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The exposure of healthcare workers to antineoplastic drugs in hospitals has been recognized to be harmful. To minimize the risk of exposure, the removal of these drugs from work environments, such as compounding facilities, has been recommended. In our previous paper, the degradation and inactivation efficacy of ozone water, which is being introduced into Japanese hospitals as a chemical decontamination agent, was reported for its effects on typical antineoplastic drugs (gemcitabine, irinotecan, paclitaxel). This article aims to further investigate the efficacy of ozone water for eight antineoplastic drugs to clarify its application limitations. A small amount (medicinal ingredient: typically ca. 1.5 μmol) of formulation containing 5-fluorouracil, pemetrexed, cisplatin, oxaliplatin, cyclophosphamide, ifosfamide, doxorubicin, or docetaxel was mixed with 50 mL of ozone water (~8 mg/L), and the resulting solutions were analyzed by high-performance liquid chromatography over time to observe the degradation. Consequently, the ozonation was overall effective for the degradation of the drugs, however this varied depending on the chemical structures of the drugs and additives in their formulations. In addition, after the parent drugs were completely degraded by the ozonation, the degradation mixtures were subjected to 1H nuclear magnetic resonance spectroscopy and evaluated for mutagenicity against Salmonella typhimurium strains and cytotoxicity against human cancer cells. The degradation mixtures of cisplatin and ifosfamide were mutagenic while those of the other drugs were non-mutagenic. Further, the ozonation resulted in clear decreases of cytotoxicity for 5-fluorouracil, oxaliplatin, and doxorubicin, but increases of cytotoxicity for pemetrexed, cisplatin, cyclophosphamide, and ifosfamide. These results suggest that the ozone water should be restrictedly used according to the situation of contamination in clinical settings because the ozonation enhances toxicity depending on the drug even if degradation is achieved.

Vibrio vulnificus can utilize the xenosiderophore desferrioxamine B (DFOB) as an iron source under iron-restricted conditions. We previously identified in V. vulnificus that transcription of the desA gene encoding the outer membrane receptor for ferrioxamine B (FOXB) is activated by the AraC-type transcriptional regulator encoded by desR together with DFOB. In this study, we overexpressed and purified DesR as a glutathione S-transferase-fused protein and examined interaction between the promoter region of desA and DesR. Electrophoretic mobility shift assay (EMSA) revealed that DesR directly binds to the regulatory region of desA, and this binding was enhanced by the presence of DFOB in a concentration-dependent manner, while the presence of FOXB did not affect the potentiation of their binding. Moreover, EMSA identified that DNA fragments lacking a probable DesR binding sequence were unable to form complexes with DesR. Finally, deoxyribonuclease I footprinting assay demonstrated that the DNA binding sequence of DesR is located between -27 and -50 nucleotides upstream of the desA transcription start site. These results strongly indicate that DesR can directly activate the transcription of desA in cooperation with DFOB, which acts as a coactivator for DesR.

Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe3+-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe3+-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe3+-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37 °C, and 8.5 and 45 °C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe3+-aerobactin, Fe3+-vibriobactin, and Fe3+-vulnibactin. When the biologically relevant substrate, Fe3+-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin.

Vibrio vulnificus can utilize the xenosiderophore desferrioxamine B (DFOB) as an iron source under iron-restricted conditions. We previously identified in V. vulnificus that transcription of the desA gene encoding the outer membrane receptor for ferrioxamine B (FOXB) is activated by the AraC-type transcriptional regulator encoded by desR together with DFOB. In this study, we overexpressed and purified DesR as a glutathione S-transferase-fused protein and examined interaction between the promoter region of desA and DesR. Electrophoretic mobility shift assay (EMSA) revealed that DesR directly binds to the regulatory region of desA, and this binding was enhanced by the presence of DFOB in a concentration-dependent manner, while the presence of FOXB did not affect the potentiation of their binding. Moreover, EMSA identified that DNA fragments lacking a probable DesR binding sequence were unable to form complexes with DesR. Finally, deoxyribonuclease I footprinting assay demonstrated that the DNA binding sequence of DesR is located between -27 and -50 nucleotides upstream of the desA transcription start site. These results strongly indicate that DesR can directly activate the transcription of desA in cooperation with DFOB, which acts as a coactivator for DesR.

Purpose Occupational exposure to antineoplastic drugs in hospital settings is recognized to be hazardous, and as such environmental decontamination including degradation and inactivation of such drugs is recommended. To data, although various agents such as oxidants have been reported to be useful for decontamination, simpler, safer, and more convenient methods are required. In this study, the degradation and inactivation efficacy of ozone water, which has newly been introduced for decontamination of antineoplastic drugs in spills, was investigated for formulations of gemcitabine, irinotecan, and paclitaxel. Methods Antineoplastic formulations (medicinal ingredient: ∼1.5 μmol) were mixed with 50 mL of ozone water (>4 mg/L). The reactions were monitored by high-performance liquid chromatography, and the degradation mixtures were analyzed by 1H nuclear magnetic resonance spectroscopy in order to obtain the structural information of the degradation products. The formulations of gemcitabine and irinotecan and those degradation mixtures were evaluated for their mutagenicity using the Ames test and cytotoxicity against human cancer cells. Results gemcitabine and irinotecan were found to be readily degraded by the ozone treatment, and their active sites were suggested to be degraded. In contrast, paclitaxel was hard to be decomposed, possibly owing to the consumption of ozone by the polyoxyethylene castor oil added as a pharmaceutical additive of the formulation. No significant mutagenic changes of Salmonella typhimurium strains used for the Ames test were observed for the samples within the concentration ranges examined. The ozone treatment showed obvious increases in cell viability for gemcitabine formulation, and mild increases for irinotecan formulation. Conclusions Ozone water was shown to be effective as a decomposition agent for the antineoplastic drug formulations examined, although the efficacy depends on the chemical structures of the drugs and the pharmaceutical additives. It was also suggested that ozone treatment has a tendency to decrease the toxicity of the antineoplastic drug formulations. As such, further studies are required in order to clarify the effects and application limitations of ozone water.

We found that Vibrio furnissii can utilize aerobactin (AERO) as a xenosiderophore. A homology search of its genome revealed that this bacterium possesses genes encoding an AERO-mediated iron acquisition system similar to that of V. vulnificus. The system consists of the ABC transporter gene vatCDB, the GntR-type transcriptional repressor gene iutR, and the outer membrane receptor gene iutA. The functions of the vatCDB operon and iutA in V. furnissii were confirmed by the inability of the corresponding deletion mutants to utilize AERO. Reverse transcription-quantitative PCR revealed that iutA transcription under iron-limiting conditions was extensively activated by the addition of AERO to the growth medium; therefore, we focused on elucidating this phenomenon. Electrophoretic mobility shift and DNase I footprinting assays revealed that glutathione S-transferase-fused IutR (GST-IutR) bound directly to a specific palindromic sequence in the iutA promoter region. However, GST-IutR did not bind to this sequence when either AERO or ferric AERO was present in the assay mixture. These in vitro findings suggest that, under iron-limiting conditions, iutA transcription in V. furnissii is artfully regulated both by IutR, acting as a direct repressor of iutA, and by AERO, acting as an effector for IutR, leading to the derepression of iutA transcription.

