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The constitutive androstane receptor (CAR) is a nuclear receptor that acts as a transcription factor for a variety of genes, including genes encoding xenobiotic, steroid, and drug-metabolizing enzymes and transporters. Transactivation of a target gene by a transcription factor is generally mediated through the concerted and stepwise recruitment of various proteins termed coregulators, including coactivators and corepressors. In this study, TRIM24 (also known as transcriptional intermediary factor 1 alpha) was found to interact with the CAR. TRIM24 enhanced the CAR-dependent transactivation in reporter assays using the direct repeat-4 motif, a binding site of the CAR. This enhancement was synergistically augmented in the presence of steroid receptor coactivator (SRC) 1 or SRC2, both of which are coactivators of the CAR. In addition, TRIM24 was recruited to the CAR-binding element of the CYP2B6 promoter together with the CAR. We also noted that knockdown of TRIM24 suppressed CAR-induced CYP2B6 mRNA expression in HepTR/CAR and HepaRG cells and suppressed CAR-induced CYP3A4 mRNA expression in HepaRG cells but not HepTR/CAR cells. From these results, we suggest that TRIM24 is a novel coactivator of the CAR that is involved in cell- and/or promoter- selective transactivation.

Human bronchial mucins, such as MUC5AC, have traditionally been defined as a family of high-molecular weight glycoproteins. Changes in the contents of sugar chains on MUC5AC are among the fundamental features in inflammatory respiratory disease. The changes have been shown to lead to unfavorable alterations in the viscosity of mucus, resulting in impairment of mucociliary transport, vulnerability to viral/bacterial infection as sugar chains play an important role in adhesion of some viruses and bacteria to the epithelium, and finally inflammatory cell infiltration in the airway. Recently, we found that expression of some glycosyltransferases associated with the contents and structure of sugar chains is regulated by phosphatidylinositol-phospholipase (PI-PL) C signaling in cells. L-Carbocisteine, a mucoregulatory drug, normalized or balanced fucosylated and sialylated sugar chains, such as sialyl Lewis x through inhibition of PI-PL C signaling. We prepared MUC5AC fusion protein with tandem repeats associated with MUC5AC, and confirmed that L-carbocisteine inhibited the increases in viscosity associated with sialyl Lewis x expression levels. In addition, the clinical study (2008) noted that L-carbocisteine reduced the frequency of common colds and exacerbation of symptoms in patients with COPD. These favorable effects in patients may be due to normalization of sugar chain contents on mucins. We suggest that the inhibitory effect on infection of airway epithelial cells by rhinoviruses, respiratory syncytial virus, and influenza viruses by treatment with L-carbocisteine may also be based on the regulation of sugar chain contents or structures on mucins.

The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. The CAR is normally located in the cytoplasmic compartment of untreated liver cells and translocates to the nucleus after exposure to phenobarbital (PB) or PB-like chemicals. Previously, we identified two nuclear localization signals (NLS) in the rat constitutive androstane/active receptor (CAR), NLS1, which is located in the hinge region, and NLS2, which overlaps with the ligand-binding domain. However, the nuclear import mechanism of CAR is unclear. In this study, we show that nuclear import of CAR is regulated by importin/Ran-GTP systems. The regulation of CAR nuclear import by a Ran-GTP concentration gradient was confirmed using the dominant negative, GTPase-deficient form of Ran (RanQ69L), suggesting the involvement of transport receptors of the importinβ family. IPO13 was shown to be involved in the PB-mediated nuclear translocation of CAR, which was found to be susceptible to inhibition by a dominant negative mutant of IPO13 in primary hepatocytes.

The role of the microtubule network in the constitutive androstane receptor (CAR)-mediated transactivation of CYP2B induced by phenobarbital (PB) in rat primary hepatocytes was investigated using the microtubule-disrupting agent nocodazole (NCZ). In human hepatocytes, it was reported that CAR mRNA expression was decreased by a microtubule-disrupting agent through the inhibition of glucocorticoid receptor (GR)-mediated transactivation. However, in the present study, we show that the rat CAR gene was unaffected by the GR-mediated pathway in rat primary hepatocytes treated with NCZ. The PB-induced expression of CYP2B mRNA was repressed in the presence of NCZ for 2 h before and during 4 h of PB treatment, whereas the CAR mRNA and protein expression levels were not affected. Furthermore, the transactivation of the PB-responsive enhancer module-luciferase reporter gene and the nuclear transport of CAR induced by PB were also repressed in the presence of NCZ. Based on these findings, microtubular integrity might be required for PB-induced nuclear translocation of CAR in rat primary hepatocytes. In the same procedures, except that NCZ was replaced with radicicol, the CYP2B mRNA expression induced by PB was also repressed. Taking these into consideration, PB-mediated nuclear translocation of rCAR might be dependent on the 90-kDa heat shock protein as well as the microtubule network.

Fluorescence recovery after photobleaching (FRAP) in spontaneous multinuclear cells shows that both rat and human constitutive active/androstane receptors (CARs) are shuttling proteins with both nuclear localization signals (NLSs) and nuclear export signals (NESs). We previously identified two NLSs in rat CAR: NLS1 in the hinge region (residues 100–108) and NLS2 in the ligand-binding domain (residues 111–320). In the present study, we compared the intracellular localization signals between rat and human CARs. There was a marked difference in their intracellular localization in COS-7 cells because, unlike rat CAR, human CAR does not contain NLS1 due to an amino acid change at position 106. A CRM1-dependent leucine-rich NES, which is sensitive to an inhibitory effect of leptomycin B, was found in the cytoplasmic retention region previously identified within the ligand-binding domain of rat CAR (residues 220–258). We found that human CAR instead has a NES in the ligand-binding domain between residues 170 and 220. Also, we detected CRM1-independent C-terminal NESs between residues 317–358 of rat and human CARs. Removal of NLS1 by N-terminal truncation and mutation of xenochemical response signal caused rat CAR to localize in the cytoplasm of COS-7 cells, which we suspect is due to the masking of NLS2.

