） 183 - 189 2016年06月

研究論文（学術雑誌）, 共著, 査読有り

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© Yosuke Nakazawa et al published by De Gruyter Open 2016. Background. Chaperone activity of α-crystallin in the lens works to prevent protein aggregation and is important to maintain the lens transparency. This study evaluated the effect of hesperetin on lens chaperone activity in selenite-induced cataracts. Methodology. Thirteen-day-old rats were divided into four groups. Animals were given hesperetin (groups G2 and G4) or vehicle (G1 and G3) on Days 0, 1, and 2. Rats in G3 and G4 were administered selenite subcutaneously 4 hours after the first hesperetin injection. On Days 2, 4, and 6, cataract grades were evaluated using slit-lamp biomicroscopy. The amount of a-crystallin and chaperone activity in water-soluble fraction were measured after animals sacrificed. Results. G3 on day 4 had developed significant cataract, as an average cataract grading of 4.6 ± 0.2. In contrast, G4 had less severe central opacities and lower stage cataracts than G3, as an average cataract grading of 2.4 ± 0.4. The a-crystallin levels in G3 lenses were lower than in G1, but the same as G4. Additionally, chaperone activity was weaker in G3 lenses than G1, but the same as in G4. Conclusions. Our results suggest that hesperetin can prevent the decreasing lens chaperone activity and a-crystallin water solubility by administered of selenite.

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Data supporting mitochondrial morphological changes by SPG13-associated HSPD1 mutants.

田村 悦臣

Data in brief 6   482 - 488 2016年03月

研究論文（学術雑誌）, 共著, 査読有り

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Coffee induces vascular endothelial growth factor (VEGF) expression in human neuroblastama SH-SY5Y cells.

田村 悦臣

Nutritional neuroscience （Nutritional Neuroscience）  20 （ 6 ） 336 - 342 2016年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1028-415X

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© 2016 Taylor & Francis. Recent evidence indicates that hypoxia-inducible vascular endothelial growth factor (VEGF) has neurotrophic and neuroprotective effects on neuronal and glial cells. On the other hand, recent epidemiological studies showed that daily coffee consumption has been associated with a lower risk of several neuronal disorders. Therefore, we investigated the effect of coffee on VEGF expression in human neuroblastoma SH-SY5Y cells. We found that even low concentration of coffee (<2%) strongly induced VEGF expression via an activation of HIF-1α. The activation of HIF-1α by coffee was attributed to the coffee-dependent inhibition of prolyl hydroxylation of HIF1α, which is essential for proteolytic degradation of HIF-1α. However, no inhibition was observed at the catalytic activity in vitro. Coffee component(s) responsible for the activation of HIF-1α was not major constituents such as caffeine, caffeic acid, chlorogenic acid, and trigonelline, but was found to emerge during roasting process. The active component(s) was extractable with ethyl acetate. Our results suggest that daily consumption of coffee may induce VEGF expression in neuronal cells. This might be related to protective effect of coffee on neural disorders such as Alzheimer's disease and Parkinson's disease.

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アクアポリン0(AQP0)の細胞間接着に関与するアミン酸の特定

田村 悦臣

日本眼科学会誌 120 （ 1 ） 50 2016年01月

研究論文（学術雑誌）, 共著

A proline-type fullerene derivative inhibits adipogenesis by preventing PPARγ activation.

田村 悦臣

Biochemistry and biophysics reports （Biochemistry and Biophysics Reports）  5   259 - 265 2016年01月

研究論文（学術雑誌）, 共著, 査読有り

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© 2016 The Authors. Obesity and its associated metabolic diseases represent some of the most rapidly expanding health issues worldwide, and, thus, the development of a novel chemical compound to suppress adipogenesis is strongly expected. We herein investigated the effects of water-soluble fullerene derivatives: a bis-malonic acid derivative and three types of proline-type fullerene derivatives, on adipogenesis using NIH-3T3 cells overexpressing PPARγ. One of the proline-type fullerene derivatives (P3) harboring three carboxy groups significantly inhibited lipid accumulation and the expression of adipocyte-specific genes, such as aP2, induced by the PPARγ agonist rosiglitazone. On the other hand, the bis-malonic acid derivative (M) and the 2 other proline-type fullerene derivatives (P1, P2), which have two carboxy groups, had no effect on PPARγ-mediated lipid accumulation or the expression of aP2. P3 fullerene also inhibited lipid accumulation induced by the combined stimulation with 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin in 3T3-L1 preadipocytes. During the differentiation of 3T3-L1 cells into adipocytes, P3 fullerene did not affect the expression of C/EBPδ, C/EBPβ, or PPARγ, but markedly inhibited that of aP2 mRNA. These results suggest that P3 fullerene exhibits anti-obesity activity by preventing the activation of PPARγ.

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Coffee Modulates the Function of Brain-Derived Neurotrophic Factor (BDNF) in Human Neuroblastoma SH-SY5Y Cells.

田村 悦臣

Neuroscience & Medicine 6   165 - 174 2015年12月

研究論文（学術雑誌）, 単著, 査読有り

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Meta-Analysis: Effects of probiotic supplementation on lipid profiles in normal to mildly hypercholesterolemic individuals

田村 悦臣

PLoS ONE （PLoS ONE）  10 （ 10 ） e0139795 2015年10月

研究論文（学術雑誌）, 共著

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© 2015 Shimizu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Recent experimental and clinical studies have suggested that probiotic supplementation has beneficial effects on serum lipid profiles. However, there are conflicting results on the efficacy of probiotic preparations in reducing serum cholesterol. Objective To evaluate the effects of probiotics on human serum lipid levels, we conducted a metaanalysis of interventional studies. Methods Eligible reports were obtained by searches of electronic databases. We included randomized, controlled clinical trials comparing probiotic supplementation with placebo or no treatment (control). Statistical analysis was performed with Review Manager 5.3.3. Subanalyses were also performed. Results Eleven of 33 randomized clinical trials retrieved were eligible for inclusion in the meta-Analysis. No participant had received any cholesterol-lowering agent. Probiotic interventions (including fermented milk products and probiotics) produced changes in total cholesterol (TC) (mean difference-0.17 mmol/L, 95% CI:-0.27 to-0.07 mmol/L) and low-density lipoprotein cholesterol (LDL-C) (mean difference-0.22 mmol/L, 95% CI:-0.30 to-0.13 mmol/ L). High-density lipoprotein cholesterol and triglyceride levels did not differ significantly between probiotic and control groups. In subanalysis, long-Term (>4-week) probiotic intervention was statistically more effective in decreasing TC and LDL-C than short-Term (4-week) intervention. The decreases in TC and LDL-C levels with probiotic intervention were greater in mildly hypercholesterolemic than in normocholesterolemic individuals. Both fermented milk product and probiotic preparations decreased TC and LDL-C levels. Gaio and the Lactobacillus acidophilus strain reduced TC and LDL-C levels to a greater extent than other bacterial strains. Conclusions In conclusion, this meta-Analysis showed that probiotic supplementation could be useful in the primary prevention of hypercholesterolemia and may lead to reductions in risk factors for cardiovascular disease.

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Nepetaefuran and leonotinin isolated from Leonotis nepetaefolia R. Br. potently inhibit the LPS signaling pathway by suppressing the transactivation of NF-κB.

田村 悦臣

International immunopharmacology （International Immunopharmacology）  28 （ 2 ） 967 - 976 2015年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1567-5769

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© 2015 Elsevier B.V. Leonotis nepetaefolia R. Br., also known as Klip Dagga or Lion's Ear, has traditionally been used as a folk medicine to treat inflammatory diseases such as rheumatism, bronchitis, and asthma; however, the components that exhibit its anti-inflammatory activity have not yet been identified. In the present study, we investigated the effects of three types of diterpenoids, nepetaefuran, leonotinin, and leonotin, which were isolated from L. nepetaefolia R. Br., on the LPS signaling pathway in order to elucidate the anti-inflammatory mechanism involved. Nepetaefuran more potently inhibited the LPS-induced production of NO and CCL2 than leonotinin by suppressing the expression of iNOS mRNA and CCL2 mRNA. On the other hand, leonotin failed to inhibit the production of NO and CCL2 induced by LPS. Although nepetaefuran and leonotinin had no effect on the LPS-induced degradation of IκBα or nuclear translocation of NF-κB p65, they markedly inhibited the transcriptional activity of NF-κB. Nepetaefuran and leonotinin also inhibited the transcriptional activity of the GAL4-NF-κB p65 fusion protein. On the other hand, nepetaefuran, leonotinin and leonotin did not affect the LPS-induced activation of MAP kinase family members such as ERK, p38, and JNK. In addition, inhibitory effect of nepetaefuran and leonotinin on NF-κB activation is well correlated with their ability to induce activation of Nrf2 and ER stress. Taken together, these results demonstrated that nepetaefuran and leonotinin could be the components responsible for the anti-inflammatory activity of L. nepetaefolia R. Br. by specifically inhibiting the LPS-induced activation of NF-κB.

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Hypomyelinating leukodystrophy-associated missense mutation in HSPD1 blunts mitochondrial dynamics.

Miyamoto Y, Eguchi T, Kawahara K, Hasegawa N, Nakamura K, Funakoshi-Tago M, Tanoue A, Tamura H, Yamauchi J

Biochemical and biophysical research communications （Biochemical and Biophysical Research Communications）  462 （ 3 ） 275 - 281 2015年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0006-291X

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© 2015 Elsevier Inc. All rights reserved. Myelin-forming glial cells undergo dynamic morphological changes in order to produce mature myelin sheaths with multiple layers. In the central nervous system (CNS), oligodendrocytes differentiate to insulate neuronal axons with myelin sheaths. Myelin sheaths play a key role in homeostasis of the nervous system, but their related disorders lead not only to dismyelination and repeated demyelination but also to severe neuropathies. Hereditary hypomyelinating leukodystrophies (HLDs) are a group of such diseases affecting oligodendrocytes and are often caused by missense mutations of the respective responsible genes. Despite increasing identification of gene mutations through advanced nucleotide sequencing technology, studies on the relationships between gene mutations and their effects on cellular and subcellular aberrance have not followed at the same rapid pace. In this study, we report that an HLD4-associated (Asp-29-to-Gly) mutant of mitochondrial heat shock 60-kDa protein 1 (HSPD1) causes short-length morphologies and increases the numbers of mitochondria due to their aberrant fission and fusion cycles. In experiments using a fluorescent dye probe, this mutation decreases the mitochondrial membrane potential. Also, mitochondria accumulate in perinuclear regions. HLD4-associated HSPD1 mutant blunts mitochondrial dynamics, probably resulting in oligodendrocyte malfunction. This study constitutes a first finding concerning the relationship between disease-associated HSPD1 mutation and mitochondrial dynamics, which may be similar to the relationship between another disease-associated HSPD1 mutation (MitCHAP-60 disease) and aberrant mitochondrial dynamics.

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Quantitation of pilsicainide in microscale samples of human biological fluids using liquid chromatography-tandem mass spectrometry

Shimizu M., Hashiguchi M., Shiga T., Nakamura K., Tamura H., Mochizuki M.

Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences （Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences）  985   172 - 179 2015年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1570-0232

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© 2015 Elsevier B.V. This paper describes a sensitive, reliable method to determine pilsicainide (PLC) levels in microscale sample volumes of human biological fluids using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). PLC and quinidine as an internal standard were extracted with diethylether from 0.1. mL of alkalinized biological fluids. The extract was injected into an analytical column (l-column 2 ODS, 75. mm. ×. 2.1. mm i.d.). The mobile phase for separation consisted of 5. mM ammonium acetate (pH 4.5)/methanol (4:1, v/v) and was delivered at a flow rate of 0.2. mL/min. The drift voltage was 100. V. The sampling aperture was heated at 120. °C and the shield temperature was 260. °C. The ion transitions used to monitor analytes were m/. z 273. →. m/. z 110 for PLC and m/. z 325. →. m/. z 79 for quinidine. The total time for chromatographic separation was less than 8. min. The validated concentration ranges of this method for PLC were 5-2000. ng/mL in plasma, 5-500. ng/mL in ultrafiltered plasma solution, and 25-2000. ng/mL in urine. Mean recoveries of PLC in plasma, ultrafiltered plasma solution, and urine were 93.2-99.7%, 91.4-100.6%, and 93.9-104.7%, respectively. Intra- and interday coefficients of variation for PLC were less than 6.0% and 4.3% in plasma, 6.1% and 3.7% in ultrafiltered plasma solution, and 5.4% and 2.5% in urine at the above concentration ranges, respectively. The lower limit of quantification for PLC in plasma, ultrafiltered plasma solution, and urine were 5. ng/mL, 5. ng/mL, and 25. ng/mL, respectively. This method can be applied to pharmacokinetic study and therapeutic drug monitoring in special populations such as neonates, infants, and the elderly by making effective use of residual samples used for general clinical laboratory testing.

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Anti-inflammatory activity of flavonoids in Nepalese propolis is attributed to inhibition of the IL-33 signaling pathway.

Funakoshi-Tago M, Okamoto K, Izumi R, Tago K, Yanagisawa K, Narukawa Y, Kiuchi F, Kasahara T, Tamura H

International immunopharmacology （International Immunopharmacology）  25 （ 1 ） 189 - 198 2015年03月

研究論文（学術雑誌）, 共著,  ISSN  1567-5769

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© 2015 Elsevier B.V. All rights reserved. Propolis has been used in folk medicine to improve health and prevent inflammatory diseases; however, the components that exhibit its anti-inflammatory activity remain unknown. We herein investigated the effects of flavonoids isolated from Nepalese propolis on the IL-33 signaling pathway to clarify the anti-inflammatory mechanism involved. Of the 8 types of flavonoids isolated from Nepalese propolis, 4 types of compounds, such as 3′,4′-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, cearoin, and chrysin, markedly inhibited the IL-33-induced mRNA expression of inflammatory genes including IL-6, TNFα and IL-13 in bone marrow-derived mast cells (BMMC). These four flavonoids also inhibited the IL-33-induced activation of nuclear factor κB (NF-κB), which was consistent with their inhibitory effects on cytokine expression. The effects of these flavonoids are attributed to inhibition of IL-33-induced activation of IKK, which leads to the degradation of IκBα and nuclear localization of NF-κB. On the other hand, other flavonoids isolated from Nepalese propolis, such as isoliquiritigenin, plathymenin, 7-hydroxyflavanone, and (+)-medicarpin, had no effect on the IL-33 signaling pathway or cytokine expression. Therefore, these results indicate that 3′,4′-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, cearoin, and chrysin are the substances responsible for the anti-inflammatory activity of Nepalese propolis.

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Effects of Coffee on Estrogen Sulfation in Human Colon Carcinoma Caco-2 Cells

Tamura H.

Coffee in Health and Disease Prevention （Coffee in Health and Disease Prevention）     545 - 552 2015年

研究論文（学術雑誌）,  ISSN  9780124095175

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© 2015 Elsevier Inc. All rights reserved. Recent epidemiological studies have shown that moderate coffee consumption is associated with a lower risk of certain types of cancers, particularly colon cancer in postmenopausal women. To elucidate the molecular basis of the preventive action of coffee toward colon cancer, we focused on the effects of coffee on the metabolism of estrogens by sulfation, a reaction that inactivates estrogens. Estrogens are sulfonated by estrogen sulfotransferase (SULT1E1), and the sulfonated estrogens are reversely hydrolyzed by steroid sulfatase (STS). Sulfonated estrogens are pumped out of the cells by an ABC transporter, breast cancer resistance protein (BCRP). Intracellular concentrations of active estrogens may be determined by the balance of these activities. In human colon cancer Caco-2 cells, coffee decreases the expression of SULT1E1 and STS, whereas it increases BCRP expression. Activities of these three proteins change according to the expression level of each gene. Active constituents of coffee extract can be generated during the roasting process of coffee beans at different times. Modulation of the expression of these genes is likely to be mediated via activation of transcription factors such as NFκB (. BCRP) and stimulating protein 1 (. SULT1E1). These results suggest that coffee consumption may modulate the inactivation of estrogens by sulfation in human colon cells.

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Coffee inhibits adipocyte differentiation via inactivation of PPARgamma.