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C.

We found that, under iron-limiting conditions, Aeromonas hydrophila ATCC 7966T could utilize the xenosiderophore desferrioxamine B (DFOB) for growth by inducing the expression of its own outer membrane receptor. Two consecutive genes, desR and desA, were selected as candidates involved in DFOB utilization. The presence of the ferric-uptake regulator boxes in their promoters suggested that these genes are under iron-dependent regulation. Mutation of desA, a gene that encodes the outer membrane receptor of ferrioxamine B, disrupted the growth of the amonabactin-deficient mutant in the presence of DFOB. β-Galactosidase reporter assays and reverse transcriptase-quantitative PCR demonstrated that desR, a gene that encodes an AraC-like regulator homolog is required for the induction of desA transcription in the presence of DFOB and under iron-limiting conditions. The functions of desA and desR were analyzed using complementation experiments. Our data provided evidence that DesA is powered primarily by the TonB2 system.

Acinetobacter haemolyticus ATCC 17906(T) is known to produce the siderophore acinetoferrin under iron-limiting conditions. Here, we show that an operon consisting of eight consecutive genes, named acbABCD and actBCAD, participates in the biosynthesis and transport of acinetoferrin, respectively. Transcription of the operon was found to be iron-regulated by a putative Fur box located in the promoter region of the first gene, acbA. Homology searches suggest that acbABCD and actA encode enzyme proteins involved in acinetoferrin biosynthesis and an outer-membrane receptor for ferric acinetoferrin, respectively. Mutants defective in acbA and actA were unable to produce acinetoferrin or to express the ferric acinetoferrin receptor under iron-limiting conditions. These abilities were rescued by complementation of the mutants with native acbA and actA genes. Secondary structure analysis predicted that the products of actC and actD may be inner-membrane proteins with 12 membrane-spanning helices that belong to the major facilitator superfamily proteins. ActC showed homology to Sinorhizobium meliloti RhtX, which has been characterized as an inner-membrane importer for ferric rhizobactin 1021 structurally similar to acinetoferrin. Compared to the parental ATCC 17906(T) strain, the actD mutant displayed about a 35 % reduction in secretion of acinetoferrin, which was restored by complementation with actD, suggesting that ActD acts as an exporter of the siderophore. Finally, the actB product was significantly similar to hypothetical proteins in certain bacteria, in which genes encoding ActBCA homologues are arranged in the same order as in A. haemolyticus ATCC 17906(T). However, the function of ActB remains to be clarified.

Aeromonas hydrophila ATCC 7966T produces a catecholate siderophore amonabactin in response to iron starvation. In this study, we determined that this strain utilizes exogenously supplied enterobactin (Ent) for growth under iron-limiting conditions. A homology search of the A. hydrophila ATCC 7966T genomic sequence revealed the existence of a candidate gene encoding a protein homologous to Vibrio parahaemolyticus IrgA that functions as the outer membrane receptor for ferric Ent. SDS–PAGE showed induction of IrgA under iron-limiting conditions. The growth of the double mutant of irgA and entA (one of the amonabactin biosynthetic genes) was restored when it was complemented with irgA in the presence of Ent. Moreover, a growth assay of three isogenic tonB mutants indicated that the tonB2 system exclusively provides energy for IrgA to transport ferric Ent. Finally, reverse transcriptase-quantitative PCR revealed that the transcription of irgA and the TonB2 system cluster genes is iron-regulated, consistently w...

Vibrio vulnificus, an opportunistic pathogen that causes a serious, often fatal, infection in humans, requires iron for its growth. This bacterium utilizes iron from the environment via the vulnibactin-mediated iron uptake system. The mechanisms of vulnibactin biosynthesis, vulnibactin export, and ferric-vulnibactin uptake systems have been reported, whereas the ferric-vulnibactin reduction mechanism in the cell remains unclear. The results of our previous study showed that VuuB, a member of the flavin adenine dinucleotide-containing siderophore-interacting protein family, is a ferric-vulnibactin reductase, but there are other reductases that can complement for the defective vuuB. The aim of this study was to identify these proteins that can complement the loss of function of VuuB. We constructed mutants of genes encoding putative reductases in V. vulnificus M2799, and analyzed their growth under low-iron conditions. Complementation analyses confirmed that IutB, which functions as a ferric-aerobactin reductase, participates in ferric-vulnibactin reduction in the absence of VuuB. This is the first genetic evidence that ferric-vulnibactin is reduced by a member of the ferric-siderophore reductase protein family. In the aerobactin-utilization system, IutB plays a major role in ferric-aerobactin reduction in V. vulnificus M2799, and VuuB and DesB can compensate for the defect of IutB. Furthermore, the expression of iutB and desB was found to be regulated by iron and a ferric uptake regulator.

In this study, we found that Acinetobacter baumannii utilized exogenously supplied desferricoprogen, rhodotorulic acid, and desferrioxamine B for growth under iron-limiting conditions. The ferric uptake regulator (Fur) titration assay method was then successfully applied to select iron-regulated genes in A. baumannii genomic libraries. Part of the nucleotide sequence homologous to Escherichia coli, fhuE, obtained from one of the positive clones allowed us to clone the entire gene, which was named fhuE. The fhuE gene had an amino acid sequence consistent with the N-terminal amino acid sequence of the 76-kDa iron-repressible outer membrane proteins in A. baumannii. Reverse transcription-polymerase chain reaction analysis demonstrated that fhuE mRNA is transcribed under iron-limiting conditions, consistent with the presence of a sequence homologous to the consensus Fur box in the promoter region. Disruption of fhuE resulted in the loss of expression of the 76-kDa protein. In addition, the double disruptant of fhuE and basD, which encodes one of the biosynthetic genes for the cognate siderophore acinetobactin, was unable to grow in the presence of desferricoprogen, rhodotorulic acid or desferrioxamine B. However, growth of the double disruptant was restored by complementation with fhuE, demonstrating that A. baumannii FhuE functions as the receptor common to coprogen, ferric rhodotorulic acid and ferrioxamine B.

We previously reported that the Vibrio parahaemolyticus pvsABCDE and psuA-pvuABCDE operons are involved in the biosynthesis and transport of its own siderophore, vibrioferrin (VF). Of these, psuA and pvuA encode TonB-dependent outer-membrane proteins (OMPs). Although pvuA was characterized as the ferric vibrioferrin receptor gene, the role of the psuA product remains unknown. In this study, a growth assay of isogenic psuA, pvuA, and psuA-pvuA double-deletion mutants followed by complementation of the double-deletion mutant with psuA or pvuA was used to identify psuA as a gene encoding an OMP involved in the uptake of ferric VF. Thus, psuA and pvuA were renamed pvuA1 and pvuA2, respectively. Moreover, we clarified the TonB specificities of PvuA1 and PvuA2, because V. parahaemolyticus has three sets of the TonB systems. The triple deletion of pvuA1, tonB1, and tonB2, and the double deletion of pvuA2 and tonB2 resulted in the complete loss of growth promotion by VF. This finding indicates that the energy required for PvuA1 and PvuA2 to transport ferric VF across the outer membrane is provided by the TonB2 system and by both the TonB1 and TonB2 systems, respectively.

