論文

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.

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.

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.

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 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.

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.

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.

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.

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.

© 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.

© 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.

© 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.

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.

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.