Published papers

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.

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).

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.

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.

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.

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.

© 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.

© 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.

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.

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.

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.

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.

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.