We found that Vibrio (V.) furnissii ATCC35016 can gain iron through a xenosiderophore desferrioxamine B (DFOB) for its growth under iron-limiting conditions, concurrent with the expression of the 79-kDa iron-repressible outer membrane protein (IROMP) in response to the presence of DFOB. Based on the sequence of the ferrioxamine B (an iron-bound form of DFOB) receptor gene in V. vulnificus, two V. furnissii genes, termed desA and desR, encoding the 79-kDa IROMP and AraC-type transcriptional regulator, respectively, were identified and cloned. Nucleotide sequences located in the promoter regions of both desR and desA predicted the presence of consensus ferric uptake regulation (Fur)-binding sequences. The transcription of both genes was negatively regulated by exogenous iron levels. Deletion of the desA gene abolished the ability of V. furnissii to utilize DFOB, and neither desA mRNA nor DesA was detected in the deletion mutant of desR regardless of the presence of DFOB. The functions of DesA and DesR as the ferrioxamine B receptor and transcriptional activator for desA, respectively, were confirmed by complementation of desA and desR deletion mutants.

During the course of our investigation on the iron acquisition systems in Vibrio parahaemolyticus, a causative agent of seafood-related gastroenteritis, we found that this species utilizes desferri-ferrichrome for growth as a heterologous siderophore (a siderophore produced by other bacteria and fungi) under iron-limiting conditions. N-Terminal amino acid sequence analysis of the iron-repressible outer membrane proteins followed by searches of the reported genomic sequences of this species identified four relevant genes (called fhuACDB) forming an operon. Deletion analysis of the fhuA and fhuD genes indicated that the most upstream gene fhuA and the three downstream genes fhuCDB encode the ferrichrome receptor and the ATP-binding cassette transport components, respectively. Moreover, it was found that the fhuCDB genes are also required for transport of ferric aerobactin which restores growth of this species under iron-limiting conditions.

Salinity stress is a major limiting factor in cereal productivity. Many studies report improvements in salt tolerance using model plants, such as Arabidopsis thaliana or standard varieties of rice, e.g., the japonica rice cultivar Nipponbare. However, there are few reports on the enhancement of salt tolerance in local rice cultivars. In this work, we used the indica rice (Oryza sativa) cultivar BR5, which is a local cultivar in Bangladesh. To improve salt tolerance in BR5, we introduced the Escherichia coli catalase gene, katE. We integrated the katE gene into BR5 plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene was actively expressed in the transgenic BR5 rice plants, and catalase activity in T1 and T2 transgenic rice was approximately 150% higher than in nontransgenic plants. Under NaCl stress conditions, the transgenic rice plants exhibited high tolerance compared with nontransgenic rice plants. T2 transgenic plants survived in a 200 mM NaCl solution for 2 weeks, whereas nontransgenic plants were scorched after 4 days soaking in the same NaCl solution. Our results indicate that the katE gene can confer salt tolerance to BR5 rice plants. Enhancement of salt tolerance in a local rice cultivar, such as BR5, will provide a powerful and useful tool for overcoming food shortage problems.

sleeping time by 1.8 to 8.0 times as compared with the control solution with 1% Tween 80-saline. The ED50 values (mg/kg, i.v.) of 9α-OH-HHC and 8-OH-iso-HHC for the antinociceptive effect were 14.1 and 39.4, respec- tively. The present study demonstrated that CBD can be converted to ∆ 9 -THC and its related cannabinoids, 9α- OH-HHC and 8-OH-iso-HHC, in artifi cial gastric juice, and that these HHCs show ∆ 9 -THC-like effects in mice, although their pharmacological effects were less potent than those of ∆ Abstract Cannabidiol (CBD), a nonpsychoactive can- nabinoid, was found to be converted to 9α-hydroxy- hexahydrocannabinol (9α-OH-HHC) and 8-hydroxy- iso-hexahydrocannabinol (8-OH-iso-HHC) together with ∆ 9 -tetrahydrocannabinol (∆ 9 -THC), a psychoactive cannabinoid, and cannabinol in artifi cial gastric juice. These cannabinoids were identifi ed by gas chromatogra- phy-mass spectrometry (GC-MS) by comparison with the spectral data of the authentic compounds. Pharma- cological effects of 9α-OH-HHC and 8-OH-iso-HHC in mice were examined using catalepsy, hypothermia, pen- tobarbital-induced sleep prolongation, and antinocicep- tion against acetic acid-induced writhing as indices. The ED50 values (effective dose producing a 50% reduction of control; mg/kg, i.v.) of 9α-OH-HHC and 8-OH-iso- HHC for the cataleptogenic effect were 8.0 and 30.4, re- spectively. 8-OH-iso-HHC (10 mg/kg, i.v.) produced a signifi cant hypothermia from 15 to 90 min after admin- istration, although 9α-OH-HHC failed to induce such an effect at the same dose. However, both HHCs (10 mg/ kg, i.v.) signifi cantly prolonged pentobarbital-induced

Gabexate (GB), a serine protease inhibitor that is widely used for the treatment of acute pancreatitis and disseminated intravascular coagulation, has been reported to be partly hydrolyzed by human serum albumin. However, other enzymes responsible for GB hydrolysis in human blood remain unclear. In this study, we examined in vitro metabolism of GB with human blood samples. The hydrolytic activities of the plasma and erythrocytes at 100µM of GB were 167 ± 26 and 151 ± 9n mol/min/ml blood fraction (mean ± S.D., n = 8), respectively. Kinetic analysis indicated that Vmax and Km values were 1.75µmol/min/ml blood fraction and 529µ Mf or the plasma and 10.6µmol/min/ml blood fraction and 7360µ Mf or the erythrocytes, respectively. The activity of human plasma wa si nhibited by ethopropazine, a butyrylcholinesterase inhibitor (27% inhibition at 100µM). Furthermore, the plasma activity was inhibited by 5,5 � -dithiobis(2-nitrobenzoic acid) (DTNB) (40% inhibition at 5000µM), suggesting the involvement of albumin in the plasma GB hydrolysis. The erythrocyte activity was also decreased by DTNB (56% inhibition at 5000µM), implying that this activity was dependent on the presence of sulfhydryl group(s), while little or no inhibition of the activity was seen with phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, and BW284C51. Butyrylcholinesterase from human serum showed GB hydrolytic activity with Vmax of 363 nmol/min/m gp rotein and Km of 263µM. These results suggest that, in addition to albumin, butyrylcholinesterase and erythrocyte sulfhydryl-dependent enzyme are responsible for GB hydrolysis in human blood.

