Published papers

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.

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.

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.

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.

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.

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.

© 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.

© 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.

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.

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.

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.

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.

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.