Ryohei Aoyagi, Megumi Funakoshi-Tago, Yosuke Fujiwara and Hiroomi Tamura*.

Biol. Pharm. Bull. 37 （ 11 ） 1820 - 1825 2014年11月

研究論文（学術雑誌）, 共著, 査読有り

Dexamemthasone suppresses neurosteroid biosynthesis via downregulation of steroidogenic enzyme gene expression in human glioma GI-1 cells.

Koibuchi F, Ritoh N, Aoyagi R, Funakoshi-Tago M, Tamura H*.

Bio Pharm Bull 37(7)   1241-1247 2014年07月

研究論文（学術雑誌）, 共著, 査読有り

New diterpenoids with estrogen sulfotransferase inhibitory activity from Leonurus sibiricus L.

Narukawa Y, Niimura A, Noguchi H, Tamura H, Kiuchi F

Journal of natural medicines （Journal of Natural Medicines）  68(1) （ 1 ） 125-131 - 31 2014年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1340-3443

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Four new diterpenoids (1-4), along with eight known diterpenoids (5-12) were isolated from the acetone extract of Leonurus herb (aerial parts of Leonurus sibiricus L.). The structures of the compounds were determined by spectroscopic methods. Among the isolated compounds, compounds 2, 3, 8, 9 and 10 showed inhibitory activity against human liver cytosol estrogen sulfotransferase (E-ST), which plays a key role in the maintenance of cellular estrogen levels. Compound 2 showed the strongest activity with an IC 50 value of 7.9 μM, which is comparable to the activity of the positive control, meclofenamic acid (IC50 5.4 μM). © The Japanese Society of Pharmacognosy and Springer Japan 2013.

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Dexamethasone suppresses neurosteroid biosynthesis via downregulation of steroidogenic enzyme gene expression in human glioma GI-1 cells.

Koibuchi F, Ritoh N, Aoyagi R, Funakoshi-Tago M, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  37 （ 7 ） 1241 - 7 2014年

研究論文（学術雑誌）,  ISSN  0918-6158

　概要を見る

Emerging evidence indicates that stress hormone glucocorticoids (GC) are an important modulator of brain development and function. To investigate whether GCs modulate neurosteroid biosynthesis in neural cells, we studied the effects of GCs on steroidogenic gene expression in human glioma GI-1 cells. The GC dexamethasone (Dex) reduced steroidogenic acute regulatory protein (StAR), CYP11A1 and 3β-hydroxysteroid dehydrogenase gene expression in a dose- and GC receptor-dependent manner. In addition to its effects on steroidogenic gene expression, Dex also reduced de novo synthesis of progesterone (PROG). Furthermore, Dex inhibited all-trans retinoic acid (ATRA) and vitamin D 3-induced steroidogenic gene expression and PROG production. This suggests that GC regulates steroidogenic gene expression in neural cells via cross-talk with the two fat-soluble vitamins, A and D. The relationship between the effects of GCs on neurosteroid biosynthesis and on cognitive behaviors and hippocampal neural activity is also discussed herein. © 2014 The Pharmaceutical Society of Japan.

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Coffee inhibits adipocyte differentiation via inactivation of PPARγ.

Aoyagi R, Funakoshi-Tago M, Fujiwara Y, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  37 （ 11 ） 1820 - 5 2014年

研究論文（学術雑誌）,  ISSN  0918-6158

　概要を見る

© 2014 The Pharmaceutical Society of Japan. Recent epidemiological studies showed that coffee consumption is associated with a lower risk of type 2 diabetes, presumably due to suppression of excess fat accumulation in adipocytes. However, the mechanism underlying the effect of coffee on adipocyte differentiation has not been well documented. To elucidate the mechanism, we investigated the effect of coffee on the differentiation of mouse preadipocyte 3T3-L1 cells. Coffee reduced the accumulation of lipids during adipocytic differentiation of 3T3-L1 cells. At 5% coffee, the accumulation of lipids decreased to half that of the control. Coffee also inhibited the expression of the peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor controlling the differentiation of adipocytes. Furthermore, coffee reduced the expression of other differentiation marker genes, aP2, adiponectin, CCAAT-enhancer-binding protein α (C/EBPα), glucose transporter 4 (GLUT4), and lipoprotein lipase (LPL), during adipocyte differentiation. Major bioactive constituents in coffee extracts, such as caffeine, trigonelline, chlorogenic acid, and caffeic acid, showed no effect on PPARγ gene expression. The inhibitory activity was produced by the roasting of the coffee beans.

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Human keratinocyte caspase-14 expression is altered in human epidermal 3D models by dexamethasone and by natural products used in cosmetics.

Kataoka S, Hattori K, Date A, Tamura H

Archives of dermatological research （Archives of Dermatological Research）  305 （ 8 ） 683-689 - 9 2013年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0340-3696

　概要を見る

Caspase-14 is a cysteinyl-aspartate-specific proteinase that is specifically expressed in epidermal keratinocytes. Dysregulation of caspase-14 expression is implicated in impaired skin barrier formation. To elucidate the regulation of caspase-14 in differentiated keratinocytes, we characterized the expression of caspase-14 in normal human epidermal keratinocytes (NHEKs) and two types of three-dimensional (3D) human epidermis culture models, EPI-200 and EPI-201, via RT-PCR and immunoblot analyses. Caspase-14 expression was absent in subconfluent NHEKs, but was present in confluent NHEKs as well as those induced to differentiate by calcium. Caspase-14 expression levels in the 3D epidermis models were almost equal to that in the Ca2+-treated differentiated NHEKs. Despite the presence of caspase-14 expression in these models, caspase-14 activity was found only in the mature 3D skin model, EPI-200. This was confirmed by detection of a 17 kDa cleaved fragment of caspase-14 present only in the EPI-200 model. Since glucocorticoid (GC) receptor is required for skin barrier competence, we investigated whether the GC dexamethasone (Dex) and various natural components of common skin moisturizers affect caspase-14 expression in keratinocytes. Dex decreased caspase-14 expression in undifferentiated, but not differentiated, NHEKs. Conversely, Dex increased caspase-14 expression in both 3D skin models, although it did not alter caspase protease activity. Similar to treatment with Dex, treatment of the premature 3D skin mode, EPI-201 with a Galactomyces ferment filtrate markedly increased expression of caspase-14. Further, these results suggest that the effect of Dex, or lack thereof, on caspase-14 expression is dependent on the stage of keratinocyte differentiation. © 2013 Springer-Verlag Berlin Heidelberg.

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Coffee reduces SULT1E1 expression in human colon carcinoma Caco-2 cells.

Isshiki M, Ohta H, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  36(2) （ 2 ） 229-304 - 304 2013年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0918-6158

　概要を見る

Recent epidemiological studies have shown that moderate coffee consumption is associated with a lower risk of certain types of cancers, particularly colon cancer in postmenopausal women. To elucidate the molecular basis for the preventive action of coffee, we investigated the effect of coffee on estrogen sulfotransferase (SULT) because sulfation is the major pathway involved in the inactivation of estrogens. We found that coffee reduced SULT1E1 gene expression in human colon carcinoma Caco-2 cells. Treatment with 2.5% (v/v) coffee for 24 h resulted in a 60% reduction of the expression of the SULT1E1 gene in Caco-2 cells. Corresponding to reduced SULT1E1 gene expression, cytosolic estrogen SULT activity toward E2 (20 nm) decreased by 25%. In addition, an accumulation of E2 sulfates in the medium, which reflects cellular activity of estrogen SULT, decreased after the cells were treated with coffee. Major bioactive constituents in coffee such as caffeine, chlorogenic acid and caffeic acid did not show any effect. The inhibitory activity was extractable by using ethyl acetate. We also found that the inhibitory activity was produced by roasting the coffee beans. Mithramycin, an inhibitor of the transcription factor stimulating protein 1 (Sp1), was able to restore coffee-reduced SULT1E1 gene expression. Our data suggest that daily coffee consumption may reduce estrogen SULT activity, thereby enhancing estrogenic activity in the colon. © 2013 The Pharmaceutical Society of Japan.

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Vitamin D3 enhances all-trans retinoic acid (ATRA)-mediated neurosteroid biosynthesis in human glioma GI-1 cells.

Yagishita T, Kushida A, Tamura H*.

J Biochem 152(3)   285-292 2012年10月

研究論文（学術雑誌）, 共著, 査読有り

Interferon gamma induces steroid sulfatase expression in human keratinocytes.

Hattori K, Yamaguchi N, Umezawa K, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  35(9) （ 9 ） 1588-1593 - 93 2012年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0918-6158

　概要を見る

Steroid sulfatase (STS) plays an important role in steroid metabolism in which estrogens and dehydroepiandrosterone (DHEA) are produced from their sulfates. However, little is known about the transcriptional regulation of the STS gene in keratinocytes. Since keratinocytes are thought to be a primary target of interferon gamma (IFNγ) in inflammatory and immune responses, we assessed the effects of this cytokine upon STS gene expression in the human keratinocyte cell line SVHK and in normal human keratinocytes (NHEK). Stimulation of SVHK cells with 50 ng/mL of IFNγ for 24 h induced an approximately three-fold increase in STS activity and in its mRNA levels compared to non-treated cells. IFNγ treatment also induced an approximately 1.5-fold increase in STS mRNA levels in NHEK cells. This induction was completely inhibited by treatment with phosphatidylinositol (PI) 3-kinase inhibitors such as LY294002 or wortmannin, and by the nuclear factor-kappa B (NF-κB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). These data suggest that activation of the PI 3-kinase signal transduction pathway mediates induction of STS gene expression by IFNγ through activation of NF-κB. The anti-inflammatory agent dexamethasone inhibited IFNγ induction of STS gene expression, suggesting involvement of a glucocorticoid receptor in the regulation of STS gene expression in keratinocytes. Regulation of STS gene expression in skin as a novel target of drugs for therapy of psoriasis in the skin is discussed. © 2012 The Pharmaceutical Society of Japan.

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Vitamin D(3) enhances ATRA-mediated neurosteroid biosynthesis in human glioma GI-1 cells.

Yagishita T, Kushida A, Tamura H

Journal of biochemistry （Journal of Biochemistry）  152 （ 3 ） 285 - 92 2012年09月

研究論文（学術雑誌）,  ISSN  0021-924X

　概要を見る

Emerging evidence indicates that vitamin D (VD) is an important modulator of brain development and function. To investigate whether VD modulates neurosteroid biosynthesis in neural cells, we investigated the effect of VD 3 on steroidogenic gene expression in human glioma GI-1 cells. We found that VD3 enhanced CYP11A1 and 3β-hydroxysteroid dehydrogenase gene expression. The induction of CYP11A1 gene expression by VD3 was dose-and incubation time-dependent. Calcipotriol, a VD 3 receptor (VDR) agonist, also induced CYP11A1 gene expression in GI-1 cells, indicating that VDR is involved in this induction. The induction of progesterone (PROG) de novo synthesis was observed along with the induction of steroidogenic genes by VD3. Furthermore, VD3 enhanced all-trans retinoic acid (ATRA)-induced CYP11A1 gene expression and PROG production. This suggests cooperative regulation of steroidogenic gene expression by the two fat-soluble vitamins, A and D. In addition, a mixed culture of neuronal IMR-32 cells and GI-1 cells treated with ATRA and VD 3 resulted in the induction of PROG-responsive gene expression in the IMR-32 cells. This result shows a paracrine action of PROG that is induced in and released by the GI-1 cells. The relationship between neurological dysfunction associated with VD deficiency and neurosteroid induction by VD is discussed. © 2012 The Authors.

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Xanthuretic acid is the endogenous substrate of silkworm cytosolic sulfotransferase bmST1.

Kushida A, Horie R, Hattori K, Sekimizu K, Tamura H*.

J Insect Physiol 58(1)   83-88 2012年01月

研究論文（学術雑誌）, 共著, 査読有り

Xanthurenic acid is an endogenous substrate for the silkworm cytosolic sulfotransferase, bmST1.

Kushida A, Horie R, Hattori K, Hamamoto H, Sekimizu K, Tamura H

Journal of insect physiology （Journal of Insect Physiology）  58 （ 1 ） 83 - 8 2012年01月

研究論文（学術雑誌）,  ISSN  0022-1910

　概要を見る

Sulfotransferase enzymes are known to regulate physiologically active substances such as steroids and catecholamines in mammals. Although invertebrates also express sulfotransferases, their biological function is mostly unclear. In a previous study, we reported that 4-nitrocatechol and the gallete ester are substrates for the silkworm sulfotransferase bmST1. The Km of bmST1 for these substrates is high. However, endogenous substrates of bmST1 have not yet been determined. We therefore investigated endogenous bmST1 substrates and carried out a detailed expression profile analysis of bmST1. We found that xanthurenic acid, a tryptophan metabolite, is a possible endogenous substrate of bmST1. The Km of bmST1 for xanthurenic acid is low, in the μM range, which is lower than that for previously reported substrates. Additionally, xanthurenic acid is a tryptophan metabolite that characteristically shows toxicity in vivo. High dose administration of xanthurenic acid resulted in inhibition of cuticular biosynthesis. The expression of the bmST1 gene reached a maximal level in the Malpighian tubule at the 4th molting stage, when amino acid metabolism might be activated. Our results suggest that bmST1 plays a role in detoxification of xanthurenic acid in the silkworm. © 2011 Elsevier Ltd.

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Coffee induces 11β-hydroxysteroid dehydrogenase type 2 activity in human placental choriocarcinoma cells, JEG-3

Arao T., Tamura H.

Journal of Health Science （Journal of Health Science）  57 （ 5 ） 436 - 441 2011年10月

研究論文（学術雑誌）,  ISSN  13449702

　概要を見る

Coffee is the beverage consumed during pregnancy that is most commonly associated with adversely affecting the developing fetoplacental unit. Since placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD-2) is an important determinant for maintenance of fetal glucocorticoid levels during pregnancy, we investigated the effect of coffee on 11β-HSD-2 expression in the human placental choriocar-cinoma cell line, JEG-3. Addition of coffee to the culture medium of JEG-3 cells increased 11β-HSD-2 gene expression after 24 hr in a dose-dependent manner. Cellular 11β-HSD-2 activity, which was calculated by the amount of cortisol that was converted to cortisone in the culture medium, also increased in the coffee-treated JEG-3 cells. None of the major constituents of coffee, such as caffeine, caffeic acid or chlorogenic acid, showed any ability to induce 11β-HSD-2 gene expression. The 11β-HSD-2-inducing activity in coffee was extractable with ethyl acetate. Increasing the degree of roasting improved the ability of the coffee extract to induce 11β-HSD-2 gene expression indicating that the active constituent(s) is produced during the roasting process. © 2011 The Pharmaceutical Society of Japan.

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Coffee induces 11b-hydroxysteroid dehydrogenase type 2 activity in human placental choriocarcinoma cells, JEG-3.

Arao T, Tamura H*.

J Health Sci 57   436-441 2011年10月

研究論文（学術雑誌）, 共著, 査読有り

Coffee induces breast cancer resistance protein expression in Caco-2 cells.

Isshiki M, Umezawa K, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  34 （ 10 ） 1624-1627 - 7 2011年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0918-6158

　概要を見る

Coffee is a beverage that is consumed world-wide on a daily basis and is known to induce a series of metabolic and pharmacological effects, especially in the digestive tract. However, little is known concerning the effects of coffee on transporters in the gastrointestinal tract. To elucidate the effect of coffee on intestinal transporters, we investigated its effect on expression of the breast cancer resistance protein (BCRP/ABCG2) in a human colorectal cancer cell line, Caco-2. Coffee induced BCRP gene expression in Caco-2 cells in a coffee-dose dependent manner. Coffee treatment of Caco-2 cells also increased the level of BCRP protein, which corresponded to induction of gene expression, and also increased cellular efflux activity, as judged by Hoechst33342 accumulation. None of the major constituents of coffee tested could induce BCRP gene expression. The constituent of coffee that mediated this induction was extractable with ethyl acetate and was produced during the roasting process. Dehydromethylepoxyquinomicin (DHMEQ), an inhibitor of nuclear factor (NF)-κB, inhibited coffee-mediated induction of BCRP gene expression, suggesting involvement of NF-κB in this induction. Our data suggest that daily consumption of coffee might induce BCRP expression in the gastrointestinal tract and may affect the bioavailability of BCRP substrates. © 2011 Pharmaceutical Society of Japan.

