Rhododendrol, a reductive metabolite of raspberry ketone, suppresses the differentiation of 3T3­L1 cells into adipocytes.

Obesity is a serious medical condition worldwide, and a major risk factor for type 2 diabetes, metabolic syndrome, cancer and cardiovascular disease. In addition to changes in dietary habits and physical activity, consuming supplements to maintain good health and prevent obesity is important in modern society. Raspberry ketone (RK) is a natural phenolic ketone found in the European red raspberry (Rubus idaeus L.) and is hypothesized to prevent obesity when administered orally. The present study found that RK was reduced to rhododendrol (ROH) in human liver microsomes and cytosol. The present study investigated whether the metabolite ROH had anti­adipogenic effects using mouse 3T3­L1 cells. The effects of ROH or RK on lipid accumulation during differentiation of 3T3­L1 pre­adipocyte into adipocyte were determined using Oil Red O staining. CCAAT enhancer­binding protein α (C/EBPα) and peroxisome proliferator­activated receptor γ (PPARγ) mRNA and protein expression were examined using reverse transcription­quantitative PCR and western blotting analysis, respectively. The present study revealed that ROH suppressed lipid accumulation in the cells, similar to RK. In addition, ROH suppressed the mRNA expression levels of C/EBPα and PPARγ in 3T3­L1 adipocytes. Furthermore, ROH suppressed PPARγ protein expression in 3T3­L1 adipocytes. These findings suggested that ROH is an active metabolite with an anti­adipogenic effect, which may contribute to the anti­obesity effect of orally administered RK. The present study indicated that it is important to understand the biological activity of the metabolites of orally administered compounds.

Carbonic anhydrase 3 increases during liver adipogenesis even in pre-obesity, and its inhibitors reduce liver adipose accumulation.

The abnormal lipid metabolism in the liver that occurs after high caloric intake is the main cause of nonalcoholic fatty liver disease (NAFLD). Differences between samples from healthy livers and livers from individuals with NAFLD indicate that changes in liver function occur during disease progression. Here, we examined changes in protein expression in a fatty liver model in the early stages of obesity to identify potential alterations in function. The proteins expressed in the liver tissue of pre-obese rats were separated via SDS/PAGE and stained with Coomassie brilliant blue-G250. Peptide mass fingerprinting indicated an increase in the expression of carbonic anhydrase 3 (CA3) relative to controls. Western blotting analysis confirmed the increase in CA3 expression, even in an early fat-accumulation state in which excessive weight gain had not yet occurred. In human hepatoma HepG2 cells, fat accumulation induced with oleic acid also resulted in increased CA3 expression. When the cells were in a state of fat accumulation, treating them with the CA3 inhibitors acetazolamide (ACTZ) or 6-ethoxyzolamide (ETZ) suppressed fat accumulation, but only ETZ somewhat reduced the fat-induced upregulation of CA3 expression. Expression of CA3 was therefore upregulated in response to the consumption of a high-fat diet, even in the absence of an increase in body weight. The suppression of CA3 activity by ACTZ or ETZ reduced fat accumulation in hepatocytes, suggesting that CA3 is involved in the development of fatty liver.

[Occupational Exposure of Pharmacists to Drugs during the Preparation of Powder Drugs in Dispensing Pharmacies].

Occupational exposure of pharmacists to drugs during powder drug preparation in dispensing pharmacies was investigated. First, we determined frequently prescribed tipepidine hibenzate and ambroxol hydrochloride suspended in the air of the dispensing room. The median concentration of the drugs in the air was 0.01 µg/m3 and 0.02 µg/m3, respectively; these values indicate that the air in the dispensing room was contaminated with powder drug. To estimate drug exposure during powder drug preparation, drug dust was collected near the nose of workers. Analysis of the active ingredients of the drugs used in the preparation revealed that eight drugs, including bethanechol, l-carbocisteine, and zonisamide, were detected in the range of 1.5-1220 µg/m3. Assuming that the respiratory volume of an adult was 0.008 m3/min, it was estimated that 0.4-36 µg of the ingredients were exposed per prescription by multiplying concentration, respiratory volume and sampling time (3-5 min). Furthermore, the effect of wearing a medical mask on the drug powder exposure was evaluated using a self-made apparatus. When the amount of drug powder collected on filters that is either covered with or without a medical mask was compared, the covered filter exhibited reduced drug powder accumulation to less than 10% the amount collected on the uncovered filter. The present data suggested that a medical mask might decrease the drug dust allergies in dispensing pharmacists.