The cyclization-carbonylation of alkynyl hydrazones (I) proceeds readily in the presence of palladous trifluoroacetate in DMSO leading to pyrazole-4-carboxylates (IV) in good to high yields.

The metabolism of methamphetamine (MA) and 4-bromo-2,5-dimethoxyphenethylamine (2C-B) in freshly isolated rat hepatocytes was investigated, and compared with in vivo results. A suspended hepatocyte culture, established from male Wistar rats using a collagenase perfusion technique, was incubated in the presence of MA or 2C-B. After enzymatic hydrolysis of the conjugated forms, the metabolites were extracted by liquid-liquid partition and analyzed by gas chromatography/mass spectrometry (GC/MS). Amphetamine, p-hydroxymethamphetamine and p-hydroxyamphetamine were detected in the culture fluids of the rat hepatocytes inoculated with MA. The alcohol derivative, carboxylic acid derivative, 2-O-desmethyl-2C-B, 2-O-desmethyl-N-acetyl-2C-B and 5-O-desmethyl-N-acetyl-2C-B were detected in the case of 2C-B. The major metabolite of MA in rat hepatocytes was p-hydroxymethamphetamine. This is in good agreement with the urinary excretion profile for rats that were fed MA. 2-O-Desmethyl-2C-B and the carboxylic acid derivative were the major recovered metabolites of 2C-B in the rat hepatocyte culture, a slight deviation from the in vivo findings, in which 5-O-desmethyl-N-acetyl-2C-B was found to be the main component. Metabolites with a hydroxy group were largely present in their conjugated forms in the culture fluids, except for 2-O-desmethyl-2C-B. Taking these results into consideration, a primary hepatocyte culture system has the potential to provide a quick and handy method for estimating the in vivo metabolic fate of abused drugs.

Both the glucocorticoid receptor (GR) agonist dexamethasone (DEX) and GR antagonist pregnenolone 16α-carbonitrile (PCN) enhanced the transcription of CYP3A1 in rats though in a different fashion. Seven-week old male Wister rats were intraperitoneally administered one to three times with corn oil (vehicle) or DEX and/or PCN (80 mg/kg each) at 24-hr intervals according to various schedules. The animals were sacrificed twenty-four hours after the last treatment, and the dissected livers and lungs were examined for mRNA contents of CYP3A1 and pregnane X receptor (PXR) by real time PCR. DEX appeared to act mainly as an inducer of PXR, that in turn transactivated the CYP3A1 gene, by activating the GR, while PCN was deduced to have a direct effect on the expression of the CYP3A1 gene via activation of PXR from the rapid increase in CYP3A1 mRNA in comparison with DEX. Sequential treatment with DEX and PCN in this order induced CYP3A1 mRNA expression more effectively than treatment in the opposite order. When DEX and PCN were administered simultaneously, the induction of PXR by DEX was reversed by PCN, resulting from their competitive effects on the GR. In the lung, an increase in the CYP3A1 mRNA level was observed in the presence of DEX but not PCN, independently of the GR-activation.

The urinary and faecal metabolic profiles of 4bromo-2,5-dimethoxyphenethylamine (2C-B) in rat were investigated. Male Wistar rats were administrated 10 mg/kg of 2C-B hydrochloride orally, and the urine and faeces were collected 0‐24 and 24‐48 hr after administration. The samples were processed by liquidliquid extraction, and the extracts analyzed by GC/ MS, after derivatization. The major metabolite excreted into the urine was 5-O-desmethyl-N-acetyl-2CB, comprising 13.2% of the administrated dose. Other metabolites detected in the urinary extracts were 2-Odesmethyl-N-acetyl-2C-B (5.8%), 2-O-desmethyl-2CB (3.5%), carboxylic acid compound (1.9%), 5-Odesmethyl-2C-B (1.2%) and an alcoholic compound (0.8%). Only 0.2% of the administrated 2C-B was excreted into the urine in its original form. On the other hand, only 5-O-desmethyl-N-acetyl-2C-B and 2-Odesmethyl-N-acetyl-2C-B were detected in the faecal extracts. The major fraction of the urinary metabolites that contained a hydroxy group were recovered as conjugates.

Chlorinated ethylenes (CEs) are known to be metabolized by CYP2C as well as CYP2E1 and CYP2B. The effects of CEs on the expression of the rat CYP2C gene in the liver and lung were comparatively studied in terms of the enzyme activity, and protein and mRNA contents. Tetrachloroethylene (PCE), trichloroethylene (TCE), and 1,1-dichloroethylene (1,1-DCE) were injected at 0.5 g/kg i.p. into male 7-week-old Wistar rats individually or in combination with phenobarbital (PB). The expression of CYP2C mRNA showed organ-specific properties without being detectable in the lung under the assay conditions employed in the present study. Individual CEs had no effect on the enzymatic activity of CYP2C estimated by 2α-hydroxylation of testosterone (2α-TSH), whereas PB markedly enhanced the enzymatic activity in the liver but not the lung microsomes, although it was highly susceptible to the suppressive effect of 1,1-DCE. A down-regulation of pulmonary enzyme activity was observed with PCE by an unknown mechanism when the animals were treated simultaneously with PB. 1,1-DCE was found to inhibit the expression of hepatic mRNA irrespective of the coexistence of PB, and the same was true for CYP2C protein in the same organ to a lesser extent. In an analogy to our previous findings with other xenobiotic-metabolizing CYP forms such as CYP2B1/2 and CYP2E1, a potent suppressive effect of 1,1-DCE was observed with the maximum around 18 hr after the treatment on both constitutive and PB-induced expression of CYP2C.

Cyclization–carbonylation of α,β-alkynic hydrazones and (o-alkynylphenyl) (methoxymethyl) sulfides with Pd(tfa)2 in DMSO/MeOH afforded methyl pyrazole-4-carboxylates and benzo[b]thiophene-3-carboxylates, respectively, in good yields. A simple change of the ligand (solvent) allowed controlled, effective switching between cyclization–carbonylation–cyclization-coupling (CCC-coupling) reactions and cyclization–carbonylation reactions.