Metabolism of nafamostat, a clinically used serine protease inhibitor, was investigated with human blood and liver enzyme sources. All the enzyme sources examined (whole blood, erythrocytes, plasma and liver microsomes) showed nafamostat hydrolytic activity. V(max) and K(m) values for the nafamostat hydrolysis in erythrocytes were 278 nmol/min/mL blood fraction and 628 microM; those in plasma were 160 nmol/min/mL blood fraction and 8890 microM, respectively. Human liver microsomes exhibited a V(max) value of 26.9 nmol/min/mg protein and a K(m) value of 1790 microM. Hydrolytic activity of the erythrocytes and plasma was inhibited by 5, 5'-dithiobis(2-nitrobenzoic acid), an arylesterase inhibitor, in a concentration-dependent manner. In contrast, little or no suppression of these activities was seen with phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), bis(p-nitrophenyl)phosphate (BNPP), BW284C51 and ethopropazine. The liver microsomal activity was markedly inhibited by PMSF, DFP and BNPP, indicating that carboxylesterase was involved in the nafamostat hydrolysis. Human carboxylesterase 2 expressed in COS-1 cells was capable of hydrolyzing nafamostat at 10 and 100 microM, whereas recombinant carboxylesterase 1 showed significant activity only at a higher substrate concentration (100 microM). The nafamostat hydrolysis in 18 human liver microsomes correlated with aspirin hydrolytic activity specific for carboxylesterase 2 (r=0.815, p

The properties of ES46.5K, an esterase from mouse hepatic microsomes, were compared with those of carboxylesterases from rabbit and porcine liver. The inhibitory profile with a serine hydrolase inhibitor (bis-p-nitrophenylphosphate) and detergents (sodium dodecylsulfate, Emulgen 911) was different between ES46.5K and the carboxylesterases. Bis-p-nitrophenylphosphate (0.1 mM) markedly inhibited the catalytic activity of the carboxylesterases but not that of ES46.5K. Emulgen 911 (0.05-0.25%) inhibited the catalytic activity of the carboxylesterases, whereas the detergent conversely stimulated that of ES46.5K by 150%. The two carboxylesterases catalyzed the hydrolysis of acetate esters of tetrahydrocannabinol (THC) analogues with different side chain lengths (C1-C5), although ES46.5K showed marginal activity only against the acetate of Delta8-tetrahydrocannabiorcol, a methyl side chain derivative of Delta8-THC. ES46.5K hydrolyzed cannabinoid esters stereospecifically and regioselectively. The esterase hydrolyzed 8alpha-acetoxy-Delta9-tetrahydrocannabinol (8alpha-acetoxy-Delta9-THC, 5.62 nmol/min/mg protein), while the enzyme did not hydrolyze 8beta-acetoxy-Delta9-THC, 7alpha-acetoxy-, and 7beta-acetoxy-Delta8-THC at all. In contrast, the carboxylesterases from rabbit and porcine liver hydrolyzed 8beta-acetoxy-Delta9-THC efficiently but not 8alpha-acetoxy-Delta9-THC. ES46.5K hydrolyzed side chain acetoxy derivatives of Delta8-THC at the 3'- and 4'-positions, and a methyl ester of 5'-nor-Delta8-THC-4'-oic acid. The enzyme, however, could not hydrolyze methyl esters of Delta8- and Delta9-THC-11-oic acid, while both carboxylesterases hydrolyzed side chain acetoxy derivatives of Delta8-THC and three methyl esters of THC-oic acids. These differences in stereospecificity and regioselectivity between ES46.5K and carboxylesterases suggest that the configurations of important amino acids for the catalytic activities of these enzymes are different from each other.

To assess the safety of the food flavoring agents, methyl anthranilate (MA) and methyl N-methylanthranilate (MNMA), their relationships with metabolism and allergenicity were examined in guinea pigs. Both MA and MNMA were hydrolyzed to anthranilic acid (AA) and N-methylanthranilic acid (N-methylAA), respectively, by guinea pig liver microsomes. These hydrolytic activities at 1000 µM were 320 and 35 nmol/min/mg protein, respectively. Moreover, MNMA was N-demethylated to MA by the liver microsomes; the oxidative activity at 1000 µM of MNMA was 2.8 nmol/min/mg protein. The N-demethylase activity for N-methylAA at 1000 µM in the liver microsomes was 3.9 nmol/min/mg protein. Kinetic analysis indicated that the Vmax/Km values of MA and MNMA hydrolyses were 15and 7.4-fold greater in guinea pig liver microsomes than in the cytosol, respectively, suggesting that these hydrolytic activities were predominantly localized in the microsomes. Liver microsomal activities for the hydrolysis of these flavoring esters were markedly inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and bis(pnitrophenyl)phosphate but not by physostigmine. These hydrolytic activities were suppressed by aspirin, a substrate of carboxylesterase, in a concentration-dependent manner. The N-demethylations of MNMA and N-methylAA were inhibited by SKF 525-A, a nonselective inhibitor of cytochrome P450. At the same time as the metabolic study described above, skin reactions in guinea pigs were investigated using MA, MNMA, N-methylAA, and AA. All compounds examined elicited positive skin reactions, although MNMA and AA exhibited relatively greater sensitizing properties. These results may provide useful information about metabolism in the toxicologic evaluations of MA and MNMA.

The progesterone 17alpha-hydroxylase activity, which is one of the steroidogenic enzymes in rat testis microsomes, was significantly inhibited by crude marijuana extracts from Delta(9)-tetrahydrocannabinolic acid (THCA)- and cannabidiolic acid (CBDA)-strains. Delta(9)-Tetrahydrocannabinol, cannabidiol and cannabinol also inhibited the enzymatic activity with relatively higher concentration (100-1000 microM). Testosterone 6beta- and 16alpha-hydroxylase activities together with androstenedione formation from testosterone in rat liver microsomes were also significantly inhibited by the crude marijuana extracts and the cannabinoids. Crude marijuana extracts (1 and 10 microg/ml) of THCA strain stimulated the proliferation of MCF-7 cells, although the purified cannabinoids (THC, CBD and CBN) did not show significant effects, such as the extract at the concentration of 0.01-1000 nM. These results indicate that there are some metabolic interactions between cannabinoid and steroid metabolism and that the constituents showing estrogen-like activity exist in marijuana.

The inhibitory effects of three major cannabinoids [Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN)] contained in marijuana, an abused drug, on progesterone 17α-hydroxylase activity in rat testis microsomes were investigated. Microsomal progesterone 17α-hydroxylase activity was significantly inhibited in the presence of more than 50 μM of Δ9-THC and CBN compared with control activity, and the IC50 values for Δ9-THC and CBN were estimated to be 42.8 and 32.9 μM, respectively. CBD showed less but significant inhibitory effects on 17α-hydroxylase activity at concentrations greater than 100 μM, and the IC50 value for the cannabinoid was estimated to be 290.9 μM. Kinetic analysis using double reciprocal plots showed that the type of inhibition by CBN was competitive, whereas that of Δ9-THC and CBD was the mixed type. These results suggest that the inhibition may be due to metabolic interactions between each cannabinoid and 17α-hydroxylase.

