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A path planning algorithm for an Autonomous Underwater Vehicle (AUV) performing docking operations in the presence of tidal current disturbances is presented. The path planner is composed a way-point generator (WPG), an Model Predictive Control based optimal planner (OP), and a tidal current estimator (TCE). The WPG defines the next way-point with respect to the vehicle position and the OP iteratively generates the trajectory over a prescribed future horizon whilst satisfying certain physical and logical constraints. The TCE estimates the tidal current disturbance speed enabling to adapt the AUV trajectory for reducing actuators power consumption. The proposed path planning policy has been evaluated in a set of simulation scenarios to illustrate the ability of the policy to drive the AUV by satisfying specifications and limiting power consumption.

AimsWe herein investigated the inducibility of cytochrome P450 1A1 (CYP1A1) by Δ9-tetrahydrocannabinol, cannabidiol (CBD), and cannabinol, three major phytocannabinoids, using human hepatoma HepG2 cells.Main methodsThe expression of CYP1A1 and the aryl hydrocarbon receptor (AhR) was measured by a quantitative real-time polymerase chain reaction and/or Western blotting.Key findingsΔ9-Tetrahydrocannabinol and CBD concentration-dependently induced the expression of CYP1A1 mRNA, whereas cannabinol showed little or no induction. Among the phytocannabinoids tested, CBD was the most potent inducer of CYP1A1 expression. The induction of CYP1A1 expression by CBD was significantly attenuated by the knockdown of AhR expression with AhR small interfering RNAs. The role of protein tyrosine kinases (PTKs) in the CBD-mediated induction of CYP1A1 was then examined using herbimycin A, a PTK inhibitor. The upregulation of CYP1A1 by CBD was significantly suppressed by herbimycin A as was the induction by omeprazole but not 3-methylcholanthrene. The inducibility of CYP1A1 by CBD-related compounds was examined to clarify the structural requirements for CBD-mediated CYP1A1 induction. Olivetol, which corresponds to the pentylresorcinol moiety of CBD, significantly induced the expression of CYP1A1, whereas d-limonene, CBD-2′-monomethyl ether, and CBD-2′,6′-dimethyl ether did not.SignificanceThese results showed that CBD may have induced human CYP1A1 expression through the activation of PTK-dependent AhR signaling, in which two phenolic hydroxyl groups in the pentylresorcinol moiety of CBD may play structurally important roles., Article, LIFE SCIENCES.136:87-93(2015)

The author developed key technologies for the next long cruising autonomous underwater vehicle. Layout is designed by CAD, the prototype vehicle is manufactured and tested in the tanks. As results, longer cruising than the conventional type underwater vehicle like Urashima is possible by low resistance shape and with hydrofoils.

The effect of α-substituent on the molecular motion and wetting behavior of poly{2-(perfluorobutyl)ethyl acrylate} [PFA-C4], poly{2-(perfluorobutyl)ethyl methacrylate} [PFMA-C4], poly{2-(perfluorobutyl)ethyl α-fluoroacrylate} [PFFA-C4], and poly{2-(perfluorobutyl)ethyl α-chloroacrylate} [PFClA-C4] films were characterized by dynamic contact angle measurement, lateral force microscopy (LFM), wide angle X-ray diffraction (WAXD), and X-ray photoelectron spectroscopy (XPS). WAXD of oriented PFClA-C4 fiber suggested the presence of rod-like chain due to the presence of bulky α-substituent. The glass transition temperature (Tg) of PFFA-C4 and PFClA-C4 were well above the room temperature. The water repellencies of PFFA-C4 and PFClA-C4 were as high as that of PFMA-C4 and their oil repellency of PFFA-C4 and PFClA-C4 was higher than the PFMA-C4. This result was originated from the low main chain mobility of PFFA-C4 and PFClA-C4 due to the presence of bulky α-substituents. The effect of molecular motion on water repellency was clarified by the results of temperature dependence studies of dynamic contact angle, LFM, and surface chemical composition measured by XPS.

We previously demonstrated that cannabidiol (CBD) was a potent mechanism-based inhibitor of human cytochrome P450 1A1 (CYP1A1). However, the moiety of CBD that contributes to the potent mechanism-based inhibition of human CYP1A1 remains unknown. Thus, the effects of compounds structurally related to CBD on CYP1A1 activity were examined with recombinant human CYP1A1 in order to characterize the structural requirements for potent inactivation by CBD. When preincubated in the presence of NADPH for 20 min, olivetol, which corresponds to the pentylresorcinol moiety of CBD, enhanced the inhibition of the 7-ethoxyresorufin O-deethylase activity of CYP1A1. In contrast, d-limonene, which corresponds to the terpene moiety of CBD, failed to inhibit CYP1A1 activity in a metabolism-dependent manner. Pentylbenzene, which lacks two free phenolic hydroxyl groups, also did not enhance CYP1A1 inhibition. On the other hand, preincubation of the CBD-2′-monomethyl ether (CBDM) and CBD-2′,6′-dimethyl ether (CBDD) enhanced the inhibition of CYP1A1 activity. Inhibition by cannabidivarin (CBDV), which possessed a propyl side chain, was strongly potentiated by its preincubation. Orcinol, which has a methyl group, augmented CYP1A1 inhibition, whereas its derivative without an alkyl side chain, resorcinol, did not exhibit any metabolism-dependent inhibition. The preincubation of CBD-hydroxyquinone did not markedly enhance CYP1A1 inhibition. We further confirmed that olivetol, CBDM, CBDD, CBDV, and orcinol, as well as CBD (kinact = 0.215 min−1), inactivated CYP1A1 activity; their kinact values were 0.154, 0.0638, 0.0643, 0.226, and 0.0353 min−1, respectively. These results suggest that the methylresorcinol structure in CBD may have structurally important roles in the inactivation of CYP1A1., Article, CHEMICO-BIOLOGICAL INTERACTIONS.215:62-68(2014)