Oral pharmacokinetics and in-vitro metabolism of metyrapone in male rats.

OBJECTIVES: The purpose of this study was to investigate the pharmacokinetics of a single oral administration of metyrapone (MP) and metabolites produced from it in male Wistar rats, and the major tissues and enzymes involved in the production of the MP metabolites. Furthermore, the MP metabolism in human liver subcellular fractions was compared with that in rats. METHODS: High-performance liquid chromatography with ultraviolet detection (HPLC-UV) was used to determine the concentrations of MP and its metabolites in plasma and urine after administration, and the production activity of MP metabolites in subcellular fractions of various tissues. KEY FINDINGS: Plasma concentration of MP was rapidly increased and decreased, and the primary metabolite, metyrapol (MPOL), was immediately produced. The production activity of MPOL was substantially inhibited by an 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitor in the rat and human liver microsomal and mitochondrial fractions. In the liver cytosolic fraction, the activity was inhibited by a carbonyl reductase inhibitor in the humans but not rats. CONCLUSIONS: In this study, we elucidated the plasma pharmacokinetics of MP and its metabolites in male rats after an oral administration. MPOL is most likely to be produced by 11ß-HSD1 in the male rats and humans.

Comparative inhibition of tetrameric carbonyl reductase activity in pig heart cytosol by alkyl 4-pyridyl ketones.

CONTEXT AND OBJECTIVE: The present study is to elucidate the comparative inhibition of tetrameric carbonyl reductase (TCBR) activity by alkyl 4-pyridyl ketones, and to characterize its substrate-binding domain. MATERIALS AND METHODS: The inhibitory effects of alkyl 4-pyridyl ketones on the stereoselective reduction of 4-benzoylpyridine (4-BP) catalyzed by TCBR were examined in the cytosolic fraction of pig heart. RESULTS: Of alkyl 4-pyridyl ketones, 4-hexanoylpyridine, which has a straight-chain alkyl group of five carbon atoms, inhibited most potently TCBR activity and was a competitive inhibitor. Furthermore, cyclohexyl pentyl ketone, which is substituted by cyclohexyl group instead of phenyl group of hexanophenone, had much lower ability to be reduced than hexanophenone. DISCUSSION AND CONCLUSION: These results suggest that in addition to a hydrophobic cleft corresponding to a straight-chain alkyl group of five carbon atoms, a hydrophobic pocket with affinity for an aromatic group is located in the substrate-binding domain of TCBR.

Elongated phytoglycogen chain length in transgenic rice endosperm expressing active starch synthase IIa affects the altered solubility and crystallinity of the storage α-glucan.

The relationship between the solubility, crystallinity, and length of the unit chains of plant storage α-glucan was investigated by manipulating the chain length of α-glucans accumulated in a rice mutant. Transgenic lines were produced by introducing a cDNA for starch synthase IIa (SSIIa) from an indica cultivar (SSIIa (I), coding for active SSIIa) into an isoamylase1 (ISA1)-deficient mutant (isa1) that was derived from a japonica cultivar (bearing inactive SSIIa proteins). The water-soluble fraction accounted for >95% of the total α-glucan in the isa1 mutant, whereas it was only 35-70% in the transgenic SSIIa (I)/isa1 lines. Thus, the α-glucans from the SSIIa (I)/isa1 lines were fractionated into soluble and insoluble fractions prior to the following characterizations. X-ray diffraction analysis revealed a weak B-type crystallinity for the α-glucans of the insoluble fraction, while no crystallinity was confirmed for α-glucans in isa1. Concerning the degree of polymerization (DP) ≤30, the chain lengths of these α-glucans differed significantly in the order of SSIIa (I)/isa1 insoluble > SSIIa (I)/isa1 soluble > α-glucans in isa1. The amount of long chains with DP ≥33 was higher in the insoluble fraction α-glucans than in the other two α-glucans. No difference was observed in the chain length distributions of the ß-amylase limit dextrins among these α-glucans. These results suggest that in the SSIIa (I)/isa1 transgenic lines, the unit chains of α-glucans were elongated by SSIIa(I), whereas the expression of SSIIa(I) did not affect the branch positions. Thus, the observed insolubility and crystallinity of the insoluble fraction can be attributed to the elongated length of the outer chains due to SSIIa(I).

Possible mechanism of superoxide formation through redox cycling of plumbagin in pig heart.