Microsomal alcohol oxygenase catalyzes the stereoselective oxidation of 7alpha- and 7beta-hydroxy-delta8-tetrahydrocannabinol (7alpha- and 7beta-hydroxy-delta8-THC) to 7-oxo-delta8-THC in monkey liver, and the activity for 7beta-hydroxy-delta8-THC is relatively higher than that for 7alpha-hydroxy-delta8-THC. We previously reported that purified P450JM-E, assumed to be CYP3A8, is a major enzyme responsible for the oxidation of 7-hydroxy-delta8-THC to 7-oxo-delta8-THC in monkey liver and is capable of catalyzing the oxidative reaction by NADH as well as NADPH. In the present study, we demonstrated that some steroids such as testosterone and progesterone stimulated both the NADH- and NADPH-dependent conversions of 7beta-hydroxy-delta8-THC to 7-oxo-delta8-THC in monkey liver microsomes. Kinetic analyses revealed that both the NADH- and NADPH-dependent 7-oxo-delta8-THC formation showed sigmoid kinetics. Testosterone caused a decrease in S50 and an increase in V(max) for the NADH-dependent activity, and resulted in a decrease in S50 without changing the V(max) for the NADPH-dependent activity. On the other hand, NADH-dependent testosterone 6beta-hydroxylation activity showed Michaelis-Menten kinetics and was also inhibited by 7beta-hydroxy-delta8-THC, resulting in a decrease in V(max) with no effect on the K(m). NADPH-dependent testosterone 6beta-hydrozylation activity was also inhibited by 7beta-hydroxy-delta8-THC, resulting in a decrease in both S50 and V(max). In order to explain the metabolic interaction between 7beta-hydroxy-delta8-THC and testosterone, we propose a kinetic model involving at least three binding sites, for the mechanism of activation by testosterone.

In order to assimilate iron,Acinetobacter baumanniiATCC 19606Tproduces a siderophore named acinetobactin (Ab) that is composed of equimolar quantities of 2,3-dihydroxybenzoic acid (DHBA),l-threonine andN-hydroxyhistamine. Application of the Fur titration assay system toA. baumanniigenomic libraries, followed by further cloning of the regions surrounding the candidate genes, led to the identification of the Ab cluster, which harbours the genetic determinants necessary for the biosynthesis and transport of the siderophore. However, anentAhomologue essential for DHBA biosynthesis was not found in this cluster. Functions of potential biosynthetic genes inferred by homology studies suggested that the precursors, DHBA,l-threonine andN-hydroxyhistamine, are linked in steps resembling those of bacterial non-ribosomal peptide synthesis to form Ab. Genes responsible for the two-step biosynthesis ofN-hydroxyhistamine from histidine were also identified in this cluster. Their genetic organization suggests that five genes involved in the transport system of ferric Ab into the cell cytosol form an operon. Construction of disruptants of some selected genes followed by phenotypic analysis supported their predicted biological functions. Interestingly, three additional genes probably involved in the intracellular release of iron from ferric Ab and the secretion of nascent Ab are contained in this cluster. Primer extension and RT-PCR analyses suggested that the Ab cluster, which includes 18 genes, is organized in seven transcriptional units originating from respective Fur-regulated promoter-operator regions.

Many oxidative metabolites of tetrahydrocannabinols (THCs), active components of Cannabis sativa L. (Cannabinaceae), were pharmacologically potent, and 11‐hydroxy‐THCs, 11‐oxo‐Δ8‐THC, 7‐oxo‐Δ8‐THC, 8β,9β‐epoxyhexahydrocannabinol (EHHC), 9α,10α‐EHHC and 3'‐hydroxy‐Δ9‐THC were more active than THC in pharmacological effects such as catalepsy, hypothermia and barbiturate synergism in mice, indicating that these metabolites are active metabolites of THCs. Cannabidiol (CBD), another major component, was biotransfomred to two novel metabolites, 6‐hydroxymethyl‐Δ9‐THC and 3‐pentyl‐6, 7, 7a, 8, 9, 11a‐hexahydro‐1, 7‐dihydroxy‐7,10‐dimethyldibenzo[b,d]oxepin (PHDO) through 8R,9‐epoxy‐CBD and 8S, 9‐epoxy‐CBD as intermediates, respectively, identified by us. Both metabolites have some pharmacological effects comparable to Δ9‐THC. Cannabinol (CBN), the other major component, was mainly metabolized to 11‐hydroxy‐CBN by hepatic microsomes of animals including humans. The pharmacological effects of the metabolite were h...

Hepatic microsomes from human liver catalyzed oxidation of the allyl aldehydes such as 11-oxo-Delta(8)-tetrahydrocannabinol and 9-anthraldehyde to the corresponding carboxylic acid metabolites. The oxygenation mechanism was confirmed by GC-MS that molecular oxygen was exclusively incorporated into Delta(8)-tetrahydrocannabinol-11-oic acid and 9-anthracene carboxylic acid formed under oxygen-18 gas. The microsomal aldehyde oxygenase (named MALDO) activities of 11-oxo-Delta(8)-tetrahydrocannabinol and 9-anthraldehyde were significantly inhibited by the antibody against CYP2C and CYP3A, respectively. MALDO activity for 11-oxo-Delta(8)-tetrahydrocannabinol was significantly inhibited by sulfaphenazole whereas that for 9-anthraldehyde was markedly inhibited by troleandomycin, but not by sulfaphenazole. CYP2C9 expressed in human B-lymphoblastoid cells catalyzed efficiently the MALDO activity for 11-oxo-Delta(8)-tetrahydrocannabinol (10.1 nmol/min/nmol P450), while the catalytic activities of other human CYPs expressed in the cells were lesser extents. In MALDO activity for 9-anthraldehyde, CYP3A4 expressed in the cells had the highest catalytic activity (7.72 nmol/min/nmol P450). These results indicate that CYP2C9 and CYP3A4 are major enzymes responsible for the MALDO activity in human liver for 11-oxo-Delta(8)-tetrahydrocannabinol and 9-anthraldehyde, respectively.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium exports vulnibactin for iron acquisition from the environment. The mechanisms of vulnibactin biosynthesis and ferric-vulnibactin uptake systems have recently been reported, while the vulnibactin export system has not been reported. Mutant growth under low-iron concentration conditions and a bioassay of the culture supernatant indicate that the VV1_0612 protein plays a crucial role in the vulnibactin secretion as a component of the resistance-nodulation-division (RND)-type efflux system in V. vulnificus M2799. To identify which RND protein(s) together with VV1_0612 TolC constituted the RND efflux system for vulnibactin secretion, deletion mutants of 11 RND protein-encoding genes were constructed. The growth inhibition of a multiple mutant (Δ11) of the RND protein-encoding genes was observed 6 h after the beginning of the culture. Furthermore, ΔVV1_1681 exhibited a growth curve that was similar to that of Δ11, while the multiple mutant except ΔVV1_1681 showed the same growth as the wild-type strain. These results indicate that the VV1_1681 protein is involved in the vulnibactin export system of V. vulnificus M2799. This is the first genetic evidence that vulnibactin is secreted through the RND-type efflux systems in V. vulnificus.