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Sulfation of estradiol in human epidermal keratinocyte.

Kushida A, Hattori K, Yamaguchi N, Kobayashi T, Date A, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  34(7) （ 7 ） 1147-1151 - 51 2011年07月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0918-6158

　概要を見る

Epidermis is one of the well-known estrogen target tissues. Information regarding estrogen metabolism in epidermis is still very limited compared to that of estrogen action. In the breast cancer tissue, 17β-estradiol (E 2) is inactivated by sulfation and the expression level of estrogen sulfotransferase (SULT1E1) is inversely correlated with its malignancy. However, there is little datum about inactivation of estradiol in skin. In order to detect and measure E 2 and its metabolites simultaneously, we established an assay method with radio HPLC. A majority of [ 3H] labeled E 2 was converted to E 2 sulfate in normal human epidermal keratinocyte (NHEK) cells. The estimated activity of sulfotransferase toward E 2 at 20 nM was 0.11±0.01 (pmol/min/mg protein). Significant induction of estrogen sulfotransferase activity was observed in calcium-differentiated NHEK cells (0.58±0.07 (pmol/min/mg protein)). The gene expression of SULT1E1 was fifteen-fold higher in differentiated keratinocyte than in proliferating keratinocyte, whereas that of steroid sulfatase was reduced. These results suggest that E 2 inactivation is primarily mediated by SULT1E1 in keratinocyte and E 2 action is likely suppressed in epidermal differentiation. © 2011 Pharmaceutical Society of Japan.

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ブラックボックスから玉手箱へ.

田村悦臣.

塾 （慶應義塾）  48   28 2011年04月

研究論文（学術雑誌）, 単著

Megadose vitamin C suppresses sulfoconjugation in human colon carcinoma cell line Caco-2.

Tsuruta H, Yagishita T, Shimizu M, Tamura H

Toxicology in vitro : an international journal published in association with BIBRA （Toxicology in Vitro）  25 （ 2 ） 500-504 - 4 2011年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0887-2333

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Intake of high doses of vitamin C has known to modulate sulfoconjugation of drugs in the intestine, but the underlying mechanisms for this effect remain to be elucidated. In the present study, we investigated the effects of vitamin C (l-ascorbic acid (AA)) on sulfation of 1-naphthol using Caco-2 cells, a model of human intestinal cells. We found that high dose of AA inhibited the accumulation of 1-naphthyl sulfate in Caco-2 culture medium within 24h in a dose-dependent manner (IC 50=42mM). Dehydroascorbic acid (DA), an oxidized form of AA, showed no inhibition. AA did not inhibit the in vitro sulfotransferase (SULT) activity toward 1-naphthol, whereas it reduced the expression of genes belonging to SULT1A family, SULT1A1 and SULT1A3. DA showed no effect on SULT1A gene expression. Consistent with the reduction in gene expression, AA reduced the cytosolic SULT activity towards 1-naphthol in the AA-treated Caco-2 cells. In addition, cAMP exerted an additive effect on AA-mediated repression of SULT1A gene expression. Our results suggest that megadose AA suppresses sulfoconjugation in the intestine mainly by downregulating the expression of SULT1A genes. © 2010 Elsevier Ltd.

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Effects of coffee on the metabolism of glucocorticoid in human syncytiotrophoblast JEG-3 cells.

Arao T, Tamura H.

Journal of Health Science （日本薬学会）  56   131 2010年10月

研究論文（学術雑誌）, 共著, 査読有り

Effects of cytochrome P450 inducers on the gene expression of ocular xenobiotic metabolizing enzymes in rats

Sugamo T., Nakamura K., Tamura H.

Journal of Health Science （Journal of Health Science）  55(6) （ 6 ） 923-929 - 929 2009年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  13449702

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In our current study, we investigated the expression profiles of the cytochromes P450 (CYPs) and transporters of the ocular tissues in Sprague-Dawley (SD) rats. Extensive expression of CYP1A1 in the cornea and CYP2E1 in the iris was observed whereas the expression of CYP2B1 and transporters was mostly ubiquitous throughout the ocular tissues. To further understand the regulation of these genes in ocular tissues, we investigated the effects of CYP inducers on the expression of the CYP and other xenobiotic-metabolizing enzyme genes in the cornea and lens. The administration of β-naphthoflavone (BNF), an agonist for aryl hydrocarbon receptor (AhR), induced CYP1A1 gene expression in the cornea, lens and liver of the rats, although the levels of induction were greatest in the liver. An AhR-sensitive UDP-glucuronosyl transferase (UGT) 1A6 gene was also induced in the cornea and the lens by BNF. Phenobarbital (PB) is a known inducer of the CYP2B genes, the expression of which is mediated by constitutive activated receptor (CAR), but did not induce CYP2B1 in the cornea or lens. This insensitivity to PB may be due to the lack of CAR expression in the ocular tissues as revealed in our present study. Pregnenolone-16α- carbonitrile (PCN) is known to induce CYP3A gene expression in the liver via the activation of pregnane X receptor (PXR). However, although PCN was found to induce the CYP3A1 gene in the rat cornea and liver, it failed to do so in the lens. In addition, another of the PXR-mediated genes, multidrug resistance-associated protein 3 (Mrp3), was not induced by PCN in either ocular region. Since the expression of the PXR gene was not detected in the rat ocular tissues, an unknown mechanism for the inducible regulation of CYP3A1 gene expression by PCN in the cornea is suggested. ©2009 The Pharmaceutical Society of Japan.

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Retinoic acids induce neurosteroid biosynthesis in human glial GI-1 cells via the induction of steroidgenic genes.

Kushida A, Tamura H*.

J Biochem 146(6)   917-923 2009年12月

研究論文（学術雑誌）, 共著, 査読有り

Retinoic acids induce neurosteroid biosynthesis in human glial GI-1 Cells via the induction of steroidogenic genes.

Kushida A, Tamura H

Journal of biochemistry （Journal of Biochemistry）  146 （ 6 ） 917 - 23 2009年12月

研究論文（学術雑誌）,  ISSN  0021-924X

　概要を見る

The steroids synthesized in the central nervous system (CNS) are the neurosteroids. Since little information is currently available concerning the roles of the retinoic acids (RAs) during steroidogenesis in the CNS, we investigated the effects of RAs upon their synthesis in our current study. Specifically, we analyzed the effects of all-trans-retinoic acid (ATRA) upon the expression of neurosteroid biosynthesis genes in the human glial cell line GI-1, in which the major steroidogenic genes are expressed. Treatment with ATRA (10 μM) induced a 4.9-fold increase in the expression of the cytochrome P450scc (CYP11A1) gene, the product of which cleaves the cholesterol side chain, a rate-limiting step during steroidogenesis. ATRA also strongly induced the expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) (an increase of 5- and 50-fold, respectively). A retinoic acid receptor (RAR)-specific agonist, TTNPB, was unable to mimic this induction whereas a retinoid X receptor (RXR)-specific agonist, methoprene acid, in addition to 9-cis-RA, could do so. These data indicate that ATRA is isomerized to 9-cis-RA in the culture medium, as reported previously, and that 9-cis-RA activates the RXR. In addition, ATRA also induced the de novo synthesis of neurosteroids such as pregnenolone and progesterone. These results suggest that ATRA might induce the de novo neurosteroid synthesis via the induction of steroidogenic genes in human glial cells. The multiple effects of vitamin A upon CNS functions might therefore be partly explained by the induction of neurosteroidogenesis by RAs, since neurosteroids have also been reported to have multiple effects in the CNS.

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ヒト消化管組織を用いたin vitro試験の有用性と課題について考える.

○田村悦臣.

HAB NEWS LETTER （エイチ・エー・ビー研究機構）  16(2)   7-8 2009年10月

研究論文（学術雑誌）, 単著

Pomegranate juice inhibits sulfoconjugation in the human colon carcinoma cell line, Caco-2.

Saruwatari A, Okamura S, Nakajima Y, Narukawa Y, Takeda Y, Tamura H*.

J Medicinal Food 11(4)   623-628 2008年12月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Coffee inhibits the glucuronidation of 1-naphthol in rat syncytiotrophoblast cells

Ueno M., Nakashima E., Tamura H.

Journal of Health Science （Journal of Health Science）  54(6) （ 6 ） 697-703 - 702 2008年12月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  13449702

　概要を見る

The placenta acts as a barrier that protects the fetus from xenobiotics in the maternal blood. Phase II conjugation reactions in the placenta are considered to play an important role in this protective process by transferring polar hydroxyl groups and thereby imparting water solubility that facilitates the excretion of xenobiotics. Since coffee consumption during pregnancy is reported to affect fetal growth and development, we examined its effects upon the conjugation reactions in the rat syncytiotrophoblast cell line TR-TBT 18d-1. We detected a high level of glucuronidation for 1-naphthol, a model compound, in both a dose- and culture time-dependent manner in the TR-TBT 18d-1 cells. However, no sulfation was detected in any of our analyses. Coffee was found to inhibit the glucuronidation of 1-naphthol with an IC50 of 4.5% (v/v). In contrast however, caffeine, which is a major bioactive constituent of coffee, did not show any inhibition at doses of up to 100 μM. Coffee was also found to inhibit UDP-glucuronosyl transferase (UGT) activity towards 1-naphthol in vitro to a similar extent [IC50 = 1.5% (v/v)] as in intact cells. Moreover, the expression of the UGT 1A6, which is known to mainly catalyze the glucuronidation of 1-naphthol, was not affected by coffee. If coffee inhibits placental glucuronidation during pregnancy, its consumption would increase the fetal plasma concentrations of harmful xenobiotics, potentially affecting normal fetal growth and development.

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Pomegranate juice inhibits sulfoconjugation in Caco-2 human colon carcinoma cells.

Saruwatari A, Okamura S, Nakajima Y, Narukawa Y, Takeda T, Tamura H

Journal of medicinal food （Journal of Medicinal Food）  11 （ 4 ） 623 - 8 2008年12月

研究論文（学術雑誌）,  ISSN  1096-620X

　概要を見る

Several fruit juices have been reported to cause food-drug interactions, mainly affecting cytochrome P450 activity; however, little is known about the effects of fruit juices on conjugation reactions. Among several fruit juices tested (apple, peach, orange, pineapple, grapefruit, and pomegranate), pomegranate juice potently inhibited the sulfoconjugation of 1-naphthol in Caco-2 cells. This inhibition was both dose- and culture time-dependent, with a 50% inhibitory concentration (IC50) value calculated at 2.7% (vol/vol). In contrast, no obvious inhibition of glucuronidation of 1-naphthol in Caco-2 cells was observed by any of the juices examined. Punicalagin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfoconjugation in Caco-2 cells with an IC50 of 45 μM, which is consistent with that of pomegranate juice. These data suggest that punicalagin is mainly responsible for the inhibition of sulfoconjugation by pomegranate juice. We additionally demonstrated that pomegranate juice and punicalagin both inhibit phenol sulfotransferase activity in Caco-2 cells in vitro, at concentrations that are almost equivalent to those used in the Caco-2 cells. Pomegranate juice, however, shows no effects on the expression of the sulfotransferase SULT1A family of genes (SULT1A1 and SULT1A3) in Caco-2 cells. These results indicate that the inhibition of sulfotransferase activity by punicalagin in Caco-2 cells is responsible for the reductions seen in 1-naphthyl sulfate accumulation. Our data also suggest that constituents of pomegranate juice, most probably punicalagin, impair the enteric functions of sulfoconjugation and that this might have effects upon the bioavailability of drugs and other compounds present in food and in the environment. These effects might be related to the anticarcinogenic properties of pomegranate juice. © 2008 Mary Ann Liebert, Inc.

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Inhibitory effects of various beverages on the sulfoconjugation of 17b-estradiol in human colon carcinoma Caco-2 cells.

Saruwatari A, Isshiki M, Tamura H*.

Biol Pharm Bull 31(11)   2131-2136 2008年11月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Inhibitory effects of various beverages on the sulfoconjugation of 17beta-estradiol in human colon carcinoma Caco-2 cells.

Saruwatari A, Isshiki M, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  31 （ 11 ） 2131 - 6 2008年11月

研究論文（学術雑誌）,  ISSN  0918-6158

　概要を見る

To investigate the possible effects of different beverages in the gastrointestinal tract on the sulfation of estrogen, which is a major hormone and prototype substrate for the human sulfotransferases (SULT), we analyzed the effects of these substances upon the sulfate conjugation of 17β-estradiol (E2) in the human colon carcinoma cell line Caco-2. Sulfoconjugation activity toward E2 was measured by incubating 20 nM E2 with Caco-2 cells in the presence (5% (v/v)) of each beverage. Among the 35 beverages analyzed, four (aronia, blueberry, coffee, and peppermint) exhibited strong inhibitory effects on E2 sulfation within Caco-2 cells (IC50 values ranging from 1.9 to 4.4% (v/v)). These active beverages also strongly inhibited the cytosolic estrogen SULT activity of Caco-2 cells in vitro (IC50 values ranging from 0.18 to 0.3% (v/v)). These inhibitory activities were extractable with ethyl acetate but not hexane or n-butanol, indicating that the molecules responsible are moderately lipophilic. Coffee was found to be the most potent inhibitor but the major constituents of this beverage, caffeic acid, caffeine, and chlorogenic acid, did not show any effects on estrogen SULT activity. Kinetic analyses further indicated that the mode of inhibition by coffee is competitive. A possible relationship between the inhibition of estrogen SULT activity by coffee in the gastrointestinal tract and the reported reduction of colon cancer incidence in women who consume coffee is discussed. © 2008 Pharmaceutical Society of Japan.

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Cloning, expression, and characterization of cytosolic sulfotransferase isozymes from Drosophila melanogaster

Hattori K., Motohashi N., Kobayashi I., Tohya T., Oikawa M., Tamura H.

Bioscience, Biotechnology and Biochemistry （Bioscience, Biotechnology and Biochemistry）  72 （ 2 ） 540-547 - 547 2008年02月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  09168451

　概要を見る

We have identified four cytosolic sulfotransferase (SULT) homologs in the genome database of Drosophila melanogaster, and have designated these genes dmST1-4. Each of these four isozymes was subsequently classified into a different and novel gene family, as determined by the low amino acid sequence homology (less than 40%) between them, and also toward their vertebrate homologs. The transcripts for these four SULT homologs were detectable at all developmental stages in D. melanogaster. In addition, three of these isozymes, the exception being dmST2, were successfully expressed and purified in Escherichia coli. These recombinant dmST1, 3, and 4 products showed high sulfating activity toward phenolic compounds such as vanillin and 4-nitrophenol, but showed no activity toward typical endogenous substrates such as tyramine and serotonin. DmST4 and dmST3 showed the lowest and highest Km values for 3′-phosphoadenosine-5′-phosphosulfate (PAPS) respectively. DmST4 also showed low but not negligible activity toward 20-hydroxyecdysone.

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Changes in the expression of cytochrome P450 genes in hemin-induced differentiated K562 cells.

Nagai F, Shimizu M, Sakamoto T, Kobayashi T, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  30 (10) （ 10 ） 1954-1957 - 7 2007年10月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  0918-6158

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We have previously reported the expression of CYP genes in human myeloblastic and lymphoid cell lines, and the induction of the CYP3A4 and GSTP1 genes by oxidative stress in the human erythroleukemia cell line, K562. To further elucidate the role of drug metabolizing enzymes in hematogenesis, we have characterized the expression of CYP genes in hemin-induced differentiated K562 cells. After incubation with 50 μM hemin for 3 d, the expression of CYP1A1 and CYP3A4 genes was induced by 2.5- and 3.5-fold, respectively. In contrast, the CYP1B1 and CYP2E1 genes were downregulated in these cells to below 10% of the control levels. Moreover, these changes correlated with the hemin dose and culture time. Metabolism of midazolam, a probe substrate for CYP3A4, in the differentiated K562 cells increased by 2-folds, suggesting that the induction of CYP3A4 activity is consistent with the mRNA level. If these changes in the CYP expression profile in hematopoietic cells occurred, the susceptibility to xenobiotics and/or the therapeutic drugs of the cells might be influenced, and it also affects the metabolism of endogenous substrates, such as steroids and prostaglandins. © 2007 Pharmaceutical Society of Japan.