Three polychlorinated ethylenes (CEs), tetrachloroethylene (PCE), trichloroethylene (TCE) and 1, 1-dichloroethylene (DCE), were comparatively studied for their effects on the expression of cytochrome P450 (CYP) mRNA forms in the liver and lung using 7-weeks-old male Wistar rats. The animals were i.p. inoculated with 0.5 g/kg of the individual CEs and sacrificed at 6-hr intervals for 30 hr for the determination of the mRNA levels of hepatic and pulmonary CYP2B and hepatic CYP2E1. A 3.5-fold increase in the expression of hepatic CYP2B mRNA was noted transiently at 6 hr in the presence of PCE. In contrast, 1, 1-DCE was suppressive and started within 6 hr and lasted for more than 30 hr, with a trough value of 15% of the control being observed around 12 to 18 hr, while there was no marked effect observed in the case of TCE-treatment. The expression of pulmonary CYP2B mRNA was severely suppressed in the presence of 1, 1-DCE during the entire observation period as was the case with its hepatic counterpart, while the temporarily enhanced expression of mRNA at 6 hr by PCE and TCE was followed by a moderate suppression, with the trough values of ca. 80 and 65% of the control, respectively, at 18 hr. Concerning the hepatic CYP2E1, the expression of mRNA was adversely affected by all the CEs with a descending order of magnitude of 1, 1-DCE, TCE and PCE with peak inhibition values of 85%, 50% and 35%, respectively. The nonselective suppressive effect of 1, 1-DCE on the expression of divergent CYP mRNAs was well correlated with the IL-1β-dependent suppression of various types of CYP mRNA in primary cultured hepatocytes previously reported. Although PCE and TCE are under stringent legislative restriction in Japan as environmental pollutants owing to their relatively stable natures, they are less toxic in terms of the inhibition of the mRNA expression of CYP forms in acute phases than short-lived 1, 1-DCE, which is inflammatory to the host animals; the shorter the environmental life span (1, 1-DCE > TCE > PCE), the more severe the acute toxicity.

The in vitro inhibitory effects of heavy metals, i.e., mercury, cadmium, lead, nickel and beryllium in their chloride forms, on rabbit pulmonary drug-metabolizing enzymes were studied comparatively. Microsomal and cytosolic fractions prepared from rabbit lungs were incubated in the presence of heavy metals prior to enzyme assays. Mercury was the most potent in reducing cytochrome P450 content and mixed-function oxidase activities including NADPH-cytochrome c reductase and benzo[a]pyrene hydroxylase. The addition of mercury to pulmonary microsomal preparations resulted in a spectral shift from 450 nm to 420 nm in an absorption maximum of cytochrome P450carbon monoxide complex with mercury chloride being more potent than methylmercury. Cadmium was also inhibitory, while the effects of nickel were noted only at higher concentrations. Neither lead nor beryllium was inhibitory. Among second-phase drug-metabolizing enzymes, UDP-glucuronyltransferase and glutathione S-transferase were found to be susceptible to the adverse effects of mercury and lead, respectively. The extent of inhibition of the latter activity by mercury were highly dependent on the concentration of glutathione, implying the complex formation between them. Cadmium was slightly inhibitory to both enzyme activities, though the other metals had no effect. The results indicate that the pulmonary toxicities of airborne heavy metals could be inferred using simple in vitro assays.

The manifestation of toxicity by certain environmental compounds requires enzymatic activation. It is well known that chlorinated ethylenes (CEs) are metabolized by intracellular enzymes represented by cytochromes P450 (CYPs) to the urinary secreted forms via epoxide intermediates, which are responsible for their toxicities by forming complexes with intracellular components including CYPs. In order to study the roles of CYPs in the metabolic activation of tetrachloroethylene (PCE), trichloroethylene (TCE) or 1, 1-dichloroethylene (1, 1-DCE), the cytotoxicity of individual CEs were tested in primary hepatocyte cultures established from animals treated with various CYP-inducers such as 3-methylchoranthrene (inducer of CYPlAl/2), phenobarbital (CYP2B1/2) and pyridine (CYP2E1). The cytotoxicity of CEs measured by lactate dehydrogenase leakage after 24 hr was enhanced in different fashions, depending on the CYP inducers used. The results are summarized as follows: CYP1A1/2 and 2B1/2 raised the cytotoxicity of PCE;CYP2B1/2 was exclusively associated with the enhanced cytotoxicity of TCE; and CYP2E1 and 2B1/2 were proved to potentiate 1, 1-DCE. Based on these observations, CEs differing in the number of chlorine substitutions were disclosed to have divergent preferences for CYPs, by which they were metabolically activated.

Many environmental chemicals (xenobiotics), including polychlorinated ethylenes (CEs), are known to be metabolically intoxicated via the formation of hazardous intermediates (referred to as biological activation), while the expression of metabolic enzymes might be interfered with by xenobiotics at various stages in a system to minimize their adverse biological effects on the host animals. Three CEs, i.e., tetrachloroethylene (PCE), trichloroethylene (TCE) and 1, 1-dichloroethylene (1, 1-DCE), were comparatively studied for their effects on the in vivo expression of CYP forms, which are responsible for their metabolic activation, as well as organospecificities between the liver and lung. Furthermore, their effects on the expression of CYP forms were studied in the animals simultaneously administered with phenobarbital (PB), known as an inducer of CYP2B. Individual CEs were administered intraperitoneally at 0.5 g/kg alone or simultaneously with PB (80 mg/kg/d) to 7-week-old male Wistar rats weighing about 200 g. The testosterone hydroxylase activities, CYP2B- and 2E1-mRNA, and CYP2B- and 2E1-proteins were measured 24 hr after the treatment. Testosterone 2β-hydroxylase (2βTSH) and 16βTSH activities are attributable to the functions of CYP3A2, and CYP2B plus CYP3A2, respectively. The induction of hepatic CYP2B in the PB-treated animals might be masked by the suppressive effect of CYP3A2 in terms of the 16βTSH activity, while the pulmonary 16βTSH activity was confined to the function of CYP2B, which was insensitive to the inducing effect of PB. The inhibition of hepatic 16βTSH activity was observed exclusively in the presence of 1, 1-DCE, especially in PB-coadministered animals, suggesting the preferable suppression of CYP2B. In the lung, however, PCE suppressed the 16βTSH activity in the absence of PB. The expression levels of hepatic CYP2B mRNA and protein were significantly lowered by 1, 1-DCE in the presence of PB. Concerning hepatic CYP2E1, no alternation was observed in the levels of mRNA and protein by any of the CEs in the absence or presence of PB, except for a marked decrease in the amount of mRNA when the rats were treated with 1, 1-DCE in combination with PB. In the lung, both mRNA and protein were not detected under the given assay conditions, as in our previous studies. Based on these results, it is suggested that 1, 1-DCE suppresses the induction of hepatic CYP2B and 2E1 in advance of the transcriptional stage. The expression of pulmonary CYP2B was obstructed by PCE posttranslationally in the absence of PB.