A pvsB-vctA-irgA triple deletion mutant of Vibrio parahaemolyticus can utilize enterobactin under iron-limiting conditions by inducing a previously undescribed receptor, PeuA (VPA0150), in response to extracellular alkaline pH and enterobactin. In silico analyses revealed the existence of a two-component regulatory system operon, peuRS, immediately upstream of peuA, which constitutes an operon with the TonB2 system genes. Both the peuRS and peuA-tonB2 operons were found to be upregulated under iron-limiting conditions in a ferric uptake regulator (Fur)-dependent manner. The involvement of peuA and peuRS in enterobactin utilization was analyzed by complementation experiments using deletion mutants. Primer extension analysis indicated that, under iron-limiting conditions, the transcription of peuA was initiated from the +1 site at pH 7.0 and from both the +1 and +39 sites at pH 8.0 in the presence of enterobactin. The +39 transcript was absent from the peuRS deletion mutant. Secondary structure prediction of their 5′-untranslated regions suggested that translation initiation is blocked in the +1 transcript, but not in the +39 transcript. Consistent with this, in vitro translation analysis demonstrated that production of PeuA was determined only by the +39 transcript. These studies establish a novel gene regulation mechanism in which the two-component regulatory system PeuRS enhances expression of the alternative +39 transcript that possesses non-inhibitory structure, allowing the peuA expression to be regulated at the translation stage.

Metabolism of cannabinol (CBN) was studied in vitro using hepatic microsomes from human livers. The metabolites formed were analyzed by thinlayer chromatography (TLC) and identified by gas chromatography-mass spectrometry as their trimethylsilyl derivatives. 11-Hydroxy-CBN, the major metabolite, was detected together with a smaller amount of another mono-hydroxylated metabolite. The minor metabolite was identified as 8-hydroxy-CBN, after comparing its Rf value by TLC, retention time by GC, and the mass spectrum with those of the authentic compound. 8-Hydroxy-CBN was confirmed to be a new metabolite of CBN formed by human hepatic microsomes.

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction-based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.

High-affinity iron acquisition in Vibrio parahaemolyticus is mediated by the cognate siderophore vibrioferrin. We have previously reported that the vibrioferrin biosynthesis operon ( pvs Op) is regulated at the transcriptional level by the iron-responsive repressor Fur (T. Tanabe, T. Funahashi, H. Nakao, S. Miyoshi, S. Shinoda, and S. Yamamoto, J. Bacteriol. 185:6938–6949, 2003). In this study, we identified the Fur-regulated small RNA RyhB and the RNA chaperone Hfq protein as additional regulatory proteins of vibrioferrin biosynthesis. We found that vibrioferrin production was greatly impaired in both the ryhB and hfq deletion mutants, and a TargetRNA search ( http://snowwhite.wellesley.edu/targetRNA/index2.html ) revealed that the 5′-untranslated region of pvs Op mRNA ( pvs Op 5′-UTR) contains a potential base-pairing region required for the formation of the RyhB- pvs Op 5′-UTR duplex. An electrophoresis mobility shift assay indicated that RyhB can directly bind to the pvs Op 5′-UTR with the aid of Hfq. Rifampin chase experiments indicated that the half-life of pvs Op mRNA in the ryhB and hfq mutants was approximately 3-fold shorter than that in the parental strain, suggesting that both RyhB and Hfq are engaged in the stabilization of pvs Op mRNA. Chrome azurol S assays followed by electrophoresis mobility shift assays and rifampin chase experiments carried out for mutant strains indicated that base pairing between RyhB and the pvs Op 5′-UTR results in an increase in the stability of pvs Op mRNA, thereby leading to the promotion of vibrioferrin production. It is unprecedented that RyhB confers increased stability on a polycistronic mRNA involved in siderophore biosynthesis as a direct target.

Aeromonas hydrophila ATCC 7966(T) produces a catecholate siderophore amonabactin in response to iron starvation. In this study, we determined that this strain utilizes exogenously supplied enterobactin (Ent) for growth under iron-limiting conditions. A homology search of the A. hydrophila ATCC 7966(T) genomic sequence revealed the existence of a candidate gene encoding a protein homologous to Vibrio parahaemolyticus IrgA that functions as the outer membrane receptor for ferric Ent. SDS-PAGE showed induction of IrgA under iron-limiting conditions. The growth of the double mutant of irgA and entA (one of the amonabactin biosynthetic genes) was restored when it was complemented with irgA in the presence of Ent. Moreover, a growth assay of three isogenic tonB mutants indicated that the tonB2 system exclusively provides energy for IrgA to transport ferric Ent. Finally, reverse transcriptase-quantitative PCR revealed that the transcription of irgA and the TonB2 system cluster genes is iron-regulated, consistently with the presence of a predicted Fur box in the promoter region.

We determined the ability of Vibrio parahaemolyticus to utilize enterobactin (Ent) as a xenosiderophore. Homology searches of the V. parahaemolyticus genomic sequence revealed the presence of genes that are homologous to the V. cholerae ferric Ent utilization genes, which consist of the iron-repressible outer-membrane protein genes irgA and vctA, and the ATP-binding cassette transport system operon vctPDGC. Moreover, the irgB and vctR genes, which encode transcriptional regulators, were also found immediately upstream of irgA and vctA, respectively. Growth assays of V. parahaemolyticus indicated that both irgA and vctA mutants grew well in the presence of Ent under iron-limiting conditions, whereas both the irgA/vctA double mutant and the vctPDGC mutant barely grew under the same conditions. In addition, growth assays of three isogenic tonB mutants demonstrated that the TonB2 system, and to a lesser extent the TonB1 system, can provide energy for both IrgA and VctA to transport ferric Ent. SDS-PAGE analysis showed that expression of both IrgA and VctA was enhanced by the presence of Ent. Complementation of the irgB and vctR mutants with their respective genes resulted in the increased expression of IrgA and VctA, respectively. Finally, reverse transcriptase-quantitative PCR revealed that transcription of the Ent utilization system genes is iron-regulated, and that transcription of irgA and vctA under iron-limiting conditions is further activated by proteins encoded by irgB and vctR, respectively, together with Ent.

The present authors have previously reported that Vibrio mimicus expresses 77-kDa and 80-kDa outer membrane proteins in response to iron-limited conditions, and that the 77-kDa protein serves as the receptor for ferriaerobactin. In this study, it was found that V. mimicus can use heme and hemoglobin as iron sources. FURTA was then applied to V. mimicus 7PT to obtain candidate gene fragments involved in utilization of heme and hemoglobin. One FURTA-positive clone was shown to contain a partial gene, whose predicted amino acid sequence correlated with the N-terminal amino acid sequence determined for the 80-kDa outer membrane protein and also shared homology with heme/hemoglobin receptors of Gram-negative bacteria. Based on this information, the entire gene (named mhuA), and a gene upstream of mhuA (named mhuB) encoding a LysR family of transcriptional activator, were cloned and analyzed. RNA analysis indicated that mhuA and mhuB are each transcribed from individual Fur-regulated promoters. Moreover, RNA analysis of an mhuB deletion mutant and a promoter reporter assay coupled with β-galactosidase suggested that MhuB could function as an activator for mhuA transcription. Finally, the role of MhuA as the heme/hemoglobin receptor was confirmed by construction of an mhuA deletion mutant and its complemented strain followed by growth assay.