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Changes in the gene expression patterns of the cytochrome P450s in selenite-induced cataracts in the rat

Nakamura K., Fujiki T., Tamura H.

Journal of Health Science （Journal of Health Science）  53(3) （ 3 ） 347-350 - 350 2007年06月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  13449702

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A single injection of an overdose of selenite is known to induce cataracts in young rat eyes. In our current study, we have investigated the gene expression profiles of the cytochrome P450s (CYPs) in the ocular tissues of rats developing such selenite-induced cataracts. Seven days after the injection of selenite, the expression levels of the CYP1A1 and CYP2E1 genes in the lenses of these animals increased and a slight reduction in the expression of the CYP3A gene family could be observed in the extralenticular tissues. When pantethine, an antioxidant, was administered 30 min before selenite injection, the extent of the CYP gene expression changes diminished. These results suggest that selenite-induced oxidative stress may play a role in both the induction and downregulation of CYP genes. In addition, the elevated expression of CYP1A1 and CYP2E1 in the lens may underlie the enhancement of cataract development induced by selenite.

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Cloning and expression of a novel sulfotransferase with unique substrate specificity from Bombyx mori

Hattori K., Hirayama M., Suzuki H., Hamamoto H., Sekimizu K., Tamura H.

Bioscience, Biotechnology and Biochemistry （Bioscience, Biotechnology and Biochemistry）  71 （ 4 ） 1044-1051 - 1051 2007年04月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  09168451

　概要を見る

We identified a cDNA encoding a putative cytosolic sulfotransferase (SULT) by searching the expressed sequence tag database of Bombyx mori, and subsequently obtained the full-length cDNA for this gene via rapid amplification of cDNA ends (RACE). We designated this gene bmST1, and showed by sequence analysis that it belongs to a novel SULT family. The tissue specificity of bmST1 mRNA expression was examined in fifth instar larvae by reverse transcriptase-polymerase chain reaction (RT-PCR), and transcripts were detectable in the silk gland, gut, fat body, and Malpighian tube. A recombinant form of bmST1 was then expressed using a gluthathione S-transferase (GST) gene fusion system, and it was purified from Escherichia coli. Purified bmST1 did not exhibit sulfating activity toward SULT substrates such as 4-nitrophenol, vanillin, hydroxysteroids, or monoamines. Surprisingly, however, recombinant bmST1 showed considerable activity toward 4-nitrocatechol and also gallate esters, although the catechins are not sulfated by this enzyme.

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A developed determination of midazolam and 1′-hydroxymidazolam in plasma by liquid chromatography-mass spectrometry: Application of human pharmacokinetic study for measurement of CYP3A activity

Shimizu M., Uno T., Tamura H., Kanazawa H., Murakami I., Sugawara K., Tateishi T.

Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences （Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences）  847 （ 2 ） 275 - 281 2007年03月

研究論文（学術雑誌）,  ISSN  15700232

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This paper describes sensitive and reliable determination of midazolam (MDZ) and its major metabolite 1′-hydroxymidazolam (1-OHMDZ) in human plasma by liquid chromatography-mass spectrometry (LC-MS) with a sonic spray ionization (SSI) interface. MDZ, 1-OHMDZ and diazepam as an internal standard were extracted from 1 ml of alkalinized plasma using n-hexane-chloroform (70:30, v/v). The extract was injected into an analytical column (YMC-Pak Pro C18, 50 mm × 2.0 mm i.d.). The mobile phase for separation consisted of 10 mM ammonium acetate and methanol (50:50, v/v) and was delivered at a flow-rate of 0.2 ml/min. The drift voltage was 100 V. The sampling aperture was heated at 120 °C and the shield temperature was 260 °C. The total time for chromatographic separation was less than 16 min. The validated concentration ranges of this method were 0.25-50 ng/ml for both MDZ and 1-OHMDZ. Mean recoveries were 93.6% for MDZ and 86.6% for 1-OHMDZ. Intra- and inter-day coefficient variations were less than 6.5 and 5.5% for MDZ, and 6.1 and 5.7% for 1-OHMDZ at 0.3, 4, 20 and 40 ng/ml. The limits of quantification were 0.25 ng/ml for both MDZ and 1-OHMDZ. This method was sensitive and reliable enough for pharmacokinetic studies on healthy volunteers, and was applied for the measurement of CYP3A activity in humans after an intravenous (1 mg) and a single-oral administration (2 mg) of subtherapeutic MDZ dose. © 2006 Elsevier B.V. All rights reserved.

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A developed determination of midazolam and 1'-hydroxymidazolam in plasma by liquid chromatography-mass spectrometry: Application to human pharmacokinetic study for measurement of CYP3A activity.

Shimizu M, Uno T*, Tamura HO, Kanazawa H, Murakami I, Tateishi T.

J Chromatogr B 847 (2)   275-281 2007年03月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Metabolism of bisphenol A in the rat syncytiotrophoblast cell line, TR-TBT 18d-1

Ikeda Y., Nakashima E., Kanazawa H., Tamura H.

Journal of Health Science （Journal of Health Science）  53 （ 1 ） 146-150 - 150 2007年02月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  13449702

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TR-TBT 18d-1 is a syncytiotrophoblast cell line established from placenta of the transgenic rat harboring temperature-sensitive simian virus 40 large T-antigen (Tg-rat). Previously we have reported high level expressions of drug metabolizing enzymes in the TR-TBT cells. To elucidate the ability of metabolizing xenobiotics in the TR-TBT cells, we measured the metabolism of bisphenol A (BPA) as a model compound in the TR-TBT 18d-1 cells. It was found that BPA was metabolized and secreted as a monoglucuronide conjugate in a manner proportional to the incubation time and the BPA concentration. To elucidate the possible effect of green tea consumption on the placental metabolism, we investigated the effect of green tea on the metabolism of BPA in the TR-TBT cells. Green tea inhibited the conjugation reaction of BPA at the low concentration (IC50=5%), however, epigallocathechin gallate (EGCG) showed only weak inhibition, suggesting another active component existence. These results suggest that TR-TBT cells are useful for the model system of rat syncytiotrophoblasts which act as a barrier to protect fetus from harmful xenobiotics.

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Expression profiling of drug metabolizing enzymes in rat syncytiotrophoblast cell lines

Ikeda Y., Nakashima E., Tamura H.

Journal of Health Science （Journal of Health Science）  52 （ 5 ） 633-636 - 636 2006年10月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  13449702

　概要を見る

TR-TBT-18d-1 and -18d-2 are syncytiotrophoblast cell lines established from the placenta of a transgenic rat harboring a temperature-sensitive simian virus 40 large T-antigen (Tg-rat). To explore the functional roles of drug metabolizing enzymes in the syncytiotrophoblasts of the placenta, we analyzed the gene expression of both cytochrome P450 (CYP) and phase II conjugation enzymes in these TR-TBT cell lines. A relatively high level of expression of CYP1A1 and CYP2C11 was detectable in the 18d-1 cells whereas CYP2E1 was more strongly expressed in the 18d-2 cells. A moderate degree of expression of CYP2B2 and CYP3A, and low expression of CYP1A2, was observed in both cell lines, but no CYP3A2 was detectable in either. Relatively high levels of SULT2 family gene expression were also evident in these TR-TBT cells, although the SULT1 family genes were found to be either absent or present at only moderate levels. Strong expression of both the UGT1 and UGT2 subfamily, and also the glutathione S-transferase (GST) subfamily, was also observed in both cell lines. These results suggest that TR-TBT cells are a useful model system for the study of syncytiotrophoblasts, which act as a protective fetal barrier against harmful xenobiotics.

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Turmeric and curcumin modulate the conjugation of 1-naphthol in Caco-2 cells.

Naganuma M, Saruwatari A, Okamura S, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  29 （ 7 ） 1476-1479 - 9 2006年06月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  0918-6158

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Turmeric, the powdered dry rhizome of the Curcuma longa plant, and curcumin, the major anti-oxidant constituent of turmeric, have been shown to possess chemopreventive activity. To elucidate the possible interaction of turmeric and curcumin with conjugation reactions, which in many cases are involved in the activation of procarcinogens, we measured their effects in the conjugation of 1-naphthol in Caco-2 cells, a human colon carcinoma cell line, within a 24 h period. Turmeric exhibits inhibitory activity toward both sulfo- and glucuronosyl conjugations of 1-naphthol at approximately the same levels (IC50=0.24 and 0.29 mg/ml, respectively). Curcumin inhibits sulfo-conjugation at lower concentrations (IC50=9.7 μg/ml), but only showed weak inhibition toward glucuronosyl conjugation of 1-naphthol in Caco-2 cells. In addition, turmeric was found to strongly inhibit in vitro phenol sulfotransferase (SULT) activity and demonstrate moderate inhibitory properties against UDP-glucuronosyl transferase (UGT) activity in Caco-2 cells (IC50=0.17 mg/ml and 0.62 mg/ml, respectively). Curcumin also strongly inhibits in vitro phenol sulfotransferase activity with an IC 50 of 2.4 μg/ml. Moreover, and in contrast to the moderate inhibition of UGT activity by turmeric and curcumin, both induce the expression of the UGT1A1 and UGT1A6 genes, revealed by real-time PCR analysis. These findings are indicative of a possible interaction of both turmeric and curcumin with conjugation reactions in the human intestinal tract and colon. This in turn may affect the bioavailability of therapeutic drugs and toxicity levels of environmental chemicals, particularly procarcinogens. © 2006 Pharmaceutical Society of Japan.

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A novel sulfotransferase abundantly expressed in the dauer larvae of Caenorhabditis elegans

Hattori K., Inoue M., Inoue T., Arai H., Tamura H.

Journal of Biochemistry （Journal of Biochemistry）  139 （ 3 ） 355-362 - 362 2006年03月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  0021924X

　概要を見る

We have isolated a gene (clone Y113G7A.11) from Caenorhabditis elegans (C. elegans), that we have designated as ceST1, and which is the only member of the cytosolic sulfotransferase (SULT) gene family present in the genome of this organism. We identified the SULT motifs of ceST1 based upon their deduced amino acid sequence, and subsequently expressed the ceST1 cDNA in Escherichia coli and characterized its enzymatic properties. The recombinant protein showed sulfation activity for 4-nitrophenol and 2-naphthol substrates, but did not catalyze the sulfation of either monoamines or hydroxysteroids. Another compound sulfated by ceST1 is bisphenol A, which is known to stimulate germ cell proliferation in C. elegans. SULT activity was not detected in the cytosol of C. elegans, probably due to heat labile inhibitors. The ceST1 protein was detectable in the cytosol of C. elegans using anti-sera raised against recombinant ceST1, and transcripts could also be detected throughout the developmental stages. Moreover, high levels of ceST1 expression were evident at both the embryonic and adult stages and were augmented in dauer larva. These findings suggest that this sulfotransferase either forms part of a defence system against xenobiotics or regulates germ cell proliferation in C. elegans. © 2006 The Japanese Biochemical Society.

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Changes in the gene expression of drug metabolizing enzymes during cataractogenesis in shumiya cataract rats

Nakamura K., Oka M., Takehana M., Kobayashi S., Tamura H.

Journal of Health Science （Journal of Health Science）  51 （ 6 ） 734-737 - 737 2005年12月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  13449702

　概要を見る

The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in both the nuclear and perinuclear regions of 10-11 week old animals. The expression of drug metabolizing enzymes, including cytochrome P450 (CYP) and conjugation enzymes, was investigated in the ocular tissues of SCRs during cataractogenesis. Significantly high levels of gene expression were detected for CYP1A1, CYP2B2, CYP2C11 and CYP2E1 in normal lenses, whereas CYP1A1 and CYP2C11 were at lower levels in the cataractous lenses. The gene expression profiles of conjugation enzymes were also compared between normal and cataractous ocular tissues in which a moderate reduction in ST1A1 and ST1B1 expression, and an extensive loss of UGT1A1 expression, were observed in the SCR animals that had developed cataracts. In contrast, the elevated expression of the GSTA family of genes was evident in cataractous lenses. The reduced expression of several drug metabolizing enzymes in the cataractous ocular tissues might therefore be related to cataractogenesis via the accumulation of endogenous or exogenous toxic substances.

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Age, gender and region-specific differences in drug metabolizing enzymes in rat ocular tissues.

Nakamura K, Fujiki T, Tamura H*.

Exp Eye Res 81   710-715 2005年12月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Age, gender and region-specific differences in drug metabolising enzymes in rat ocular tissues

Nakamura K., Fujiki T., Tamura H.

Experimental Eye Research （Experimental Eye Research）  81 （ 6 ） 710 - 715 2005年12月

研究論文（学術雑誌）,  ISSN  00144835

　概要を見る

Previously we have reported our analyses of cytochrome P450 (CYP) expression in rat ocular tissues and in our current study we have extended these analyses to the different stages of growth in both male and female rats. Additionally, we have examined the expression levels of the different conjugation enzymes, sulfotransferases (SULTs), UDP-glucuronosyl transferases (UGTs) and glutathione S-transferases (GSTs), in ocular tissues. In 5-week-old animals, the CYP genes, CYP2B2 and CYP3A1, were abundantly expressed in the lens, with higher CYP1A1 expression detectable in the extra-lenticular tissues, of both genders. However, the expression of CYP1A2 and CYP2E1 in female ocular tissues was more extensive than in male. Moreover, in females the expression of the phenol-sulfating SULTs, ST1A1, ST1B1 and ST1C1, was more abundant in the extra-lenticular tissues, whereas the levels of the hydroxysteroid-sulfating SULTs, ST2A1 and ST2A5, predominated in the lens. The expression levels of the UGTs, UGT1A1, UGT1A6 and UGT2B1, were also higher in the female extra-lenticular tissues but the expression of the GSTA subfamily (GSTA1 and GSTA2) was very weak in the ocular tissues of both genders. The overall gene expression profiles were found to change with the age of the animals (from 3 to 8 weeks) and, in general, the expression levels of the CYPs and SULTs declined with age, whereas the levels of the UGTs and GSTs increased. These results demonstrate that the expression profiles of drug metabolising enzymes show both region- and gender-specific patterns in rat ocular tissues. © 2005 Elsevier Ltd. All rights reserved.

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ポリエチレンテレフタレート再生材中のホルムアルデヒド,アセトアルデヒドおよびオリゴマーの分析.

大門由佳, 河村葉子*, 六鹿元雄, 田村悦臣, 棚元憲一.

食品衛生学雑誌 46   46 2005年10月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Analysis of formaldehyde, acetaldehyde and oligomers in recycled polyethylene terephthalate

Ohkado Y., Kawamura Y., Mutsuga M., Tamura H., Tanamoto K.

Journal of the Food Hygienic Society of Japan （Journal of the Food Hygienic Society of Japan）  46 （ 5 ） 218 - 223 2005年10月

研究論文（学術雑誌）,  ISSN  00156426

　概要を見る

Formaldehyde (FA), acetaldehyde (AA) and oligomers in recycled polyethylene terephthalate (PET) were analyzed by HPLC. All of the physically recycled PET contained detectable levels of FA, AA and oligomers, and the levels were almost the same as in used bottles. Most superclean-like and chemically recycled PET contained lower levels than new pellets. These compounds showed no decrease upon physical recycling, but showed a marked decrease upon superclean-like recycling. In PET sheets made using physically recycled PET, FA was decreased, though AA was increased by the sheeting process as same as new one. FA, AA and oligomers originated from PET resin and their levels in recycled products were almost equivalent to those in new products. It was concluded that there is no particular safety concern about their presence in recycled PET.

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ポリエチレンテレフタレート再生材中の残存金属と再生材使用の判別法.

大門由佳, 河村葉子, 六鹿元雄, 田村悦臣, 棚元憲一*.

食品衛生学雑誌 46   46 2005年06月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Metals in recycled polyethylene terephthalate and discrimination method for its use

Ohkado Y., Kawamura Y., Mutsuga M., Tamura H., Tanamoto K.