Almost all of fucose and sialic acid in mucus are found on the mucus glycoproteins (mucins), and these sugar components on mucins are known to be associated with the viscous property of mucus. We have reported some aspects of carbocisteine, a mucoregulatory drug, correcting fucose and sialic acid contents in mucus. At present, carbocisteine's expectorant action of airway mucus is postulated to involve - the regulation of fucose and sialic acid contents on mucins. However little information is available about the relationship between the viscosity and sugar contents on mucins when treated with carbocisteine. To investigate further the mechanism behind the action of carbocisteine, the present study prepared MUC5AC fusion protein which has tandem repeat regions associated with MUC5AC, and evaluated the effects of carbocisteine on tumor necrosis factor (TNF)-alpha-induced increases of mucus viscosity and sialyl-Lewis x-epitopes antigen, an antigen which consists of fucosylated and sialylated sugar chains on the MUC5AC fusion proteins. Carbocisteine inhibited the TNF-alpha-induced increases of the viscosity and sialyl-Lewis x-epitopes on MUC5AC fusion protein. These findings suggest that carbocisteine may normalize the viscosity of mucus through "balancing" of fucose and sialic acid contents on airway mucins.

The efficacy of the extraction of polychlorinated biphenyls (PCBs) from varnish-infiltrated insulating papers as a model of solid materials with an aqueous sodium hydroxide (NaOH) by autoclaving at 121°C for 30 min was compared with those for the conventional procedures, i.e., saponification with 1 N ethanolic NaOH in a boiling water bath for 60 min and extraction with benzene in a Soxhlet apparatus. The newly invented autoclaving method was found to be superior to the conventional ones, yielding approximately 5- to 6-fold cumulative PCB congeners without being accompanied by extended decomposition. Therefore, the autoclave-based sample treatment is recommended for more accurate determination of PCBs in the case of PCB-impregnated solid materials such as hardened oils and resin-coated or -infiltrated papers instead of being treated conventionally.

Chlorinated ethylenes (CEs) such as tetrachloroethylene (PCE), trichloroethylene (TCE), 1, 1-dichloroethylene (1, 1-DCE), cis-1, 2-dichloroethylene (cis-DCE), and trans-1, 2-dichloroethylene (trans-DCE) are members of the class of volatile halogenated hydrocarbons. Each was administered intraperitoneally at 0.5g/kg alone or simultaneously with phenobarbital (PB, 80mg/kg/d) to male Wistar rats weighing about 200g (7-weeks-old). Microsomal fractions of livers and lungs removed from animals sacrificed 24h after treatment were tested for monooxygenase activities and protein content of 4 cytochrome P450 (CYP) forms, i.e., CYP1A1/2, 2B1/2, 2E1 and 3A1/2. In terms of constitutive expression, they were compensatory in liver and lung with CYP1A1/2, 2E1 and 3A1/2 being detected only in liver and CYP2B1 only in lung. All 5 CEs employed in this work suppressed hepatic CYP1A1/2, 2E1 and 3A1/2 in the descending order of 1, 1-DCE > cis-DCE > trans-DCE > TCE > PCE. The magnitude of suppression of pulmonary CYP2B1 by CEs were compared as follows; PCE > trans-DCE > 1, 1-DCE > cis-DCE > TCE. CYP1A1/2, CYP2E1 and CYP3A1/2 in hepatic microsomes from PB-treated animals responded to CEs similarly to those from PB-untreated animals. These 3 enzyme activities could not be detected in pulmonary microsomal fractions, irrespective of the PB-treatment. Hepatic CYP2B1/2 could be detected only when treated with PB in marked contrast to the constitutive expression of pulmonary CYP2B1. The suppression of PB-induced hepatic CYP2B1/2 was observed with all 5 CEs, with special emphasis on complete inhibition with 1, 1-DCE. The adverse effects of TCE and PCE on pulmonary CYP2B1 from PB-treated rats were comparable with those from PB-untreated animals. In contrast, 3 dichloro-isomers were more suppressive to the enzyme in the absence than in the presence of PB. The protein levels of hepatic CYP forms were generally proportional to the enzyme activities in the case of 1, 1-DCE-treatment. The amounts of pulmonary CYP2B1 apoprotein were in good accordance with the enzyme activities when treated with CEs individually.

Trichloroethylene (TCE) is a highly toxic compound which belongs to the class of volatile halogenated hydrocarbons, which are well-known pollutants of drinking water and the atmosphere. The effects of moderately toxic concentration of TCE on the levels of cytochrome P450 (CYP) isoforms and on their phenobarbital (PB) induction in rats were studied. Monooxygenase activities associated with individual CYP isoforms, i.e., CYP1A, 2B, 2E1 and 3A, were measured in microsomal fractions prepared from both lungs and livers of male and female Wistar rats (7-weeks old). Differences in constitutive levels of CYP isoforms were observed between liver and lung of mock-treated rat ; microsomal CYP2B activity was solely detected in lung while the activities of CYP1A, 2EI and 3A, but not 2B were detected in liver. Among these, the pulmonary CYP2B and hepatic CYP2E1 activities were reduced by TCE-treatment. PB-treatment resulted in the detection of increased levels of hepatic CYP1A and 2E1 and of pulmonary CYP2B activities, and also the appearance of hepatic CYP2B activity. Coadministration of TCE was suppressive against the activities of CYP isoforms except for hepatic CYP2E1 in PB-treated rats. The lowered CYP2B activity in the presence of TCE was accompanied by the reduction in the amount of CYP2B apoprotein.