N3-(2',5 '-Dimethoxyphenacyl)arabinofuranosyluracil (N3-(2',5 '-DiMeOPhAc)AraU) is a pyrimidine nucleoside derivative which possesses antinociceptive effect by intracerebroventricular (i.c.v.) administration to mice. The compound (100 microM) significantly decreased the specific binding of [3H]D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO) and [3H]D-Pen2, D-Pen5-enkephalin (DPDPE) at the mu- and delta-opioid receptor, respectively, but has no effect on the binding of [3H]U-69,593 at the kappa-opioid receptor of rat brain slices. The autoradiographic studies also demonstrated that [3H]DAMGO and [3H]DPDPE bindings on the rat brain slice were displaced by N3-(2',5'-DiMeOPhAc)AraU. These results indicate that N3-(2',5'-DiMeOPhAc)AraU interacts with mu- and delta-opioid receptors.

Although the authors recently reported that nafamostat, a clinically used serine protease inhibitor, was mainly hydrolysed by carboxylesterase in human liver microsomes, the involvement of human liver cytosol has not been elucidated. The current study examined the in vitro metabolism of nafamostat with human liver cytosols. Kinetic analysis indicated that the Vmax and Km values in the liver cytosols were 9.82 nmolmin(-1) mg(-1) protein and 197 microM for a liver sample HL-1, and 15.1 nmolmin(-1) mg(-1) protein and 157 microM for HL-2, respectively. The Vmax/Km values in both cytosols were at least threefold higher than those in the corresponding microsomes. The liver cytosolic activity for nafamostat hydrolysis was inhibited by phenylmethylsulfonyl fluoride (PMSF) (43% inhibition at 100 microM), whereas diisopropyl fluorophosphate (DFP) and bis(p-nitrophenyl)phosphate (BNPP) failed to inhibit the activity. Furthermore, the hydrolytic activity was also reduced by palmitoyl-CoA (67% inhibition at 100 microM) but not by acetyl-CoA. Effects of PMSF, DFP and BNPP on cytosolic palmitoyl-CoA hydrolytic activity were comparable with those of the cytosolic nafamostat hydrolytic activity. In addition, the palmitoyl-CoA hydrolytic activity was competitively inhibited by nafamostat with the apparent Ki value of 164 microM for the liver cytosol from HL-2. These results suggest that an isoform of long-chain acyl-CoA hydrolase may be responsible for the nafamostat hydrolysis in human liver cytosol.

The cytotoxicity of Vibrio parahaemolyticus has been related to the type III secretion system 1 effector protein VP1680, which is secreted and translocated into host cells with the help of the specific chaperone protein, VP1682. This study sought to confirm the in silico analysis, which predicted that a small regulatory RNA (Spot 42) could base pair with the region encompassing the ribosomal-binding site and initiation codon of the vp1682 mRNA. Electrophoresis mobility shift assays indicated that Spot 42 could bind to the vp1682 mRNA with the help of Hfq. Consistent with these results, the translation of the vp1682 mRNA was inhibited when both Hfq and Spot 42 were added to the in vitro translation reaction. The cytotoxic activity against infected Caco-2 cells was significantly increased in the Spot 42 deletion mutant (Δ spf ) at 4 h after infection as compared with the parental strain. Additionally, we observed that both VP1682 and VP1680 were more highly expressed in Δ spf mutants than in the parental strain. These results indicate that Spot 42 post-transcriptionally regulates the expression of VP1682 in V. parahaemolyticus , which contributes to cytotoxicity in vivo.

In this study, tetrahydrocannabinols (THCs) were mainly oxidized at the 11-position and allylic sites at the 7alpha-position for Delta(8)-THC and the 8beta-position for Delta(9)-THC by human hepatic microsomes. Cannabinol (CBN) was also mainly metabolized to 11-hydroxy-CBN and 8-hydroxy-CBN by the microsomes. The 11-hydroxylation of three cannabinoids by the microsomes was markedly inhibited by sulfaphenazole, a selective inhibitor of CYP2C enzymes, while the hydroxylations at the 7alpha-(Delta(8)-THC), 8beta-(Delta(9)-THC) and 8-positions (CBN) of the corresponding cannabinoids were highly inhibited by ketoconazole, a selective inhibitor of CYP3A enzymes. Human CYP2C9-Arg expressed in the microsomes of human B lymphoblastoid cells efficiently catalyzed the 11-hydroxylation of Delta(8)-THC (7.60 nmol/min/nmol CYP), Delta(9)-THC (19.2 nmol/min/nmol CYP) and CBN (6.62 nmol/min/nmol CYP). Human CYP3A4 expressed in the cells catalyzed the 7alpha-(5.34 nmol/min/nmol CYP) and 7beta-hydroxylation (1.39 nmol/min/nmol CYP) of Delta(8)-THC, the 8beta-hydroxylation (6.10 nmol/min/nmol CYP) and 9alpha,10alpha-epoxidation (1.71 nmol/min/nmol CYP) of Delta(9)-THC, and the 8-hydroxylation of CBN (1.45 nmol/min/nmol CYP). These results indicate that CYP2C9 and CYP3A4 are major enzymes involved in the 11-hydroxylation and the 8-(or the 7-) hydroxylation, respectively, of the cannabinoids by human hepatic microsomes. In addition, CYP3A4 is a major enzyme responsible for the 7alpha- and 7beta-hydroxylation of Delta(8)-THC, and the 9alpha,10alpha-epoxidation of Delta(9)-THC.

We had previously reported that Vibrio hollisae produces aerobactin in response to iron starvation. In the present study, we identified in V. hollisae ATCC33564 the aerobactin system cluster which consists of eight genes, hatCDB, iucABCD and iutA. The hatCDB genes encode proteins homologous to components of bacterial ATP binding cassette transport systems for ferric aerobactin. The iucABCD and iutA orthologs code for aerobactin biosynthesis enzymes and the ferric aerobactin receptor, respectively. In accordance with their iron-regulated expression, putative Fur box sequences were found within the respective promoter regions of hatC, iucA and iutA. The monocistronic iutA transcript was detected by northern blotting. Moreover, phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions that were expected for the respective operons and genes on the basis of the homology search. The arrangement of the aerobactin gene clusters thus far found in Vibrio and enterobacterial species was compared and discussed from an evolutionary point of view.

Seventy eight N(3)-substituted derivatives of uridine (1), thymidine (2), 2'-deoxyuridine (3), 6-azauridine (4), 2',3'-O-isopropylideneuridine (5), and arabinofuranosyluracil (6) were synthesized and their antinociceptive effects were evaluated. N(3)-(2',4'-Dimethoxyphenacyl)uridine (1l), N(3)-(2',4'-dimethoxyphenacyl)2'-deoxyuridine (3l), and N(3)-(2',5'-dimethoxyphenacyl)arabinofuranosyluracil (6m) possessed 93, 86, and 82% of the antinociceptive effects tested by hot plate, respectively. The antinociceptive effects of three derivatives were 5.8, 5.4, and 5.1-folds of the effect of N(3)-phenacyluridine (1h) (16%), respectively. The structure-activity relationship of N(3)-substituted pyrimidine nucleosides was also discussed.