Journal of the Food Hygienic Society of Japan （Journal of the Food Hygienic Society of Japan）  46 （ 3 ） 109 - 115 2005年06月

研究論文（学術雑誌）,  ISSN  00156426

　概要を見る

Metals in recycled polyethylene terephthalate (PET) were analyzed by ICP-MS following microwave digestion with nitric acid. Physically and superclean-like recycled PET contained both Ge and Sb, and sometimes contained Co, P or Si. In contrast, the chemically recycled PET contained only Ge or Sb, and some samples contained Co. The recycled PETs did not contain Pb or Cd. Ge and Sb were catalysts of the polymerization, and the other metals also originated from the PET resin as additives. It was concluded that there is no safety concern about metals in recycled PET. It became clear that the presence of both Ge and Sb could identify products formed using physically or superclean-like recycled PET. According to this discrimination method, about half of the sheet molding products used recycled PET.

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ポリエチレンテレフタレート再生材中の残存揮発性物質の分析.

大門 由佳, 河村 葉子, 六鹿 元雄, 田村 悦臣, 棚元 憲一*.

食品衛生学雑誌 46   46 2005年02月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

The effects of coffee on conjugation reactions in human colon carcinoma cells

Okamura S., Suzuki K., Yanase M., Koizumi M., Tamura H.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  28 （ 2 ） 271-274 - 274 2005年02月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  09186158

　概要を見る

We examined the effect of coffee on conjugation reactions in the human colon carcinoma cell line, Caco-2. After supplementing Caco-2 cultures with both 1-naphthol (200 μM) and various concentrations of coffee, the accumulation of 1-naphthyl sulfate and glucuronide in the growth medium was determined by analytical HPLC over a 24-h period. A strong reduction in sulfo-conjugation (<50% of the control value) was observed in cells treated with coffee (IC50=4.3%), but no effect on glucuronic acid conjugation (glucuronidation) was observed. Coffee was also found to inhibit sulfotransferase (SULT) activity towards 1-naphthol in vitro to a similar extent (IC50=5.1%) as in intact Caco-2 cells, but exhibited no effect upon UDP-glucuronosyl transferase (UGT) activity in vitro. PCR analyses showed no significant changes in the expression of either SULT genes (SULT1A1 and SULT1A3) or UGT genes (UGT1A1 and UGT1A6) following treatment with coffee solutions of up to 5% in concentration. These results suggest that the consumption of coffee can modify sulfo-conjugation reactions within intestinal epithelial cells, which may possibly affect the bioavailability of therapeutic drugs and the toxicity of environmental chemicals. © 2005 Pharmaceutical Society of Japan.

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Analysis of residual volatiles in recycled polyethylene terephthalate

Ohkado Y., Kawamura Y., Mutsuga M., Tamura H., Tanamoto K.

Journal of the Food Hygienic Society of Japan （Journal of the Food Hygienic Society of Japan）  46 （ 1 ） 13 - 20 2005年02月

研究論文（学術雑誌）,  ISSN  00156426

　概要を見る

The residual volatiles in recycled polyethylene terephthalate (PET) were analyzed using headspace/GC/MS. Recycled PET samples were made from PET bottles used for beverages, alcohol and soy sauce, and they were recycled in physical recycling plants, chemical recycling plants and superclean-like recycling trials. The physically recycled PET flakes contained small amounts of volatiles such as ethanol, limonene, 2-methyl-1,3-dioxolane, acetone, octanal and nonanal. Most of them originated from foods packed in bottles, and only 2-methyl-1,3-dioxolane was derived from polymer impurities. In contrast, the superclean-like or chemically recycled PET contained no detectable volatiles, like new PET pellets. The PET sheets shaped from physically recycled PET had no detectable volatiles. Not only the chemically and superclean-like recycled PET, but also the physically recycled PET contained no hazardous volatiles. It was concluded that there is no safety concern about volatiles in recycled PET, for the present use.

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Region- and isoform-specific expression of hydroxysteroid sulfotransferase in rat brain.

Shimizu M, Nakano Y, Yamazaki T, Kimoto T, Kawato S, Tamura H*.

J Health Sci 50   689-692 2004年12月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Region- and isoform-specific expression of hydroxysteroid sulfotransferases in rat brain

Shimizu M., Nakano Y., Yamasaki T., Kimoto T., Kawato S., Tamura H.

Journal of Health Science （Journal of Health Science）  50 （ 6 ） 689 - 692 2004年12月

研究論文（学術雑誌）,  ISSN  13449702

　概要を見る

Hydroxysteroid sulfotransferase (HS-ST) is thought to be a key enzyme in the synthesis of neurosteroid sulfates, which are known to act as potent regulators of neuronal activity within the brain. In rat liver, three isoforms of HS-ST (ST2A1, ST2A2 and ST2A5) have now been identified and to further elucidate the precise contribution of each of these variants in neurosteroid sulfation in rat brain, we analyzed the expression of their transcripts in different brain regions using isoform-specific RT-PCR. The expression of ST2A1 was found exclusively in the olfactory bulb and ST2A2 expression levels were most abundant in the hippocampus, but no ST2A5 expression was detectable in any brain region. We then measured the HS-ST activity in these same regions and detected low but significant activity levels in the olfactory bulb and hippocampus, but less activity in the cortex. These activity levels were found to correlate with the mRNA levels of the HS-ST isoforms. Our findings will further our understanding of the physiological role of HS-ST enzymes within the brain, particularly how they affect neurosteroid metabolism and modulate neuronal activity.

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Effect of herbal teas on conjugation reaction in a human colon carcinoma cell line, Caco-2.

Okamura S, Tamura H*.

J Health Sci 50   189-192 2004年04月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Oxidative stress induces GSTP1 and CYP3A4 expression in the human erythroleukemia cell line, K562

Nagai F., Kato E., Tamura H.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  27 （ 4 ） 492-495 - 495 2004年04月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  09186158

　概要を見る

To explore the effects of oxidative stress on metabolizing enzymes in blood cells, we examined the effect of hydrogen peroxide (H2O2) on glutathione S-transferase (GST) and cytochrome P450 (CYP) expression in a human erythroleukemic cell line, K562. After adding H2O2 (up to 100 μM) to the culture medium of K562 cells, the expression levels of GST and CYP enzymes were monitored by RT-PCR. The expression of GSTP1 and CYP3A4 was induced by oxidative stress. Quantitative PCR and immunoblot analysis revealed a 3- to 4-fold increase in GSTP1 and CYP3A4 mRNA, and a 2- to 3-fold increase in GSTP1 and CYP3A4 protein levels, 3 d after the addition of 100 μM H2O2. Induction was H2O2 dose-dependent and also depended on the length of culture. Our results suggest that oxidative stress may affect GST and/or CYP expression in human blood cells, which may alter the metabolism of drugs and xenobiotics and thus, the toxicity of these compounds to the blood cells. © 2004 Pharmaceutical Society of Japan.

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Effect of herbal teas on conjugation reactions in a human colon carcinoma cell line, Caco-2

Okamura S., Tamura H.

Journal of Health Science （Journal of Health Science）  50 （ 2 ） 189 - 192 2004年04月

研究論文（学術雑誌）,  ISSN  13449702

　概要を見る

We examined the effect of herbal teas (including black and green teas) on conjugation reactions within a human colon carcinoma cell line, Caco-2. After adding herbal tea to the culture medium of Caco-2 cells, accumulation of 1-naphthyl sulfate and glucuronide was determined within the medium by analytical HPLC prior to 24 hr. A reduction in sulfoconjugation (< 50% of the control value) was observed in cells treated with 5% solutions of green tea, jasmine tea or black tea. Mild induction of sulfoconjugation (20-30%) was detected in cells exposed to 5% solutions of hibiscus or lemongrass tea. In addition, a slight reduction (20-30%) in glucuronic acid conjugation (glucuronidation) was observed in cells treated with rose red, peppermint, lavender or St. John's wort. At low concentrations, ranging from 0.1-1.0%, several herbal teas resulted in weak but significant induction (10-20%) of both types of conjugation reactions. These results suggest that herbal teas modify conjugation reactions within intestinal epithelial cells, thereby potentially affecting the bioavailability and toxicity of therapeutic drugs and environmental chemicals.

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Identification and purification of sulfotransferases for 20-hydroxysteroid from the larval fat body of a freshfly, Sarcophaga peregrina.

Matsumoto E, Matsui M, Tamura H*.

Biosci Biotechnol Biochem 67   1780-1785 2003年08月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Sulfoconjugation of bisphenol A in a human neuroblastoma cell line, NB-1

Ishii Y., Tamura H.

Journal of Health Science （Journal of Health Science）  49 （ 4 ） 319-323 - 323 2003年04月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  13449702

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Bisphenol A (BPA) is a common environmental endocrine disruptor and BPA sulfate has been demonstrated as one of metabolites of BPA. Since it has been observed to have effects on the central nervous system (CNS), we examined the effects of sulfoconjugation on the toxicity of BPA in a human neuroblastoma cell line, NB-1. NB-1 cells showed limited but significant sulfotransferase activity toward BPA in vitro (Km = 74 μM, Vmax = 7.8 pmol/min/mg). Accumulation of BPA sulfate was observed for 12 hr to 4 days after 10 μM BPA was added to a culture of NB-1 cells. These results suggest that BPA is partly detoxified by the sulfoconjugation reaction in human neuronal cells.

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Identification and purification of sulfotransferases for 20-hydroxysteroid from the larval fat body of a fleshfly, sarcophag peregrina

Matsumoto E., Matsui M., Tamura H.

Bioscience, Biotechnology and Biochemistry （Bioscience, Biotechnology and Biochemistry）  67 （ 8 ） 1780 - 1785 2003年01月

研究論文（学術雑誌）,  ISSN  09168451

　概要を見る

Sulfotransferase (ST) activity for 20-hydroxyecdysone (20E) was identified in a larval fat body lysate of the fleshfly, Sarcophaga peregrina, but not in the hemolymph. The activity was highly sensitive to 2,6-dichloro-4-nitrophenol (DCNP) (IC50=0.61 μM), a specific inhibitor of phenol ST (P-ST), but insensitive to triethylamine, a hydroxysteroid ST inhibitor. These results suggest that 20E-specific ST enzymes belong to the P-ST family, despite the fact that 20E is a hydroxysteroid. In addition to 20E ST activity, a relatively high level of 2-naphthol ST activity was detected in the fat body lysate. The ST activity for both substrates transiently decreased to the 50% of maximal levels, 6 hrs after induction of pupation. The ST enzymes were separated on a DEAE-cellulose column. The 20E-ST enzymes were eluted around 50 mM KCl as two separate peaks of close proximity and the P-ST was eluted at 0.1 M KCl. The 20E ST enzymes were further purified using 3′-phosphoadenosine 5′-phosphate (PAP)-agarose affinity column chromatography. Both of the eluted active fractions demonstrated 43-kDa proteins on SDS-polyacrylamide gel. Photoaffinity labeling with [35S]-3′-phosphoadenosine 5′-phosphosulfate (PAPS) showed 43-kDa bands in the fat body lysate, as well as in the purified fractions. These results suggest that the 43-kDa proteins catalyze 20E sulfation within the fat body of S. peregrina. © 2003 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

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Cytochrome P450-dependent neurosteroid synthesis in the rat brain hippocampal neurons

Kimoto T., Tsurugizawa T., Ohta Y., Makino J., Tamura H., Hojo Y., Enami T., Kawato S.

International Congress Series （International Congress Series）  1233 （ C ） 127 - 137 2002年11月

研究論文（学術雑誌）,  ISSN  05315131

　概要を見る

We have demonstrated the neuronal localization of neurosteroidogenic systems as well as the regulation of neurosteroidogenic activity in an adult rat hippocampus. The expressions of steroidogenic enzymes (cytochrome P450scc, adrenodoxin (ADX), NADPH-adrenodoxin reductase (ADR), hydroxysteroid sulfotransferase and steroidogenic acute regulatory protein (StAR)) were observed principally in pyramidal neurons and granule neurons by means of immunohistochemical staining. The expression of P450scc, redox partners, sulfotransferase and StAR was also confirmed by Western blot analysis.Exposing the neurons to N-methyl-D-aspartate (NMDA) stimulated the process of active neurosteroidogenesis. Upon stimulation with NMDA, the significant net production of pregnenolone (PREG) and pregnenolone sulfate (PREGS) was observed in the hippocampus. This neurosteroid production was considerably suppressed by the addition of antagonists of NMDA receptors, by Ca2+ depletion or by the addition of an inhibitor of P450scc. Upon stimulation with NMDA, the processing of full-length StAR (37-kDa) to the truncated 30-kDa StAR was observed. Taken together, these observations imply that hippocampal neurons synthesize neurosteroids. This synthesis may be stimulated and regulated by glutamate-mediated synaptic communication. We also discuss the possibility that PREGS may act as a paracrine modulator of NMDA receptors. © 2002 Elsevier Science B.V.

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Identification and localization of two hydroxysteroid sulfotransferase in the human brain.

Shimizu M, Tamura H*.

J Health Sci 48   467-472 2002年10月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Identification and localization of two hydroxysteroid sulfotransferases in the human brain

Shimizu M., Tamura H.

Journal of Health Science （Journal of Health Science）  48 （ 5 ） 467 - 472 2002年10月

研究論文（学術雑誌）,  ISSN  13449702

　概要を見る

Hydroxysteroid sulfotransferase (SULT2) is thought to be a key enzyme in the synthesis of neurosteroid sulfates, which are known to act as potent regulators of neuronal activity within the brain. We detected two SULT2 mRNAs (SULT2A1 and SULT2B1b) in the human brain. We isolated their cDNAs, and determined the location of their expression in the brain by Northern blot hybridization. Exclusive SULT2A1 expression was detected in the thalamus and hypothalamus, whereas SULT2B1b expression was detected throughout the brain in varying amounts, reaching levels two to three times greater in some regions than in others. These findings further our understanding of the physiological role of SULT2 enzymes within the human brain, particularly how they affect neurosteroid metabolism, and thus, modulate neuronal activity within the brain.

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Cytochrome P450 expression in rat ocular tissues and its induction by phenobarbital

Tanaka H., Hirayama I., Takehana M., Kobayashi S., Tamura H.

Journal of Health Science （Journal of Health Science）  48 （ 4 ） 346-349 - 349 2002年09月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  13449702

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Composition and inducibility of cytochrome P450 (CYP) in ocular tissues were investigated using reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot techniques. Composition of ocular CYPs is more restricted than that of hepatic forms. High levels of CYP2B1/2 and reduced levels of CYP2C11 expression were detected in rat ocular tissues. Phenobarbital induced CYP2B1/2 expression in the lens but not in the rest of the eye, whereas CYP2C11 induction was observed in both regions. In addition, CYP2B1/2 proteins were found to be prominent in the retina. Our data indicate eye-specific regulation of CYP expression.

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Cytochrome P450 (CYP) expression in human myeloblastic and lymphoid cell lines

Nagai F., Hiyoshi Y., Sugimachi K., Tamura H.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  25 （ 3 ） 383・385 - 385 2002年03月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  09186158

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To explore the physiological roles of cytochrome P450 (CYP) in peripheral blood cells, we examined which isoforms of CYP families were expressed in human myeloid leukemia cell lines (U937, HL-60 and K562) and lymphoid cell lines (BALL-1, MOLT-4 and Jurkat) by RT-PCR. We observed relatively high expression of CYP1A1, CYP1B1, CYP2A6, CYP2A7, CYP2D6, and CYP2E1 in all cell types, but CYP2A13 and CYP2C9 expression was not detected. Expressions of aryl hydrocarbon (Ah) receptor and Ah receptor nuclear translocator (ARNT), which mediate induction of the CYP1 family, were also detected in all cell types. Cell-type specific expression of CYP3A4 and CYP3A5 was observed in MOLT-4 and K562 cells. Weak, but significant, expression of CYP3A7 was detected in K562 cells. The profile of CYP expression in the culture cells reported here provides information that furthers our understanding of the physiological roles of CYP enzymes in human blood cells.

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Expression profiling of drug metabolizing enzymes in human myeloblastic cell lines.

Tamura H*, Nagai F.