The effect of wearing of sand in the furnaces of fluidized bed incinerators (FBIs) on the formation of polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran (PCDF) and their related compounds from polychlorinated biphenyl (PCB) and 1,2,3-trichlorobenzene (1,2,3-T3CBz) was studied experimentally using a pyrolysis apparatus consisting of an injection unit; a stainless steel tube, in which packed was either unused sand (virgin sand) or used for two months in FBI (worn-out sand), heated to 650°C by an electric furnace; and a sampling unit. Furthermore, polychlorinated diphenyl ether (PCDE) was pyrolyzed on worn-out sand in order to compare the formation mechanisms of PCDD and PCDF with those from PCBs. Besides the generation of PCDD and PCDF, the hydroxylation of the starting compounds occurred. PCB was detected in the pyrolyzed samples of 1,2,3-T3CBz on both types of sand. In all cases, PCDF was in much higher yield than PCDD and pyrolysis on worn-out sand was more favorable for the formation of PCDD, PCDF and their relatives than pyrolysis on virgin sand. There was marked difference in metal concentrations between virgin and worn-out sand, implicating that the increased metal concentrations in sand would promote the formation of PCDD, PCDF and their related compounds in FBIs.

The risk of daily exposure to volatile halogenated hydrocarbons (VHH) in ambient air was assessed by the urinary concentration of VHH metabolites. Thirty residents in the urban Tokyo area, 13 males and 17 females, aged at 7 to 78, were enrolled in the present study. They were requested to carry passive gas samplers adsorbing chemicals in the ambient air during daily activities. The mean levels (mean±S.D.) of 1, 1, 1-trichloroethane (1, 1, 1-TCE), trichloroethylene (TCE) and tetrachloroethylene (PCE) in the breathing air for 12 h were 3.4±2.4μg/m3, 2.0±1.3μg/m3 and 1.1±0.8μg/m3, respectively. The daily intake of individual VHH was calculated to be 68, 40 and 23μg/person for 1, 1, 1-TCE, TCE and PCE, respectively, while women inhaled twice as much VHH as men. Since total VHH levels in the breathing air correlated well with the levels of urinary metabolites (γ=0.80), the latter could be employed as a biological index representing the level of exposure to VHH.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a major cellular energy sensor and master regulator of metabolic homeostasis; thus, AMPK plays a central role in studies on diabetes and related metabolic diseases. From the rhizomes of Polygonatum odoratum (Mill.) Druce, six homoisoflavonoids (1-6) and one dihydrochalcone (7) were isolated, and the structures of polygonatones A-D (4-7) were elucidated by various spectroscopic analyses. Compounds 1-7 were evaluated for their effect on AMPK activation. The amount of active phosphorylated AMPK and acetyl-CoA carboxylase in rat liver epithelial IAR-20 cells increased when the cells were incubated with the aforementioned compounds. Specifically, (3R)-5,7-dihydroxyl-6-methyl-8-methoxyl-3-(4'-hydroxylbenzyl)-chroman-4-one (1), (3R)-5,7-dihydroxyl-6,8-dimethyl-3-(4'-hydroxylbenzyl)-chroman-4-one (2), (3R)-5,7-dihydroxyl-6-methyl-3-(4'-hydroxylbenzyl)-chroman-4-one (3), and polygonatone D (7) exhibited significant activation effects.

The macrocyclic polyamines, cyclen and cyclam, and their derivatives have been tested for inhibitory activity against the cytopathogenic effect (CPE) of human immunodeficiency virus type 1 strain HTLV-IIIB (HIV-1IIIB) on CD4+ human lymphoblastoma MT-4 cells. Cyclam and its derivatives were complexed with a variety of transition metal ions NiII, ZnII, CuII, FeIII and CoIII. The divalent metal complexes effected lower toxicity and greater anti-HIV-1 activity, while the trivalent metal complexes had no effect on HIV-1-dependent CPE. When dimerized, the anti-HIV activity of monomers was significantly enhanced. A potent inhibition of CPE by biscyclam was transiently observed 4 d after the virus infection, but was not seen at 6 d due to severe toxicity. The toxicity of biscyclam, referred to as delayed toxicity, could be overcome by a metal complexation. The strain specificities of biscyclams were further studied by testing their effects on syncytium formation between HIV-infected and uninfected human acute lymphoblastic leukemia MOLT-4 cells. The 50% inhibitory concentrations of biscyclams against HIV-2GH-1-dependent syncytium formation were less than one hundredth those for the other HIV strains (HIV-1IIIB, HIV-1RF and HIV-1SF-2).

Palladium(II) catalyzed carbonylation of 1,2-allenyl ketones 1 in the presence of p-benzoquinone (1 equiv) under a CO atmosphere (balloon) afforded difuranylketones 4 in moderate to good yields. Mechanistically, the electron-withdrawing nature of the acyl group should enhance the electrophilicity of the acylpalladium species B, and thus promote the oxypalladation of an additional molecule of 1, leading to the difuranyl ketone 4.

The purification and physico-chemical properties of the enactin congeners are concerned in this paper. We proposed the group name hydroxamic acid antimycotic antibiotics (HAAA) for enactin, neoenactin and the related antibiotics. Streptomyces roseoviridis, the enactin-producing microbe, was cultured to prepare an inoculum seed.