The antinociceptive activity of N3-phenacyluridine, a novel hypnotic, was examined with tail pinch, hot plate and acetic acid-induced abdominal constriction methods by intracerebroventricular (i.c.v.) injection to mice. In the first method, N3-phenacyluridine exerted antinociceptive activity after the i.c.v. injection at a dose of 0.5 micromol/mouse, although this activity was comparable to the activity of morphine (0.01 micromol/mouse, i.c.v.). In the second method, the activity of N3 -phenacyluridine was continued until 120 min after the injection at a dose of 0.5 micromol/mouse. In last, N3-phenacyluridine (0.1 micromol/mouse, i.c.v.) significantly decreased in the numbers of acetic acid-induced abdominal constriction as compared to the control (1% Tween 80 saline). The ED50 value of the antinociceptive activity of N3-phenacyluridine was 0.02 imol/mouse, i.c.v. The present paper demonstrated for the first time that N3-phenacyluridine belonging to oxopyrimidine nucleoside analogue possesses not only the hypnotic and sedative activities previously reported, but also antinociceptive activity.

In response to low iron availability, Vibrio parahaemolyticus synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, l -alanine, ethanolamine, and citric acid. We have previously reported the cloning and characterization of the pvuA gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin. In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the pvuA gene. The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis. Five of the genes, named pvsABCDE , constitute an operon that is expressed under iron-limiting conditions. Homology searches of their predicted protein products suggested that the four genes pvsABDE are implicated in the biosynthesis of the siderophore. Another gene in the same operon, pvsC , encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps. The remaining four genes, named pvuBCDE , encode proteins strongly homologous to Escherichia coli FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate. Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, psuA and pvuA . Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.

A previous investigation using the Fur titration assay system showed that Vibrio parahaemolyticus possesses a gene encoding a protein homologous to IutA, the outer-membrane receptor for ferric aerobactin in Escherichia coli. In this study, a 5·6 kb DNA region from the V. parahaemolyticus WP1 genome was cloned and two entire genes, iutA and alcD homologues, were identified which are absent from Vibrio cholerae genomic sequences. The V. parahaemolyticus IutA and AlcD proteins share 43 % identity with the Escherichia coli IutA protein and 24 % identity with the Bordetella bronchiseptica AlcD protein of unknown function, respectively. Primer extension analysis revealed that the iutA gene is transcribed in response to low-iron availability from a putative promoter overlapped with a sequence resembling a consensus E. coli Fur-binding sequence. In agreement with the above finding, V. parahaemolyticus effectively utilized exogenously supplied aerobactin for growth under iron-limiting conditions. Moreover, insertional inactivation of iutA impaired growth in the presence of aerobactin and incapacitated the outer-membrane fraction from iron-deficient cells for binding 55Fe-labelled aerobactin. These results indicate that the V. parahaemolyticus iutA homologue encodes an outer-membrane protein which functions as the receptor for ferric aerobactin. Southern blot analysis revealed that the iutA homologues are widely distributed in clinical and environmental isolates of V. parahaemolyticus. However, additional genes required for ferric aerobactin transport across the inner membrane remain to be clarified.

We previously reported that Vibrio parahaemolyticus expresses two outer membrane proteins of 78 and 83 kDa concomitant with production of siderophore vibrioferrin in response to iron starvation stress and that these proteins are the ferric vibrioferrin receptor and heme receptor, respectively (S. Yamamoto, T. Akiyama, N. Okujo, S. Matsuura, and S. Shinoda, Microbiol. Immunol. 39:759-766, 1995; S. Yamamoto, Y. Hara, K. Tomochika, and S. Shinoda, FEMS Microbiol. Lett. 128:195-200, 1995). In this study, the Fur titration assay (FURTA) system was applied to isolate DNA fragments containing a potential Fur box from a genomic DNA library of V. parahaemolyticus WP1. Sequencing a 3.2-kb DNA insert in one FURTA-positive clone revealed that an amino acid sequence deduced from a partial gene, which was preceded by a full-length gene ( psuA ) encoding a receptor for a siderophore of unknown origin, was consistent with the N-terminal amino acid sequence of the 78-kDa ferric vibrioferrin receptor. Then, the full-length gene ( pvuA ) encoding the ferric vibrioferrin receptor was cloned and characterized. The deduced protein encoded by pvuA displayed the highest similarity (31% identity; 48% similarity) to RumA, a ferric rhizoferrin receptor of Morganella morganii . Primer extension and Northern blot analyses indicated that psuA and pvuA constitute an operon which is transcribed from a Fur-repressed promoter upstream of psuA . The product of the pvuA gene and its function were confirmed by generating a pvuA -disrupted mutant, coupled with genetic complementation studies. A mutant with disruption in the upstream psuA gene also displayed a phenotype impaired in the utilization of ferric vibrioferrin.

Sequencing of Fur titration assay-positive clones obtained from genomic DNA libraries of Vibrio parahaemolyticus, V. mimicus and V. vulnificus revealed open reading frames encoding proteins of 202, 205 and 202 amino acid residues, respectively. Each open reading frame was preceded by a predicted Fur box which overlaps a likely promoter with similarity to the -10 and -35 consensus sequence of Escherichia coli. The deduced amino acid sequences shared considerable homology with bacterial Mn-containing superoxide dismutases (MnSODs). Consistent with this, these Vibrio strains produced proteins with SOD activity resistant to inhibition by H2O2 and KCN only when grown under iron-limiting conditions. Primer extension analysis of the total RNA from these vibrios revealed iron-repressible expression of the genes. Furthermore, when grown under iron-limiting conditions, E. coli carrying a plasmid with each cloned gene overexpressed protein with the same electrophoretic mobility and insensitivity of SOD activity to H2O2 and KCN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing revealed that proteins (MnSODs) having N-terminal amino acid sequences consistent with those deduced from the corresponding genes were present in cell lysates of the vibrios grown under these iron-limited conditions. These results demonstrate that the genes cloned in this study are sodA homologs encoding MnSODs, whose expression is regulated by the iron status of the growth medium. PCR using a primer set based on the V. parahaemolyticus sodA sequence revealed the presence of homologous genes in certain other Vibrio species.

In many bacteria, the ferric uptake regulatory protein (Fur) has a central role in the negative regulation of genes affected by iron limitation. In this study, Vibrio parahaemolyticus strains carrying mutations in the fur gene encoding Fur were isolated by the manganese selection method to assess the function of Fur in connection with alternations in the coordinate expression of the siderophore vibrioferrin (VF) and iron-repressible outer membrane proteins (IROMPs). Ten out of 25 manganese-resistant mutants constitutively produced VF and expressed at least two IROMPs irrespective of the iron concentration in the medium. PCR-direct DNA sequencing of the fur genes in these mutants identified four different point mutations causing amino acid changes. Moreover, a fur overexpressing plasmid was constructed to prepare antiserum against V. parahaemolyticus Fur. Western blotting with this antiserum revealed that the intracellular abundance of the wild-type Fur was not significantly affected by the iron concentrations in the growth medium, and that the Fur proteins of the mutant strains occurred at substantially smaller amounts and/or migrated more rapidly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the wild-type Fur. These data afford an additional insight into the structure-function relationship of Fur and imply its involvement in the iron acquisition systems of V. parahaemolyticus, although it is yet unknown whether its action on the target genes is direct or indirect.