The purpose of this study is to elucidate the possible mechanism of superoxide formation through redox cycling of plumbagin (PLG) in pig heart. Of four 1,4-naphthoquinones tested in this study, PLG was most efficiently reduced in the cytosolic fraction of pig heart. On the other hand, lawsone (LAS) was little reduced. Thus, whether or not PLG and LAS induce the formation of superoxide anion radical in pig heart cytosol was examined, by using the methods of cytochrome c reduction and chemiluminescence. PLG significantly induced the formation of superoxide anion radical, even though LAS had no ability to mediate superoxide formation. PLG was a significant inhibitor for the stereoselective reduction of 4-benzoylpyridine (4-BP) catalyzed by tetrameric carbonyl reductase (TCBR) in pig heart cytosol. Furthermore, PLG was confirmed to competitively inhibit the 4-BP reduction, and the optimal pH for the PLG reduction was around 6.0 similar to that for the 4-BP reduction. These results suggest that PLG mediates superoxide formation through its redox cycling involved in the two-electron reduction catalyzed by TCBR, and induces oxidative stress in pig heart.

Structures of starches from rice mutants deficient in the starch synthase isozyme SSI or SSIIIa.

Amylose and amylopectin of rice mutants deficient in a starch synthase (SS) isozyme in the endosperm, either SSI (ΔSSI) or SSIIIa (ΔSSIIIa), were structurally altered from those of their parent (cv. Nipponbare, Np). The amylose content was higher in the mutants (Np, 15.5%; ΔSSI, 18.2%; ΔSSIIIa, 23.6%), and the molar ratio of branched amylose and its side chains was increased. The chain-length distribution of the ß-amylase limit dextrins of amylopectin showed regularity, which appeared consistent with the generally accepted cluster structure, and the degrees of polymerization found at the intersections were taken as the boundaries of the B-chain fractions. The mole % of the B(1)-B(3) fractions was changed slightly in ΔSSI, which is consistent with the proposed role of SSI in elongating the external part of clusters. In ΔSSIIIa, a significant increase in the B(1) fraction and a decrease in the B(2) and B(3) fractions were observed. The internal chain length of the B(2) and B(3) fractions appeared to be slightly altered, suggesting that the deficiency in SS affected the actions of branching enzyme(s).

Thrombomodulin suppresses invasiveness of HT1080 tumor cells by reducing plasminogen activation on the cell surface through activation of thrombin-activatable fibrinolysis inhibitor.

Cell malignancy is negatively correlated with the expression of thrombomodulin (TM), a thrombin receptor expressed on the surface of various cells, including tumor cells. TM accelerates thrombin-activatable fibrinolysis inhibitor (TAFI) activation catalyzed by thrombin. The active form of TAFI (TAFIa) contributes to inhibition of plasmin formation through its carboxypeptidase B (CPB)-like activity. Here, we examined whether TM- and tumor cell-dependent TAFI activation participates in controlling pericellular fibrinolysis and cell invasion. Human fibrosarcoma HT1080 cells retained the ability to activate both prothrombin and plasminogen, but did not express TM. HT1080 cells mediated activation of TAFI in plasma in the presence of soluble-type TM (sTM) through cell-dependent prothrombin activation. HT1080 cells stably expressing TM (TM-HT1080) mediated plasma TAFI activation without added sTM, but HT1080 (wild-type) and Mock-transfected HT1080 cells (Mock) did not. Production of TAFIa suppressed cell-mediated plasminogen activation. Matrigel invasion by wild-type and Mock cells was enhanced two-fold by diluted plasma (10%), whereas the plasma-induced invasion of TM-HT1080 cells (65% of wild-type invasion) was lower than those of wild-type and Mock cells. Cell invasion by TM-HT1080 was partially enhanced by addition of a TAFIa/CPB inhibitor. These results suggest that TM suppresses pericellular fibrinolysis and plasma-induced tumor cell invasion, and that it is mediated, at least in part, by plasma TAFI activation.

Characterization of pullulanase (PUL)-deficient mutants of rice (Oryza sativa L.) and the function of PUL on starch biosynthesis in the developing rice endosperm.