Carbocisteine is a mucoregulatory drug normalizing sialic acid and fucose contents in mucins through the regulation of glycosyltransferase activities. Tumor necrosis factor (TNF)-alpha-induced overexpression of sialyl-Lewis x epitopes, containing sialic acid and fucose, in mucins were previously reported to be regulated by glycosyltransferase mRNAs expression through phosphatidyl inositol-specific phospholipase C (PI-PLC) signaling pathways [Ishibashi, Y., Inouye, Y., Okano, T., Taniguchi, A., 2005. Regulation of sialyl-Lewis x epitope expression by TNF-alpha and EGF in an airway carcinoma cell line. Glycoconj. J. 22, 53-62]. To investigate the mechanism behind the mucoregulatory action of carbocisteine, the present study evaluated the effects of carbocisteine on TNF-alpha-induced overexpression of sialyl-Lewis x epitopes in NCI-H292 cells. 100 mug/ml of carbocisteine was able to inhibit the TNF-alpha-induced expression of hST3GallV mRNA, FUT3 mRNA, C2/4GnT mRNA and sialyl-Lewis x epitopes as well as the TNF-alpha-induced activity of PI-PLC in NCI-H292 cells. These findings suggest that carbocisteine may normalize the sialyl-Lewis x epitopes expression in mucins through the inhibition of cellular PI-PLC activity in vivo.

1, 1'-(m-Xylenediyl)-bis (1, 4, 7, 10-tetraazacyclododecane)-ZnII2 complex (m-xylenediyl-bicyclen-ZnII2), a potent inhibitor of human immunodeficiency virus (HIV), was obtained from cyclen by a combination of dimerization and metal complexation. The ratio of median cytotoxic concentration against host cells (CC50) and median effective concentration against HIV cytopathogenicity (EC50), referred to as the selectivity index (SI), was regarded to be a measure of anti-HIV activity. These two chemical modifications contributed to a potent, in vitro anti-HIV activity of m-xylenediyl-bicyclen-ZnII2 by respectively increasing the CC50 and decreasing the EC50 in comparison with those of cyclen.

Sialyl-Lewis x epitopes and MUC5AC protein are known to be overexpressed in mucins secreted by patients suffering from various respiratory diseases. To investigate the mechanisms by which airway inflammatory agents mediate the expression of sialyl-Lewis x epitopes and MUC5AC mucin, we examined the effects of tumor necrosis factor (TNF)-α and epidermal growth factor (EGF) in the human lung carcinoma cell line, NCI-H292. Basal expression levels of hST3GalIV, FUT3 and C2/4GnT mRNA, involved in the biosynthesis of sialyl-Lewis x, were higher than those of other glycosyltransferases in NCI-H292 cells. TNF-α induced expression of hST3GalIV, FUT3, C2/4GnT and MUC5AC mRNAs in NCI-H292 cells. When cells were pretreated with U73122, a phosphatidylinositol-phospholipase C (PI-PLC) inhibitor, the expression of these glycosyltransferase mRNAs was suppressed. Treating cells with EGF induced the down-regulation of these glycosyltransferase mRNAs and sialyl-Lewis x epitopes, while inducing an increase in expression of MUC5AC mRNA. These EGF-mediated effects on the glycosyltransferase and MUC5AC mRNAs were blocked when cells were first exposed to AG1478, an EGF receptor tyrosine kinase inhibitor. These findings suggest that the expression of sialyl-Lewis x epitopes, which is regulated separately from the expression of MUC5AC protein, may be controlled through pathways such as the EGF receptor tyrosine kinase and PI-PLC signaling cascades in NCI-H292 cells. Published in 2005.

One isopolyoxometalate and 42 heteropolyoxometalates consisting of 3 compounds with the trivacant Keggin structure, 2 with the lacunary Keggin structure, 30 with the Keggin structure, one with the Wells-Dawson structure and 6 with miscellaneous structures were tested for their suppressive effect on the cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) in vitro and inhibitory activity against HIV-1 reverse transcriptase. In contrast to the leading interpretations which attribute the suppressive effect of polyoxometalates on the cytopathogenicity of HIV-1 to the inhibition of HIV-1 reverse transcriptase by these compounds, there was no distinct correlation observed between these two functions of polyoxometalates.

Fifteen heteropolyoxotungstates were tested for their effects on the proliferation of human immunodeficiency virus type 1 (HIV-1) using an in vitro system consisting of MT-4 cells and HTLV-III b . Eight heteropolyoxotungstates (HPOTs) with the Keggin structure or dimerized deficient Keggin structure proved to be potent inhibitors of HIV-1. In contrast, seven non-Keggin HPOTs including HPA 23 did not have significant effects on HIV-1 proliferation at non-toxic doses. [PTi 2 W 10 O 40 ] 7− (PM-19) was the most potent inhibitor of HIV-1 among the 15 HPOTs tested. The inhibition of HIV-1 replication by PM-19 presumably results from impaired virus adsorption and/or penetration into target cells. Viral spread of HIV-1 and HIV-2 on cell-to-cell basis was also susceptible to PM-19. In combination, PM-19 and 3′-azido-3′-deoxythymidine were synergistic in inhibiting HIV-1 proliferation.

Phyto-remediation by kenaf was attemptedto apply to improve eutrophic water quality. The growth of kenaf was satisfactorily achieved in water environment by using Kanuma-soil(sedimentary rocks and clays of volcanic ashes obtained primarily in northern region of Kanto Plain in Japan). Nitrate was removed from eutrophic water through growing kenaf, while phosphate was mainly absorbed on Kanuma-soil. Accordingly, the combination of kenaf and Kanuma-soil was found to be effective for remediation of eutrophic water environment.

BACKGROUND: The constitutive androstane receptor (CAR, NR1I3) plays a key role in the transcriptional activation of genes that encode xenobiotic/steroid and drug metabolizing enzymes. RESULTS: The expression of CAR mRNA throughout the circadian rhythm is reported for the first time in phase with the clock gene Bmal1 and in antiphase with the clock-controlled gene Rev-erbalpha mRNAs, with a peak at Zeitgeber time (ZT) 20 and a trough at ZT8, and a peak/trough ratio of 2.0. The diurnal difference in CAR mRNA expression might underlie the 1.7-fold difference in the magnitude of the PB-dependent induction of CYP2B1/2 mRNA. CONCLUSION: The circadian oscillation of xenosensor gene CAR mRNA expression is partially responsible for chronopharmacokinetics and chronopharmacology in disease.