Drug Metabol Rev 33   158 2001年11月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Expression profiling of sulfotransferases in human cell lines derived from extra-hepatic tissues

Tamura H., Taniguchi K., Havashi E., Hiyoshi Y., Nagai F.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  24 （ 11 ） 1258・1262 - 1262 2001年11月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  09186158

　概要を見る

To explore the physiological roles of sulfotransferases (SULTs) in extra-hepatic tissues, we examined the expression of eight SULT genes by reverse transcription (RT)-PCR in human cell lines that were established from various tissues. Expression levels of SULTs were low in neural cell lines such as NB-1 and GI-1, and high in epithelial cell lines, such as Caco-2 and BeWo. SULT1C2 expression was abundant in all cell types, whereas that of SULT1E1, SULT1B1 or SULT2B1 was restricted to a specific cell type. SULT1C1, which can catalyze the sulfation of N-hydroxy-2-acetylaminofluorence, was expressed in Caco-2, BeWo and KB562. Induction of differentiation did not generally affect SULT expression, although that of SULT1C2 was reduced after differentiation of the neuroblastoma cell line, NB-1, was induced. The profile of SULT expression in the culture cells obtained here gives clues to understanding the physiological roles of SULT enzymes in extra-hepatic tissues or organs.

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Effects of phenolic environmental estrogens on the sulfotransferase activity of the mouse intestine and a human colon carcinoma cell line, Caco-2

Tamura H., Matsui M.

Journal of Health Science （Journal of Health Science）  47 （ 5 ） 468・472 - 472 2001年10月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  13449702

　概要を見る

In order to explore the possible role of sulfation in the inactivation of environmental estrogens at gastrointestinal sites and their subsequent removal, we investigated the effects of phenolic environmental estrogens on sulfotransferase (ST) activity. The mouse intestine and a human colon carcinoma cell line, Caco-2, were studied. ST enzymes were found to have a high affinity for diethylstilbestrol (DES) and bisphenol A (BPA), whereas phenol ST (PST) activity was strongly inhibited by nonylphenol and genistein in both mice and humans. Kinetic analysis showed that this inhibition was competitive. These observations suggest that nonylphenol and genistein compounds might inhibit PST activity in the human intestine and that they might escape detoxification by sulfation.

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Epigallocatechin gallate (EGCG) inhibits the sulfation of 1-naphthol in a human colon carcinoma cell line, Caco-2

Isozaki T., Tamura H.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  24 （ 9 ） 1076・1078 - 1078 2001年09月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  09186158

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We have previously reported that epigallocatechin gallate (EGCG) strongly inhibits the in vitro phenol sulfo-transferase (P-ST) activity of a human colon carcinoma cell line, Caco-2. In the present study, we examined the ability of EGCG to inhibit the sulfation of 1-naphthol in intact Caco-2 cells. Sulfation of 1-naphthol was detected in Caco-2 cells after 2 h of incubation, and was observed to continue for 24 h, resulting in an accumulation of sulfated 1-naphthol. Sulfation was strongly inhibited by the addition of EGCG to the culture medium. The IC50 of EGCG was calculated to be 20 μM; this value is similar to that obtained from in vitro assays (14 μM) [Ref. Tamura et al., Biol. Pharm. Bull., 23, 695, (2000)]. These results indicate that catechins are capable of inhibiting P-ST activity in intact cells as well as in vitro. We believe that the inhibitory activity of catechins might be the mechanism by which catechins (and green tea) exert anti-carcinogenic activity against procarcinogenic compounds that require P-ST activation in vivo.

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Neurosteroid synthesis by cytochrome P450-containing systems localized in the rat brain hipocampal neurons:N-methyl-D-aspartate and calcium-dependent synthesis.

Kimoto T, Tsurugizawa T, Ohta Y, Makino J, Tamura H, Hojo Y, Takata N, Kawato S*.

Endocrinology 142   3578・3589 2001年08月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Neurosteroid synthesis by cytochrome P450-containing systems localized in the rat brain hippocampal neurons: N-methyl-D-aspartate and calcium-dependent synthesis

Kimoto T., Tsurugizawa T., Ohta Y., Makino J., Tamura H., Hojo Y., Takata N., Kawato S.

Endocrinology （Endocrinology）  142 （ 8 ） 3578 - 3589 2001年

研究論文（学術雑誌）,  ISSN  00137227

　概要を見る

Neurosteroidogenesis has not been well elucidated due to the very low level of steroidogenic proteins in the brain. Here we report the first demonstration of the neuronal localization of neurosteroidogenic systems as well as the regulation of neurosteroidogenic activity in the adult rat hippocampus. Significant localization of cytochrome P450scc was observed in pyramidal neurons and granule neurons by means of immunohistochemical staining of slices. We also observed the colocalization, in hippocampal neurons, of P450scc with redox partners, hydroxysteroid sulfotransferase and steroidogenic acute regulatory protein. The distributions of astroglial cells and oligodendroglial cells showed very different patterns from that of the P450scc-containing cells. The expression of P450scc, redox partners, the sulfotransferase, and steroidogenic acute regulatory protein was also confirmed by Western blot analysis. The process of active neurosteroidogenesis was stimulated by exposing neurons to N-methyl-D-aspartate. Upon stimulation with N-methyl-D-aspartate, Ca2+ influx through the N-methyl-D-aspartate subtype of glutamate receptors occurred, and significant net production of pregnenolone and pregnenolone sulfate was observed in the hippocampus. This neurosteroid production was considerably suppressed by the addition of antagonists of N-methyl-D-aspartate receptors, by Ca2+ depletion, or by the addition of an inhibitor of P450scc. Upon stimulation with N-methyl-D-aspartate, the processing of full-length steroidogenic acute regulatory protein (37-kDa) to the truncated 30-kDa steroidogenic acute regulatory protein was observed. Taken together, these observations imply that hippocampal neurons synthesize neurosteroids. This synthesis may be stimulated and regulated by glutamate-mediated synaptic communication.

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Sulfotransferases in a human coln carcinoma cell line, Caco-2.

Satoh T, Matsui M, Tamura H*.

Biol. Pharm. Bull. 23(7)   810-814 2000年07月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Sulfotransferases in a human colon carcinoma cell line, Caco-2

Satoh T., Matsui M., Tamura H.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  23 （ 7 ） 810 - 814 2000年07月

研究論文（学術雑誌）,  ISSN  09186158

　概要を見る

We investigated the expression of sulfotransferases (STs) in the human colon carcinoma cell line, Caco-2, which is widely used as a human intestine model for orally administered drugs. By reverse transcription-polymerase chain reaction (RT-PCR), we could detect mRNA expressions of phenol STs (P- ST), hydroxysteroid ST (HS-ST) and estrogen ST (E-ST). Treatment with 1,25- dihydroxyvitamine D3, which induces CYP3A4 expression in Caco-2 cells, did not affect the ST expression of the cells. We detected significant P-ST activity toward 2··naphthol and dopamine in the cell extract. We also observed detectable HS-ST activity toward dehydroepiandrosterone (DHEA), but no E-ST activity toward β-estradiol was observed in the extract. Analyses by RT-PCR and separation of P-ST activities on ion-exchange column chromatography showed that the thermolabile monoamine phenol sulfotransferase (M-PST) is dominant in Caco-2 cells.

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Inhibitory effects of green tea and grape juice on the phenol sulfotransferase activity of mouse intestines and human colon carcinoma cell line, Caco-2

Tamura H., Matsui M.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  23(6) （ 6 ） 695-699 - 699 2000年06月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り,  ISSN  09186158

　概要を見る

Tea and fruit juices are beverages consumed daily all over the world. The present study reports the inhibitory effects of these beverages on the activity of mammalian intestinal phenol sulfotransferases (P-STs). Green tea strongly inhibited the E. coli-expressed mouse intestinal P-ST activity in vitro. (-)-Epigallocatechin gallate (EGCG) was found to be the most potent inhibitor among the catechins tested (IC50=0.93 μM). (-)EGCG also inhibited the P-ST activity of the human colon carcinoma cell line, Caco-2. Kinetic analysis showed that the inhibition was competitive. Among fruit juices examined (apple, grape, grapefruit and orange), grape juice exhibited the most potent inhibitory action on the P-ST activity of mouse intestines and human colon carcinoma cells. The inhibitory activity of grape juice was located mainly in the skin and seeds. Flavonols, such as quercetin and kaempferol, inhibited the P-ST activity at low concentrations. These observations suggest the possible inhibition of P-ST activity in human intestines by green tea or grape juice.

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書評「生活・環境化学物質と安全性」梅津・北島著.

田村悦臣.

月間薬事 （/薬業時報社）  ４２巻５号   128 2000年05月

研究論文（学術雑誌）, 単著

Assignment of rat estrogen sulfotransferase gene (Ste) to rat chromosome band 14p21.3 → p21.2

Nagai F., Satoh H., Matsui M., Tamura H.

Cytogenetics and Cell Genetics （Cytogenetics and Cell Genetics）  88 （ 1-2 ） 74・75 - 75 2000年04月

研究論文（研究会，シンポジウム資料等）, 単著, 査読有り,  ISSN  03010171

Chromosomal assignments of rat phenol sulfotransferase genes, ST1A1 (Sult1a1) and ST1C1 (Sult1c1) by fluorescence in situ hybridization.

Nagai F, Satoh H, Usui T Tamura H, Matsui M*.

Cytogenet Cell Genet 84   145-147 1999年10月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Chromosome assignments of rat phenol sulfotransferase ST1A1 and ST1C1 genes (Sult1a1 and Sult1c1) by fluorescence in situ hybridization

Nagai F., Satoh H., Usui T., Tamura H., Matsui M.

Cytogenetics and Cell Genetics （Cytogenetics and Cell Genetics）  84 （ 3-4 ） 145 - 147 1999年07月

研究論文（学術雑誌）,  ISSN  03010171

Roles of Asp126 and Asp156 in the enzyme function of sphingomyelinase from Bacillus cereus

Fujii S., Ogata K., Inoue B., Inoue S., Murakami M., Iwama S., Katsumura S., Tomita M., Tamura H., Tsukamoto K., Ikezawa H., Ikeda K.

Journal of Biochemistry （Journal of Biochemistry）  126 （ 1 ） 90 - 97 1999年07月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021924X

　概要を見る

To elucidate the roles of conserved Asp residues of Bacillus cereus sphingomyelinase (SMase) in the kinetic and binding properties of the enzyme toward various substrates and Mg2+, the kinetic data on mutant SMases (D126G and D156G) were compared with those of wild type (WT) enzyme. The stereoselectivity of the enzyme in the hydrolysis of monodispersed short-chain sphingomyelin (SM) analogs and the binding of Mg2+ to the enzyme were not affected by the replacement of Asp126 or Asp156. The pH-dependence curves of kinetic parameters (1/K(m) and k(cat)) for D156G-catalyzed hydrolysis of micellar SM mixed with Triton X-100 (1:10) and of micellar 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP) were similar in shape to those for WT enzyme-catalyzed hydrolysis. On the other hand, the curves for D126G lacked the transition observed for D156G and WT enzymes. Comparison of the values and the shape of pH-dependence curves of kinetic parameters indicated that Asp126 of WT SMase enhances the enzyme's catalytic activity toward both substrates and its binding of HNP but not SM. The deprotonation of Asp126 enhances the substrate binding and slightly suppresses the catalytic activity toward both substrates. Asp156 of WT SMase acts to decrease the binding of both substrates and the catalytic activity to HNP but not SM. From the present study and the predicted three-dimensional structure of B. cereus SMase, Asp126 was thought to be located close to the active site, and its ionization was shown to affect the catalytic activity and substrate binding.

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Molcecular cloning, expression and characterization of a phenol sulfotransferase cDNA from mouse intestine.

Tamura H*, Miyawaki A, Yoneshima H, Mikoshiba K, Matsui M.

Biol Phram Bull 22   234-239 1999年03月

研究論文（研究会，シンポジウム資料等）, 共著, 査読有り

Molecular cloning, expression and characterization of a phenol sulfotransferase cDNA from mouse intestine

Tamura H., Miyawaki A., Yoneshima H., Mikoshiba K., Matsui M.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  22 （ 3 ） 234 - 239 1999年03月

研究論文（学術雑誌）,  ISSN  09186158

　概要を見る

A sulfotransferase (ST) cDNA was isolated from a mouse intestinal cDNA library using a probe which was generated by reverse transcription (RT)-PCR based on the conserved amino acid sequences of the ST molecules. The isolated cDNA (1.1 kb) contained an 858 bp open reading frame encoding a 286 amino acid polypeptide with molecular weight of 33439. The deduced amino acid sequence shares 55.1% and 40.2% identity with mouse liver aryl/phenol (mSTp1) and alcohol (mSTa1 or mSTa2) STs, respectively, and it is highly similar to those of rat and human liver phenol ST (P-ST) isozymes, ST1B1 (87.8%) and ST1B2 (71.0%), respectively. RT-PCR analyses showed abundant expression of the P-ST mRNA in the intestine as well as in the liver in the mouse tissues examined (brain, heart, intestine, kidney, liver and lung) of both sexes. E. coli-expressed enzyme is capable of catalyzing the sulfation of 2-naphthol at K(m)=3.3 μM and V(max)=3.33 nmol/min/mg protein and also showed sulfation activity for L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine. Among food constituents tested, tannic acid and epigallocatechin gallate strongly inhibited the P-ST activity in vitro.

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Mg2+ binding and catalytic function of sphingomyelinase from Bacillus cereus.

S. Fujii*, B. Inoue, H. Yamamoto, K. Ogata, T. Shinki, S. Inoue, M. Tomita, H. Tamura, K. Tsukamoto, H. Ikezawa and K. Ikeda

J. Biochem. 124 （ 6 ） 1178 - 1187 1998年12月

研究論文（学術雑誌）, 共著, 査読有り

Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase

Tamura H., Harada Y., Miyawaki A., Mikoshiba K., Matsui M.

Biochemical Journal （Biochemical Journal）  331 （ 3 ） 953 - 958 1998年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  02646021

　概要を見る

Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme.,PSTG. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT-PCR).

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Effects of gonadal hormones on zonal expression of rat hepatic phenol sulfotransferases.

M. Matsui*, T. Nakamura, M. Tada, S. Yamagata, H. Tamura and H. Homma

Biol Pharm Bull 21 （ 2 ） 97 - 101 1998年02月

研究論文（学術雑誌）, 共著, 査読有り

On the nature of rat hepatic and mouse olfactory sulfotransferases

Matsui M., Tamura H., Nagai F., Homma H., Miyawaki A., Mikoshiba K.

Chemico-Biological Interactions （Chemico-Biological Interactions）  109 （ 1-3 ） 69 - 80 1998年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00092797

　概要を見る

Rat hydroxysteroid sulfotransferase (HS-SULT) cDNAs, ST-40 and ST-20 are 90% identical in amino acid sequences and show different substrate specificities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS). ST-40 enzyme is active toward the three substrates, whereas ST-20 enzyme is preferentially active for CS. First we prepared mutants of well conserved histidine, lysine and asparagine by site-directed mutagenesis. Secondly we constructed 20 chimeric HS-SULTs by reciprocal exchange of five protein domains between ST-20 and ST-40 enzymes. The studies on the expressed mutant and chimetic enzymes indicate the importance of the C-terminal region for the substrate specificity and the involvement of multiple regions for the enzyme activities. Next we determined the genetic loci of ST-40 and ST-20 by fluorescence in situ hybridization. Biotinylated ST-20 and ST-40 probes gave a pair of fluorescent spots on the same region of rat chromosome 1 and the loci of these genes were localized to the same chromosomal region of 1q21.3→q22.1. Finally we studied mouse olfactory phenol SULT (P-SULT). It was immunolocalized in the cytoplasm of mouse olfactory sustentacular cells and mouse nasal cytosols show high SULT activities toward phenolic aromatic odorants. We subsequently isolated a mouse P-SULT cDNA from mouse olfactory cDNA library. It encodes 304 amino acid polypeptide and is 94% identical with rat STlC1 in amino acid sequences.