Rice (Oryza sativa) allelic sugary1 (sug1) mutants defective in isoamylase 1 (ISA1) accumulate varying levels of starch and phytoglycogen in their endosperm, and the activity of a pullulanase-type of a debranching enzyme (PUL) was found to correlate closely with the severity of the sug1 phenotype. Thus, three PUL-deficient mutants were generated to investigate the function of PUL in starch biosynthesis. The reduction of PUL activity had no pleiotropic effects on the other enzymes involved in starch biosynthesis. The short chains (DP < or = 13) of amylopectin in PUL mutants were increased compared with that of the wild type, but the extent of the changes was much smaller than that of sug1 mutants. The alpha-glucan composition [amylose, amylopectin, water-soluble polysaccharide (WSP)] and the structure of the starch components (amylose and amylopectin) of the PUL mutants were essentially the same, although the average chain length of the B(2-3) chains of amylopectin in the PUL mutant was approximately 3 residues longer than that of the wild type. The double mutants between the PUL-null and mild sug1 mutants still retained starch in the outer layer of endosperm tissue, while the amounts of WSP and short chains (DP < or = 7) of amylopectin were higher than those of the sug1 mutant; this indicates that the PUL function partially overlaps with that of ISA1 and its deficiency has a much smaller effect on the synthesis of amylopectin than ISA1 deficiency and the variation of the sug1 phenotype is not significantly dependent on the PUL activities.

Sp1 is an essential transcription factor for LPS-induced tissue factor expression in THP-1 monocytic cells, and nobiletin represses the expression through inhibition of NF-kappaB, AP-1, and Sp1 activation.

Nobiletin is a citrus polymethoxylated flavonoid extracted from Citrus depressa, and has several reported biological effects. In this study, we investigated the effect of nobiletin on bacterial lipopolysaccharide (LPS)-induced expression of tissue factor (TF), a trigger protein for the blood coagulation cascade, and studied the possible mechanism of TF transcriptional regulation. THP-1 monocytic cells stimulated with LPS showed an increased expression of both TF protein and mRNA levels. However, pretreatment with nobiletin resulted in inhibition of LPS-induced expression of both TF protein and mRNA in a dose-dependent manner. Electrophoretic mobility shift assays revealed that binding of nuclear proteins from LPS-stimulated THP-1 cells to the NF-kappaB or AP-1 binding motif was increased as compared to non-stimulated control cells. Such increased binding activities were significantly reduced by pretreatment with nobiletin. Binding activity of nuclear proteins to the Sp1 binding motif was observed irrespective of LPS stimulation, but Sp1 activation was inhibited by nobiletin treatment of the cells. Treatment of THP-1 cells with Sp1-specific small interfering RNA (Sp1 siRNA) abolished the ability of LPS to induce TF activity. A similar reduction in the level of TF mRNA was also observed upon treatment of cells with Sp1 siRNA. These studies reveal that constitutive Sp1 activation is an essential event for transcriptional activation of TF, and nobiletin prevents LPS-induced TF expression by inhibiting NF-kappaB, AP-1, and Sp1 activation.

Verotoxin-1 stimulation of macrophage-like THP-1 cells up-regulates tissue factor expression through activation of c-Yes tyrosine kinase: Possible signal transduction in tissue factor up-regulation.

Verotoxin (VT)-producing Escherichia coli (E. coli) O157:H7 infections are frequently complicated by thrombotic angiopathy, hemolytic uremic syndrome (HUS) and neurological symptoms. The present data demonstrate that VT-1 (Shiga toxin) stimulation of macrophage-like THP-1 cells up-regulates the activity, antigen and mRNA levels of tissue factor (TF), a key cofactor of the coagulation-inflammation-thrombosis circuit. This up-regulation is accompanied by phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase), IkappaB kinase beta (IKKbeta) and extracellular signal-regulated kinase 2 (ERK2). Changes in TF mRNA levels were in parallel with the activation of NF-kappaB/Rel and Egr-1 activation, but not with AP-1. Inhibition of PI3-kinase attenuated VT-1-induced phosphorylation of IKKbeta and ERK2, and the up-regulation of TF mRNA levels. VT-1 stimulation rapidly activated c-Yes tyrosine kinase, a member of the Src family. Treatment of the cells with c-Yes antisense oligos attenuated the VT-1-induced phosphorylation of PI3-kinase, IKKbeta and ERK2, activations of NF-kappaB/Rel and Egr-1, and up-regulation of TF mRNA levels. These results suggest that VT-1-induced macrophage stimulation activates c-Yes, which then up-regulates TF expression through activation of the IKKbeta/proteasome/NF-kappaB/Rel and MEK/ERK2/Egr-1 pathways via activation of PI3-kinase. Induction of macrophage TF expression by VT-1 may play an important role in the acceleration of the coagulation-inflammation-thrombosis circuit during infections by VT-producing E. coli.