Details of a Researcher - Tamura, Hiroomi

Papers - Tamura, Hiroomi

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

Tamura H*, Taniguchi K, Hayashi E, Hiyoshi Y, Nagai F.

Biol Pharm Bull (Biological and Pharmaceutical Bulletin) 24 ( 11 ) 1258・1262 - 1262 2001.11

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 09186158

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.

J. Health Sci (Journal of Health Science) 47 ( 5 ) 468・472 - 472 2001.10

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 13449702

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*.

Biol Pharm Bull (Biological and Pharmaceutical Bulletin) 24 ( 9 ) 1076・1078 - 1078 2001.09

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 09186158

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 2001.08

Research paper (conference, symposium, etc.), Joint Work, Accepted

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

Research paper (scientific journal), ISSN 00137227

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 2000.07

Research paper (conference, symposium, etc.), Joint Work, Accepted

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 2000.07

Research paper (scientific journal), ISSN 09186158

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.

Biol. Pharm. Bull. (Biological and Pharmaceutical Bulletin) 23(6) ( 6 ) 695-699 - 699 2000.06

Research paper (conference, symposium, etc.), Joint Work, Accepted, ISSN 09186158

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 2000.05

Research paper (scientific journal), Single Work

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

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

Cytogenet Cell Genet (Cytogenetics and Cell Genetics) 88 ( 1-2 ) 74・75 - 75 2000.04

Research paper (conference, symposium, etc.), Single Work, Accepted, 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 1999.10

Research paper (conference, symposium, etc.), Joint Work, Accepted

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 1999.07

Research paper (scientific journal), ISSN 03010171

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

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

J Biochem (Journal of Biochemistry) 126 ( 1 ) 90 - 97 1999.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021924X

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 1999.03

Research paper (conference, symposium, etc.), Joint Work, Accepted

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 1999.03

Research paper (scientific journal), ISSN 09186158

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 1998.12

Research paper (scientific journal), Joint Work, Accepted

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

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

Biochem J (Biochemical Journal) 331 ( 3 ) 953 - 958 1998.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 02646021

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 1998.02

Research paper (scientific journal), Joint Work, Accepted

On the nature of rat hepatic and mouse olfactory sulfotransferases.

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

Chem-Biol Interact (Chemico-Biological Interactions) 109 ( 1-3 ) 69 - 80 1998.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 00092797

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.

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

Biol Pharm Bull (Biological and Pharmaceutical Bulletin) 21 ( 1 ) 10 - 15 1998.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 09186158

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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