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Identification of three hydroxysteroid sulfotransferase isoenzymes in the rat liver

Takahashi M., Tamura H., Kondo S., Kobayashi K., Matsui M.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  21 （ 1 ） 10 - 15 1998年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09186158

　概要を見る

Isolation of several hydroxysteroid sulfotransferase (HS-ST) cDNAs from the rat liver cDNA library has demonstrated the possible expression of these HS-ST isoforms in rat liver. We devised a method to detect unequivocally ST- 20 and ST-40 mRNAs by a reverse transcription-polymerase chain reaction, using specific primers. ST-40 mRNA was expressed only in liver, but ST-20 mRNA was present predominantly in the liver and slightly in extrahepatic tissues. On chromatofocusing, expressed ST-20 or ST-40 enzymes were eluted at approx. pH 8.2 and 5.7-4.7 or at pH 6.4-5.4, respectively. Chromatofocusing of adult female rat liver cytosols resolved HS-ST isoenzymes in a broad range of pH, and ST fractions A, B, C, D and E were eluted at approx. pH 8.2, 7.6, 7.5-6.8, 6.2 and 6.1-5.5, respectively. After PAP-agarose affinity column chromatography and SDS-polyacrylamide gel electrophoresis (PAGE), their N- terminal amino acid sequences were determined. ST isoenzymes present in fractions B and E showed identical N-terminal amino acid sequences with those of ST-21 and ST-20, respectively, whereas the ST isoenzymes present in fractions C and D had the same N-terminal amino acid sequence as those of ST- 40 (and/or ST-41). The results demonstrated the presence of at least three HS-ST isoenzymes in adult female rat liver.

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Effects of gonadal hormones on the zonal expression of rat hepatic phenol sulfotransferases

Matsui M., Nakamura T., Tada M., Yamagata S., Tamura H., Homma H.

Biological and Pharmaceutical Bulletin （Biological and Pharmaceutical Bulletin）  21 （ 2 ） 97 - 101 1998年

研究論文（学術雑誌）,  ISSN  09186158

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Estradiol benzoate (EB) and testosterone propionate (TP) were administered to male and female Wistar rats, and their effects on the zonal expression of hepatic phenol sulfotransferase (P-ST) activities were determined at pH 5.5 and 7.4. Cytosolic fractions from periportal (PP) and perivenous (PV) hepatocytes were prepared by the dual-digitonin-pulse perfusion technique. In control rats, P-ST activities assayed at pH 5.5 and 7.4 were higher in males than in females, and were higher in the PV fraction than in the PP fraction in both sexes. P-ST activities were increased by the administration of TP in the PP and PV fractions of females, whereas the same treatment diminished the enzyme activities in both fractions of the males. EB administration gave reduced P-ST activity at pH 5.5 of both fractions, irrespective of sex, but not a marked difference at pH 7.4. Chromatofocusing of PP and PV fractions revealed the presence of P-ST isoforms eluted at approx. pH 8.0 (peak 1), 7.5 (peak 2), 7.0 (peak 3) and 6.0 (peak 4). In male rats, peak 3, which showed high enzyme activity at pH 5.5 in the PP and PV fractions, was markedly decreased by EB treatment, whereas in females, peak 3 was present only in the PV fraction and was not affected by EB administration. TP treatment did not show remarkable changes in P-ST peaks in the males, while peaks 2 and 3 were increased in the females. Immunoblot analysis revealed the presence of multiple P-ST isoforms which showed different immunoreactivity and electrochemical properties.

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Mg²⁺ binding and catalytic function of Sphingomyelinase from Bacillus cereus

Fujii S., Inoue B., Yamamoto H., Ogata K., Shinki T., Inoue S., Tomita M., Tamura H., Tsukamoto K., Ikezawa H., Ikeda K.

Journal of Biochemistry （Journal of Biochemistry）  124 （ 6 ） 1178 - 1187 1998年

研究論文（学術雑誌）,  ISSN  0021924X

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The modes of Mg2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg2+ with low and high affinities. The effects of Mg2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/K(m) for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of k(cat) for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.

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Construction and expression of chimeric enzymes of rat liver hydroxysteroid sulfotransferase isozymes.

田村 悦臣

Arch Biochem Biophys 341 （ 2 ） 309 - 314 1997年05月

研究論文（学術雑誌）, 共著, 査読有り

Construction and expression of chimeric rat liver hydroxysteroid sulfotransferase isozymes

Tamura H., Morioka Y., Homma H., Matsui M.

Archives of Biochemistry and Biophysics （Archives of Biochemistry and Biophysics）  341 （ 2 ） 309 - 314 1997年05月

研究論文（学術雑誌）,  ISSN  00039861

　概要を見る

The ST-20 and ST-40 cDNAs encode rat liver hydroxysteroid sulfotransferases (HS-ST) that are 90% identical in amino acid sequence but exhibit different substrate preferences for dehydroepiandrosterone (DHEA), androsterone (AD), and cortisol (CS), ST-40 is active for all three substrates, whereas ST-20 is mainly active for cortisol. To determine the domain responsible for the substrate preferences of the HS-STs, 20 chimeric HS-STs were constructed by reciprocal exchanges of DNA fragments derived from the cDNAs and were expressed in Escherichia coli. Some chimeric enzymes were enzymatically active for all three substrates, and some displayed reduced or lost CS-ST activity, with retention of DHEA- and AD-ST activities. Others lost all HS-ST activity. Analysis revealed that a central region (region III spanning amino acids 102164 with five amino acid differences between ST-20 and ST-40) is essential for HS-ST activity, whereas regions II (amino acids 65-101) and IV (amino acids 165219) are unimportant with regard to substrate preference. It was also shown that the parental combination of regions I (amino acids 1-64) and V (amino acids 220-284) is essential for CS-ST activity. Photoaffinity labeling with [35S]3'-phosphoadenosine 5'- phosphosulfate (PAPS) revealed that some inactive chimeras lost affinity for PAPS. These results suggested that an ordered structure formed by regions I, III, and V is required for HS-ST activity, especially for substrate preference and PAPS binding.

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High sulfotransferase activity for phenolic aromatic odorants present in the mouse olfactory organ

Tamura H., Miyawaki A., Inoh N., Harada Y., Mikoshiba K., Matsui M.

Chemico-Biological Interactions （Chemico-Biological Interactions）  104 （ 1 ） 1 - 9 1997年04月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00092797

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Mouse nasal cytosols show high sulfotransferase (ST) activities toward phenolic aromatic odorants, but have little activities for most alcoholic aromatic odorants. Most ST activities toward the phenolic odorants preferred slightly acidic pH (6.4) and were sensitive to 2,6-dichloro-4-nitrophenol, a specific inhibitor for phenol ST (P-ST) but were not inhibited by triethylamine and tetra-n-butylammonium chloride, which are specific inhibitors for hydroxysteroid ST (HS-ST). These results suggested that P-ST activities are responsible for sulfation of the phenolic odorants. The spectra of the ST activities for these odorants were similar in mouse nasal and liver cytosols, however, nasal cytosols showed much higher ST activity toward cinnamyl alcohol than liver cytosols. This activity preferred higher pH (7.4) compared to the phenolic odorant-ST activities and was inhibited by both types of inhibitors, specific for P-ST and HS-ST. These results appear to indicate the participation of multiple ST isoforms for the sulfation of odorants in mouse nasal cytosols. The existence of P-ST(s) active for the phenolic odorants in olfactory cytosols suggests a role in odorant perception, in particular, in the signal termination process.

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A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I

Matsuo Y., Yamada A., Tsukamoto K., Tamura H., Ikezawa H., Nakamura H., Nishikawa K.

Protein Science （Protein Science）  5 （ 12 ） 2459 - 2467 1996年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09618368

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The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method: the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase 1 structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center; are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues. Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.

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High-level expression of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C by the Bacillus brevis host-vector system

Kobayashi T., Tamura H., Taguchi R., Udaka S., Ikezawa H.

Japanese Journal of Medical Science and Biology （Japanese Journal of Medical Science and Biology）  49 （ 3 ） 103 - 112 1996年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00215112

　概要を見る

We succeeded in hyperproduction of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC), using a Bacillus brevis 47 expression system. The recombinant B. thuringiensis PIPLC was expressed under the control of the middle wall protein gene promoter in B. brevis expression vector pNU211. A large amount of recombinant PIPLC (0.4 g per liter culture) was secreted into the medium as a mature enzyme, and the enzymatic properties of purified recombinant PIPLC were similar to those of the enzyme from wild-type B. thuringiensis. This system provides a useful approach to the three-dimensional structure-function relationship of PIPLC.

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Zonal distribution of sulfotransferase for phenol in olfactory sustentacular cells

Miyawaki A., Homma H., Tamura H., Matsui M., Mikoshiba K.

EMBO Journal （EMBO Journal）  15 （ 9 ） 2050 - 2055 1996年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  02614189

　概要を見る

We have immunolocalized phenol sulfotransferase (PST)G, an isoform of PST in sustentacular cells which reside in the dorso-medial portion of the nasal cavity of the mouse. The same topographical pattern of gene expression has been reported for some olfactory neuron-specific genes. When several established (phenol-containing) odorants were used as substrate, mouse nasal tissue cytosol showed a significant level of PST activity, as does mouse liver cytosol. This study is the first to demonstrate that gene expression in the olfactory sustentacular cells is also organized zonally, and indicates the involvement of sulfo-conjugation in olfactory perireceptor processes, such as odorant clearance and xenobiotic detoxification.

Studies on the active sites of Bacillus cereus sphingomyelinase substitution of some amino acids by site-directed mutagenesis

Ikezawa H., Tameishi K., Yamada A., Tamura H., Tsukamoto K., Matsuo Y., Nishikawa K.

Amino Acids （Amino Acids）  9 （ 3 ） 293 - 298 1995年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  09394451

　概要を見る

Chemical modifications suggested that acidic amino acids such as aspartic and glutamic acids are involved in the active sites of Bacillus cereus sphingomyelinase. Among aspartic acid residues in the conserved regions of this enzyme, Asp-126, Asp-156, Asp-233 and Asp-295 were converted to glycine by site-directed mutagenesis. According to prediction on structural similarity to pancreatic DNase I, His-151 and His-296 were also converted to alanine. The Asp and His mutants, D126G, D156G, D233G, D295G, H151A and H296A, were produced in Bacillus brevis 47, a protein-hyperproducing strain. The catalytic activities of D295G, H151A and H296A were completely abolished, and sphingomyelin-hydrolyzing activity of D126G or D156G was reduced by more than 50%. The activity of D126G toward p-NPPC was comparable to that of the wild-type, while D156G catalyzed the hydrolysis of HNP and p-NPPC more efficiently than the wild-type. Hemolytic activities of the mutants were parallel to their sphingomyelin-hydrolyzing activities. © 1995 Springer-Verlag.

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Mutation in aspartic acids modifies catalytic and hemolytic activities of Bacillus cereus sphingomyelinase.

H. Tamura*, K. Tameishi, A. Yamada, M. Tomita, Y. Matsuo, K. Nishikawa and H. Ikezawa

Biochem J 309 （ 3 ） 757 - 764 1995年08月

研究論文（学術雑誌）, 共著, 査読有り

Studies on catalytic sites of glycosylphosphatidylinositol-anchoring aminopeptidase N from larval midgut of the silkworm, Bombyx mori and its expression in virus-infected silkworm.

田村 悦臣

Protein Engng 8   966 1995年

研究論文（学術雑誌）, 共著, 査読有り

Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase

Tamura H., Tameishi K., Yamada A., Tomita M., Matsuo Y., Nishikawa K., Ikezawa H.

Biochemical Journal （Biochemical Journal）  309 （ 3 ） 757 - 764 1995年

研究論文（学術雑誌）,  ISSN  02646021

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Four aspartic acid residues (Asp126, Asp156, Asp233 and Asp295) of Bacillus cereus sphingomyelinase (SMase) in the conservative regions were changed to glycine by in vitro mutagenesis, and the mutant SMases [D126G (Asp126 → Gly etc.), D156G, D233G and D295G] were produced in Bacillus brevis 47, a protein-producing strain. The sphingomyelin (SM)-hydrolysing activity of D295G was completely abolished and those of D126G and D156G were reduced by more than 80%, whereas that of D233G was not so profoundly affected, Two mutant enzymes (D126G and D156G) were purified and characterized further. The hydrolytic activities of D126G and D156G toward four phosphocholine-containing substrates with different hydrophobicities, SM, 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP), lysophosphatidylcholine (lysoPC) and p-nitrophenylphosphocholine (p-NPPC), were compared with those of the wild-type. The activity of D126G toward water-soluble p-NPPC was comparable with that of the wild-type. On the other hand, D156G catalysed the hydrolysis of hydrophilic substrates such as HNP and p-NPPC more efficiently (> 4-fold) than the wild-type. These results suggested that Asp126 and Asp156, located in the highly conserved region, may well be involved in a substrate recognition process rather than catalytic action. Haemolytic activities of the mutant enzymes were found to be parallel with their SM-hydrolysing activities. Two regions, including the C-terminal region containing Asp295, were found to show considerable sequence identity with the corresponding regions of bovine pancreatic DNase I. Structural predictions indicated structural similarity between SMase and DNase I. An evolutionary relationship based on the catalytic function was suggested between the structures of these two phosphodiesterases.

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Purification of glycosylphophatidylinositol-anchoring aminopeptidase N from the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori.

M. Tomita*, H. Obara, Y. Takesue, H. Tamura, S. Miyajima, R. Taguchi and H. Ikezawa

Int J Biochem 26 （ 8 ） 977 - 986 1994年08月

研究論文（学術雑誌）, 共著, 査読有り

Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori

Masahiro T., Hiroshi O., Yoshiki T., Hiro-Omi T., Shigetoshi M., Ryo T., Hiroh I.

International Journal of Biochemistry （International Journal of Biochemistry）  26 （ 8 ） 977 - 986 1994年08月

研究論文（学術雑誌）,  ISSN  0020711X

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1. 1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies. 2. 2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3. 3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met-p-nitroanilide as substrates. Especially, Leu-p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm. 4. 4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut. © 1994.

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Inhibition of NGF-induced neurite outgrowth of PC12 cells by Bacillus cereus sphingomyelinase, a bacterial hemolysin

Tamura H., Noto M., Kinoshita K., Ohkuma S., Ikezawa H.

Toxicon （Toxicon）  32 （ 5 ） 629 - 633 1994年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00410101

　概要を見る

H-o. Tamura, M. Noto, K. Kinoshita, S. Ohkuma and H. Ikezawa. Inhibition of NGF-induced neurite outgrowth of PC12 cells by Bacillus cereus sphingomyelinase, a bacterial hemolysin. Toxicon 32, 629-633, 1994.-Sphingomyelinase of Bacillus cereus, a bacterial hemolysin, reduced nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells in a dose-dependent manner. At 200 mU/ml, sphingomyelinase repressed half the neurite outgrowth of the cells at 250 ng/ml NGF. The c-fos superinduction, one of the early responses induced by NGF, was not influenced by this treatment, suggesting that the repression by sphingomyelinase occurred via a protein kinase C-independent pathway. © 1994.

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Mutational analysis of the COOH-terminal hydrophobic domain of 5'-nucleotidase as a signal for the glycosylphosphatidylinositol (GPI)-anchor modification.

Y. Furukawa, H. Tamura* and H. Ikezawa

Biochim Biophys Acta 1190 （ 2 ） 273 - 278 1994年03月

研究論文（学術雑誌）, 共著, 査読有り

Mutational analysis of the COOH-terminal hydrophobic domain of bovine liver 5′-nucleotidase as a signal for glycosylphosphatidylinositol (GPI) anchor attachment

Furukawa Y., Tamura H., Ikezawa H.