The title compounds were readily prepared in one-pot by phase-transfer reaction of a methanol adduct of 2-( F -methyl)- F - propene with acetamidine or benzamidine in the presence of aqueous sodium hydroxide in CH2Cl2. Various 5-trifluoromethyl- pyrimidines were also synthesized via nucleophilic substitution of the 4-fluorine of the title compounds.

The in situ reaction of triphenylphosphine with tetrakis(trifluoromethyl)-1,3-dithietane in the presence of aliphatic or aromatic aldehydes gives good to excellent yields of bis-trifluoromethyl olefins.

Novel streptonigrin 2-amide derivatives have been efficiently synthesized without protection by using 3,3′-(phenylphosphoryl)bis(1,3-thiazolidine-2-thione)(PPBTT) as a chemoselective condensing agent.

Sarcosine oxidase (EC 1.5.3.1) produced by Streptomyces sp. KB210-8SY was purified by ion exchange chromatography on DEAE-sepharose CL-6B, affinity chromatography on sarcosyl-AH-sepharose 4B and gel filtration on sephadex G-150 to electrophoretic homogeneity.The molecular weight of the enzyme was estimated to be 44000 by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis and gel filtration on Sephadex G-150. The enzyme showed the maximum activity at pH 8 and was stable at pH 7-9. In addition to sarcosine, the enzyme oxidized N-methyl-L-leucine, N-methyl-DL-alanine and N-methyl-DL-valine to lesser extents. The apparent Km values for sarcosine, N-methyl-L-leucine, N-methyl-DL-alanine and N-methyl-DL-valine were 0.91, 0.58, 1.6 and 6.7 mM, respectively. The enzyme was inactivated by N-bromosuccinimide, hydroxylamine hydrochloride, SDS, Zn2+, Ni2+ and Hg2+ but not by ethylenediaminetetraacetate, p-chloromercuribenzoate or p-toluenesulfonyl chloride.The taxonomic characteristics of strain KB210-8SY are closely related to those of Streptomyces flavovirens and S. misakiensis.

Thirteen heterocyclic quinones (5 quinoline quinones, 7 isoquinoline quinones, 1 indole quinone) were tested for their effects on avian myeloblastosis virus reverse transcriptase, growth of murine lymphoblastoma L5178Y cells, respiration of rat liver mitochondria and oxidation of NADH by Clostridium kluyveri diaphorase in comparison with those of streptonigrin, in which the quinoline quinone moiety is considered to play a crucial role. Most of the quinoline quinones and isoquinoline quinones inhibited reverse transcriptase to the same extent as streptonigrin with the ID50 values ranging between 1 and 5 micrograms/ml, whereas the ID50 value of the indole quinone derivative, 4,7-dihydro-2,3-dimethylindole-4,7-dione, was 80 micrograms/ml. The cytotoxicities of the quinones were much lower than that of streptonigrin; the ID50 values of the quinones were higher than 0.15 micrograms/ml. In particular, the ID50 value of the ortho-quinoline quinone derivative, 8-methoxy-7-methyl-5,6-dihydroquinoline-5,6-dione, was as high as 16 micrograms/ml, while the 50% inhibition of cell growth was seen in the presence of 0.0025 micrograms/ml streptonigrin. The membrane transport of the quinones was evaluated by comparing the effects on oxygen consumption by mitochondria and oxidation of NADH by bacterial diaphorase, being proven not to be responsible for their lower cytotoxicities.

In contrast to the formation of 2,2,4,4-tetrakis(trifluoromethyl)-1,3-dithietane from hexafluoropropene, potassium fluoride, and sulfur, perfluoro-2-butene and perfluoro-pentenes react with potassium fluoride and sulfur to form perfluoro-1,3-dithioles via intermediate thioenolate anions.

The usefulness of 2-hydryl-2-( F -methyl)- F -propanoyl fluoride as a building block for the synthesis of trifluoro methylated compounds has been shown in the synthesis of the title compounds by the reaction of the acid fluoride with 1,3-dimethylthiourea and 1,3-dimethylurea followed by intramolecular cyclization.

Inhibition of avian myeloblastosis virus (AMV) reverse transcriptase by natural and synthetic quinones including antibiotics could be accounted for by an oxidation-reduction reaction. The quinones were shown to function as electron acceptors as revealed by the catalytic oxidation of NADH by Clostridium kluyveri diaphorase which was in excellent agreement with enzyme inhibition activity. The kinetics of inhibition of AMV reverse transcriptase by three synthetic quinones with different core structures, i.e., 6-methoxy-5, 8-dihydroquinoline-5, 8-dione, 5, 8-dihydroisoquinoline-5, 8-dione and 1, 4-naphthoqumone, were studied. These quinones inhibited reverse transcriptase in the same manner as streptonigrin (STN) and were shown to act at a single class of reaction site(s) on the enzyme molecule. In contrast, the quinones with bulky substituents, i.e., 7-(2-nitrophenethylamino)-5, 8-dihydroisoquinoline-5, 8-dione and 7-methoxy6-methyl-3-piperidino-5, 8-dihydroisoquinoline-5, 8-dione, were inactive as inhibitors of reverse transcriptase, whereas they retained competent catalytic activities in the oxidation of NADH by C. kluyveri diaphorase. Based on these observations, the existence of a specific site of interaction on the enzyme molecule, referred to as a quinone pocket, was proposed. The quinone pocket might play a crucial role in the early sequence of events leading to the inhibition of reverse transcriptase by quinones including STN and sakyomicin A (SKM). Access of SKM to a quinone pocket might be restricted due to its bulky structure in the vicinity of the quinone group. This is inferred from unsuccessful inhibition of reverse transcriptase by the quinones with bulky substituents, resulting in much poorer inhibition of reverse transcriptase in spite of more potent electron acceptor activity in the oxidationreduction system as compared with those of STN.