BBA - Biomembranes （BBA - Biomembranes）  1190 （ 2 ） 273 - 278 1994年03月

研究論文（学術雑誌）,  ISSN  00052736

　概要を見る

In order to address the minimum domain of the COOH-terminal hydrophobic region responsible for GPI modification of bovine liver 5′-nucleotidase, we constructed a series of the deletion mutants of the COOH-terminus and expressed them in COS cells. Cells transfected by the deletion mutant of 6 amino acids (-IIILYQ) from the hydrophobic domain (-FSLIFLSVLAVIIILYQ) did not show any elevation of cell surface-associated 5′-nucleotidase activity, whereas the 2 (-YQ) or 4 (-ILYQ) amino acid deletion mutant retained the bovine liver-derived activity on the cell surface as a GPI-anchored protein. Loss of half the hydrophobic domain (6 or 8 amino acids) resulted in accumulation of the activity in the cell. On the other hand, deletion of the whole hydrophobic domain (17 amino acids) or the entire cleaved-off domain (25 amino acids) made the product secreted into the medium. In conclusion, the hydrophobicity of 13 amino acids in length was enough for the GPI modification of the bovine liver 5′-nucleotidase. © 1994.

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Studies on the GPI-anchored enzyme, hepatic 5'-nucleotidase. Microheterogeneity of the anchor, processing and localization.

Ikezawa H., Taguchi R., Tamura H., Kobayashi T.

Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.] （Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.]）  27 （ 2 ） 395 - 399 1994年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0100879X

　概要を見る

Hepatic 5'-nucleotidases of vertebrates were investigated for localization in the lysosomes and the plasma membrane, microheterogeneity of the glycosylphosphatidylinositol (GPI)-anchor moiety and minimal requirement of the C-terminal signal peptide for GPI attachment. Using PIPLC of Bacillus thuringiensis and subcellular fractionation by Percoll gradient centrifugation, we found that chicken liver 5'-nucleotidase can be transferred from plasma membrane to lysosomes in the GPI-anchored or soluble form. Bovine liver ecto 5'-nucleotidase was solubilized by PIPLC, purified to a homogeneous state, and analyzed for the structures of GPI-anchor isoforms by HPLC and ESI-MS in combination with glycosidase treatments, after peptide-bond cleavage by CNBr or trypsin. Several isomers of the GPI anchor were thus characterized; major components contained two phosphorylethanolamine residues, whereas the component containing three phosphorylethanolamine residues was present only as a small percentage of the total. The cleavage/attachment site of the GPI anchor in the C-terminal of 5'-nucleotidase was shown to be Ser523. The peptide region cleaved off at the posttranslational processing has a length of 25 amino acid residues which contains a hydrophobic stretch of 17 amino acids. By site-directed mutagenesis, we determined the minimal length of the hydrophobic peptide to be 13 amino acids for expression of 5'-nucleotidase as a GPI-anchored form on the COS cell surface. When peptide length was shortened to less than 13 amino acids, the expressed enzyme was not sorted to the cell surface but present within, or secreted out of the cells.

The role of acidic amino acid residues in catalytic and adsorptive sites of Bacillus cereus sphingomyelinase.

M. Tomita*, Y. Ueda, H. Tamura, R. Taguchi and H. Ikezawa

Biochim Biophys Acta 1203 （ 1 ） 85 - 92 1993年11月

研究論文（学術雑誌）, 共著, 査読有り

The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus spingomyelinase

Tomita M., Ueda Y., Tamura H., Taguchi R., Ikezawa H.

Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular （Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular）  1203 （ 1 ） 85 - 92 1993年11月

研究論文（学術雑誌）,  ISSN  01674838

　概要を見る

By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3′-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90%. Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol cause a drasic decrease in enzymatic activity, whereas the spingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid. Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the unchanged amino-acid residues Ser and Thr. The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes. However, the extent of inhibition in the enzyme adsorption, 20-50%, was less than that observed in the sphingomyelinase activity. These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites. Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards crythrocyte membranes. © 1993.

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Inhibition of cell growth by bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase.

S. Ohkuma*, S. Shimizu, M. Noto, Y. Sai, K. Kinoshita and H. Tamura

In Vitro Cell Dev Biol 29A （ 11 ） 862 - 866 1993年11月

研究論文（学術雑誌）, 共著, 査読有り

Inhibition of cell growth by bafilomycin A<inf>1</inf>, a selective inhibitor of vacuolar H⁺-ATPase

Ohkuma S., Shimizu S., Noto M., Sai Y., Kinoshita K., Tamura H.

In Vitro Cellular &amp; Developmental Biology - Animal: Journal of the Society for In Vitro Biology （In Vitro Cellular &amp; Developmental Biology - Animal: Journal of the Society for In Vitro Biology）  29 （ 11 ） 862 - 866 1993年11月

研究論文（学術雑誌）,  ISSN  10712690

　概要を見る

Bafilomycin A1, a potent selective inhibitor of vacuolar H+-ATPase, inhibited the growth of a variety of cultured cells dose-dependently, including golden hamster embryo and NIH-3T3 fibroblasts, whether or not they were transformed, and PC12 and HeLa cells. The concentration of bafilomycin A1 for 50% inhibition of cell growth ranged from 10 to 50 n M. The dose response was nearly parallel with that of the bafilomycin A1-induced lysosomal pH increase. The degree of pH increase for growth inhibition produced by bafilomycin A1 was similar to that produced by NH4Cl in which little difference was recognized in effect among cell types. © 1993, Tissue Culture Association. All rights reserved.

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Purification and cDNA molecular cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells.

田村 悦臣

J Biochem 113 （ 3 ） 607 - 613 1993年05月

研究論文（学術雑誌）, 共著, 査読有り

Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47 and its purification.

H. Tamura*, K. Tameishi, H. Yamagata, S. Udaka, T. Kobayashi, M. Tomita and H. Ikezawa

J Biochem 112 （ 4 ） 488 - 491 1992年10月

研究論文（学術雑誌）, 共著, 査読有り

Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47, and its purification

Tamura H., Tameishi K., Yamagata H., Udaka S., Kobayashi T., Tomita M., Ikezawa H.

Journal of Biochemistry （Journal of Biochemistry）  112 （ 4 ） 488 - 491 1992年10月

研究論文（学術雑誌）,  ISSN  0021924X

　概要を見る

Sphingomyelinase (sphingomyelin cholinephosphohydrolase) [EC 3.1.4.12] of Bacillus cereus was overproduced in a protein-hyperproducing strain, B. brevis 47, by cloning the gene into an expression vector pNU211, which has been developed to express a foreign gene utilizing a promoter and a signal sequence of an outer cell wall protein gene. From 1 liter of culture, about 10 mg of protein was purified to near-homogeneity by two steps of column chromatography; this is almost 500 times higher production compared to the conventional preparation from the original strain, B. cereus IAM 1208. The N-terminal amino acid sequence of the secreted enzyme was identical to that of the authentic enzyme, indicating that the signal sequence for secretion of B. cereus was processed properly in B. brevis 47. © 1992 BY THE JOURNAL OF BIOCHEMISTRY.

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Molecular cloning of a rat liver cDNA encoding the 16KDa subunit of vacuolar H+-ATPase; Organella and tissue distribution of 16 KDa proteolipids.

J. Nezu, K. Motojima, H. Tamura and S. Ohkuma*

J Biochem 112 （ 2 ） 212 - 219 1992年08月

研究論文（学術雑誌）, 共著, 査読有り

Molecular cloning of a rat liver cDNA encoding the 16 kDa subunit of vacuolar H⁺-ATPases: Organellar and tissue distribution of 16 kDa proteolipids

Nezu J., Motojima K., Tamura H., Ohkuma S.

Journal of Biochemistry （Journal of Biochemistry）  112 （ 2 ） 212 - 219 1992年08月

研究論文（学術雑誌）,  ISSN  0021924X

　概要を見る

A cDNA (T3-L) encoding the 16 kDa subunit of vacuolar H+-ATPase was cloned from a cDNA library of rat liver. A polypeptide of 156 amino acids with a molecular mass of 15,807 Da (pI=9.5) having four hydrophobic stretches was predicted. T3-L polypeptide was 92% and 100% identical with the 16 kDa proteolipid of bovine chromaffin granule and that of mouse, respectively. Antisera raised against the NH2-terminal of the T3-L polypeptide reacted positively with the membrane ghosts of rat liver tritosomes and the partially purified H+-ATPase thereof. Western blotting of subcellular fractions with the antisera showed high abundance of 16 kDa protein in the lysosomes, although a significant amount was also detected in the Golgi apparatus. Western blotting of rat tissues revealed high levels of 16 kDa proteolipid in the brain and the kidney. Northern blots with T3-L similarly showed considerably high expression of T3-L mRNA in the brain and the kidney. Southern hybridization of rat genomic DNA with T3-L showed at most three distinct bands, regardless of the stringency of hybridization and whether hybridization was performed with its subfragments. This suggests the possibility of multiple (at least three) homologous/identical genes encoding 16 kDa proteolipid. The possible presence and significance of isoforms of 16 kDa proteolipid in rats are discussed. © 1992 THE JOURNAL OF BIOCHEMISTRY.

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Induction of neurite outgrowth of PC12 cells by an inhibitor of vacuolar H+-ATPase, bafilomycin A1.

H. Tamura* and S. Ohkuma

FEBS Lett 294 （ 1-2 ） 51 - 55 1991年12月

研究論文（学術雑誌）, 共著, 査読有り

Induction of neurite outgrowth of PC12 cells by an inhibitor of vacuolar H⁺-ATPase, bafilomycin A<inf>1</inf>

Tamura H., Ohkuma S.

FEBS Letters （FEBS Letters）  294 （ 1-2 ） 51 - 55 1991年12月

研究論文（学術雑誌）,  ISSN  00145793

　概要を見る

Bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase, induced neurite outgrowth of PC12 cells dose- and time-dependently: more than 50% of the cells extended neurite-like spikes after 24 h treatment with 100 nM bafilomycin A1. Its dose-response ran roughly parallel to that of a bafilomycin A1-induced lysosomal pH increase. It was inhibited by LiCl, an inhibitor of the phosphorylation of microtubule-associated proteins and, like nerve growth factor (NGF)-induced neurite outgrowth, it was also inhibited by cycloheximide and actinomycin D. But, unlike the NGF-effect, it was not associated with rapid induction of c-fos. © 1991.

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Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites

Tamura H., Kohchi C., Yamada R., Ikeda T., Koiwai O., Patterson E., Keene J., Okada K., Kjeldsen E., Nishikawa K., Andoh T.

Nucleic Acids Research （Nucleic Acids Research）  19 （ 1 ） 65 - 75 1991年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  03051048

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Camptothecin (CPT), a plant alkaloid with antltumor activity, is a specific Inhibitor of eukaryotlc DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 μM, whereas the parental fusion protein was sensitive to CPT as low as 1 μM. The resistance index (>125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT. © 1991 Oxford University Press.

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Mode of Inhibition of Eukaryotic DNA Topoisomerase I Inhibitors

Tamura H., Ikegami Y.

nippon nogeikagaku kaishi （nippon nogeikagaku kaishi）  65 （ 6 ） 1008 - 1010 1991年

研究論文（学術雑誌）,  ISSN  00021407

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Acidic phospholipids directly inhibit DNA binding of mammalian DNA topoisomerase I

田村 悦臣

FEBS Letters （FEBS Letters）  261 （ 1 ） 151 - 154 1990年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00145793

　概要を見る

Inhibition of mammalian DNA topoisomerase I by phospholipids was investigated using purified enzyme. Acidic phospholipids inhibited the DNA relaxation activity of topoisomerase I whereas neutral phospholipid, phosphatidylethanolamine, did not. Accumulation of a protein-DNA cleavable complex, an intermediate which is known to accumulate upon inhibition by a specific inhibitor camptothecin, did not occur. The filter binding assay revealed that the DNA binding activity of the enzyme was inhibited by acidic phospholipids. Moreoever, direct binding of phosphatidylglycerol to topoisomerase I was demonstrated. These results indicated that the inhibitory effect of acidic phospholipids on topoisomerase I was due to the loss of the DNA binding of the enzyme as a result of direct interaction between phospholipids and the enzyme. © 1990.

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Identification of the DNA binding subunit of RNA polymerase II from ehrlich ascites tumor cells

Horikoshi M., Tamura H., Sekimizu K., Obinata M., Natori S.

Journal of Biochemistry （Journal of Biochemistry）  94 （ 6 ） 1761 - 1767 1983年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021924X

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The DNA binding subunits of RNA polymerase II from Ehrlich ascites tumor cells were investigated in the following three ways.RNA polymerase II was dissociated in urea and the binding of the dissociated subunits to DNA was investigated.The RNA polymerase II: DNA complex was dissociated progressively with various concentrations of urea, and the subunits firmly attached to DNA were investigated.RNA polymerase II was dissociated into subunits in a SDS-polyacrylamide gel containing urea and blotted onto a nitrocellulose filter. The filter was then incubated with32P-nick-translated DNA to identify the DNA binding subunits.These procedures all showed that the largest subunit a of RNA polymerase II had strong affinity to DNA. It was found that a portion of subunits b and c could be recovered in DNA fraction when analyzed by procedures (1) and (2), but no significant DNA binding activity was detected when analyzed by procedure (3), suggesting that these subunits have either a much weaker affinity toward DNA compared to a or have affinity to a itself. © 1983 THE JOURNAL OF BIOCHEMISTRY.

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Differential expression of two abundant messenger RNAs during development of Sarcophaga peregrina

Tamura H., Tahara T., Kuroiwa A., Obinata M., Natori S.

Developmental Biology （Developmental Biology）  99 （ 1 ） 145 - 151 1983年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  00121606

　概要を見る

Expression of two abundant mRNAs in the fat body of third instar larvae of Sarcophaga peregrina was studied using genomic clones of storage protein and 25K protein as probes. It was found that these two genes were expressed sequentially in the third instar with a 20-hr interval between their expressions. No amplification of genes was detected during larval development, suggesting that the high level of mRNA was due to efficient transcription. The contents of the two mRNAs in third instar larvae were about the same, although the synthesis of storage protein was much more than that of 25K protein. © 1983.

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Tissue-Specific Distribution of DNA-Binding Nuclear Proteins

Tamura H., Natori S., Obinata M.

Differentiation （Differentiation）  23 （ 1 ） 87 - 90 1982年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  03014681

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The DNA-binding capacity of nuclear proteins of mouse cells was examined by the protein-blotting method. Under conditions in which the lac repressor specifically binds to the lac operator, the DNA-binding nuclear proteins from different tissues showed a tissue-specific distribution, suggesting that the species and amounts of nuclear proteins with DNA binding activity differ in different tissues. When cloned eukaryotic genes were used for binding, eukaryotic DNA showed stronger binding than prokaryotic DNA. Competition experiments suggested that many nuclear proteins have different DNA binding properties from that of the prokaryotic repressor. © 1982, International Society of Differentiation. All rights reserved.

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Purification of a new protein stimulating RNA polymerase II from ehrlich ascites tumor cells: Comparison with proteins purified before

Nakanishi Y., Sekimizu K., Tamura H., Natori S.

Journal of Biochemistry （Journal of Biochemistry）  90 （ 3 ） 805 - 814 1981年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021924X

　概要を見る

A protein factor named S-I(b) that stimulates the activity of RNA polymerase II was purified from Ehrlich ascites tumor cells. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein has a molecular weight of 24,000 and is significantly smaller than S-II and S-II', stimulatory proteins of RNA polymerase II purified previously. However, S-I(b) cross-reacted with antibody against S-II and its peptide map obtained after radio-iodination was identical with that of S-II' (a phosphorylated form of S-II), indicating that S-I(b) and S-II share a common primary structure. S-I(b) is suggested not to be a degradation product of S-II produced during the purification process, and the structural relations between S-II and S-II' are discussed. © 1981, by the Japanese Biochemical Society.

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Measurement of a stimulatory protein of RNA polymerase II in various mouse organs by the complement fixation test

Tamura H., Sekimizu K., Obinata M., Natori S.

Journal of Biochemistry （Journal of Biochemistry）  88 （ 5 ） 1475 - 1480 1980年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021924X

　概要を見る

The amount of S-II, a protein specifically stimulating RNA polymerase II, was measured in various mouse organs by a micro complement fixation assay. The amount was almost the same in brain, liver, kidney, spleen, and Ehrlich ascites tumor cells on the basis of DNA, being about 3.8 μg/mg DNA, which corresponds to 3.6 × 105molecules/cell. However, the amount of S-II decreased greatly during erythro-differentiation of Friend leukemia cells and no S-II was detected in matured erythrocytes. © 1980, by the Japanese Biochemical Society.

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