Sarcosine oxidase (EC 1.5.3.1) produced by Bacillus sp. B-0618 was purified by ion exchange chromatography on diethyl aminoethyl-cellulose and gel filtration on Sephadex G-100 and G-150. The molecular weight of the enzyme was estimated to be 42000 by gel filtration on Sephadex G-150 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited an absorption spectrum characteristic of flavoprotein. The enzyme showed the maximum activity at pH 8.5-9 and was stable at pH 7-10. The pI value was 4.7 as determined by isoelectric focusing. Although sarcosine is the preferred substrate, the enzyme also oxidized N-methyl-DL-alanine, N-methyl-L-leucine and N-methyl-DL-valine to lesser extents. The apparent Km values of sarcosine, N-methyl-DL-alanine, N-methyl-L-leucine and N-methyl-DL-valine were 12.2, 6.8, 106 and 173 mM, respectively. The enzyme was inactivated by N-bromosuccinimide, Zn2+, Fe3+ and Hg2+, but not by ethylenediaminetetraacetate, p-chloromercuribenzoate, monoiodoacetate or p-toluenesulfonylchloride.

A neutral metalloendopeptidase, produced by Streptomyces, which has anti-inflammatory activity against carrageenan-induced edema in rats was purified by affinity chromatography on N-benzyloxycarbonylglycyl-D-leucylaminohexyl-Sepharose (Z-Gly-D-Leu-AH-Sepharose) to electrophoretic homogeneity. The enzyme was adsorbed on a Z-Gly-D-Leu-AH-Sepharose column equilibrated with phosphate buffer (pH 7.0). The column was washed with phosphate buffer (pH 5.6) containing 2 M urea and eluted with acetate buffer (pH 4.1) supplemented with 2 M urea. The caseinolytic activity of the enzyme, which was lost on treatment with ethylenediaminetetraacetate, was recovered by the addition of ZnCl2 to the reaction mixture. The enzyme showed the maximum caseinolytic activity at pH between 7.0 and 8.0, and was stable in the pH range of 5.0-10.0. The molecular weight was estimated to be 59000. The enzyme preferentially hydrolyzed N-benzyloxycarbonylglycyl-L-leucine amide, N-benzyloxycarbonylglycyl-L-phenylalanine amide and N-benzyloxycarbonylglycyl-L-leucyl-L-tyrosine among the synthetic substrates tested in this work. The producing microbe was identified as Streptomyces mauvecolor based on the results of taxonomic studies.

3β-Hydroxysteroid oxidase activity was detected in the broth filtrate of strain H 1109 MY 12. Based on taxonomic studies, this strain was shown to be a new species of genus Streptoverticillium and the name Stv. cholesterolicum is proposed for this strain. The enzyme was purified by ammonium sulfate precipitation and affinity column chromatography on crystalline cholesterol, and the product showed a single band on SDS-polyacrylamide gel electrophoresis. The enzyme showed optimum activity at pH 7.0-7.5 and was stable over a rather wide pH range of 4.0 to 12.5. Cholesterol and dihydrocholesterol were oxidized rapidly. The Km value for the oxidation of cholesterol by this enzyme was about 0.4mM. The enzyme activity was greately inhibited by HgCl2, and AgNO3. FeCl3 and CuSO4 were also inhibitory though to lesser extents. Iodine and N-bromosuccinimide completely inhibited the enzyme activity at concentrations of less than 0.01 mM. Neither metal-binding agents nor p-chloromercuribenzoic acid had any inhibitory effect on the oxidation of cholesterol by this enzyme. The molecular weight of the enzyme was estimated to be 56000 by SDS-polyacrylamide gel electrophoresis. The enzyme was proved to be a flavoprotein containing flavin adenine dinucleotide as a prosthetic group.

The title compounds were readily synthesized in one pot by the reaction of methyltriethylammonium 2-( F -methyl)- F -propenolate, formed directly by the reaction of methyl 2-( F -methyl)-2-hydryl- F -propyl ether with two equivalents of triethylamine in DMF, and S-alkylisothiourea hydrogen halide, followed by the reaction with triethylamine again. Derivatizations via nucleophilic substitution of the fluorine atom at the 6-position were performed after alkylation of the title compounds leading to the preparation of various trifluoromethyl substituted pyrimidines.

Antitumor antibiotic streptonigrin (STN-COOH) is a potent inhibitor of avian myeloblastosis virus (AMV) and human immunodeficiency virus reverse transcriptases. The carboxyl group at 2'-position of STN-COOH was modified to give esters, hydrazide, amides and amino acid derivatives for biological studies. Against AMV reverse transcriptase, the hydrazide, amides and amino acid derivatives showed inhibitory activity, which compared favorably to that of STN-COOH, with the ID50 values ranging 2-8 micrograms/ml. In contrast, the esters lacked this activity except for those having a dimethylamino group in the substituent. Splenomegaly caused by Friend leukemia virus infection was significantly inhibited by STN-COOH and STN-COO(CH2)3N(CH3)2, but not STN-CONH(CH2)3N(CH3)2. Doxorubicin-resistant murine lymphoblastoma L5178Y cells showed collateral sensitivity to both STN-COOH and STN-COO(CH2)3N(CH3)2 not only in vitro but also in vivo.

The naphthoquinone moiety was proven to be essential to the biological activities of sakyomicin A using various naphthoquinone derivatives. Among the naphthoquinones tested, juglone (5-hydroxy-1, 4-naphthoquinone) which resembles the partial structure of sakyomicin A was the most active in cytotoxicity against murine lymphosarcoma L5178Y cells, electron acceptor function in the oxidation of NADH by Clostridium kluyveri diaphorase or rat liver mitochondria and inhibition against avian myeloblastosis virus reverse transcriptase. The significantly lower cytotoxicity of sakyomicin A as compared with juglone was attributable to its poor membrane transport. The inhibition of reverse transcriptase activity may result from the interaction between a sulfhydryl group in the active center of the enzyme and quinone groups of the naphthoquinones and sakyomicin A.