Tetrahydrocannabinol (THC), a psychoactive component of marijuana, is known to exert cytotoxicity in immune cells. In the present study, we examined the cytotoxicity of Δ⁸-THC in mouse macrophage J774-1 cells and a possible involvement of cannabinoid receptors and stress-responsive mitogen-activated protein kinases (MAPKs) in the cytotoxic process. J774-1 cells were treated with Δ⁸-THC (0-20 μM) for up to 6 h. As measured by the MTT and LDH assays, Δ⁸-THC induced cell death of J774-1 cells in a concentration- and/or exposure time-dependent manner. Δ⁸-THC-induced cell damage was associated with vacuole formation, cell swelling, chromatin condensation, and nuclear fragmentation. The cytotoxic effect of Δ⁸-THC was significantly prevented by a caspase-1 inhibitor Ac-YVAD-cmk but not a caspase-3 inhibitor z-DEVD-fmk. The pretreatment with SR144528, a CB₂ receptor-selective antagonist, effectively suppressed Δ⁸-THC-induced cytotoxicity in J774-1 cells, which exclusively expressed CB₂ receptors as indicated by real-time polymerase chain reaction analysis. In contrast, AM251, a CB₁ receptor-selective antagonist, did not affect the cytotoxicity. Pertussis toxin and α-tocopherol significantly attenuated Δ⁸-THC-induced cytotoxicity suggesting that G(i/o) protein coupling signal transduction and oxidative stress are responsible for the cytotoxicity. Δ⁸-THC stimulated the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) in J774-1 cells, which were effectively antagonized by the pretreatment with SR144528. In addition, SB203580, a p38 MARK inhibitor, significantly attenuated the cytotoxic effect of Δ⁸-THC, whereas SP600125, a JNK inhibitor, significantly enhanced the cytotoxicity. These results suggest that the cytotoxicity of Δ⁸-THC to J774-1 cells is exerted mediated through the CB₂ receptor followed by the activation of p38 MAPK.

Aims Cannabidiol (CBD), one of the major constituents in marijuana, has been shown to be extensively metabolized by experimental animals and humans. However, human hepatic enzymes responsible for the CBD metabolism remain to be elucidated. In this study, we examined in vitro metabolism of CBD with human liver microsomes (HLMs) to clarify cytochrome P450 (CYP) isoforms involved in the CBD oxidations. Main methods Oxidations of CBD in HLMs and recombinant human CYP enzymes were analyzed by gas chromatography/mass spectrometry. Key findings CBD was metabolized by pooled HLMs to eight monohydroxylated metabolites (6α-OH-, 6β-OH-, 7-OH-, 1″-OH-, 2″-OH-, 3″-OH-, 4″-OH-, and 5″-OH-CBDs). Among these metabolites, 6α-OH-, 6β-OH-, 7-OH-, and 4″-OH-CBDs were the major ones as estimated from the relative abundance of m/z 478, which was a predominant fragment ion of trimethylsilyl derivatives of the metabolites. Seven of 14 recombinant human CYP enzymes examined (CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) were capable of metabolizing CBD. The correlations between CYP isoform-specific activities and CBD oxidative activities in 16 individual HLMs indicated that 6β-OH- and 4″-OH-CBDs were mainly formed by CYP3A4, which was supported by inhibition studies using ketoconazole and an anti-CYP3A4 antibody. The correlation and inhibition studies also showed that CBD 6α-hydroxylation was mainly catalyzed by CYP3A4 and CYP2C19, whereas CBD 7-hydroxylation was predominantly catalyzed by CYP2C19. Significance This study indicated that CBD was extensively metabolized by HLMs. These results suggest that CYP3A4 and CYP2C19 may be major isoforms responsible for 6α-, 6β-, 7-, and/or 4″-hydroxylations of CBD in HLMs.

15-Lipoxygenase (15-LOX) is one of the key enzymes responsible for the formation of oxidized low-density lipoprotein, a major causal factor for atherosclerosis. Δ(9)-Tetrahydrocannabinol (Δ(9)-THC), a major component of marijuana, has suggested to suppress atherosclerosis. Although Δ(9)-THC seems to be attractive for the prevention of atherosclerosis, there is no information about whether or not 15-LOX isoform can be inhibited by Δ(9)-THC. In the present study, Δ(9)-THC was found to be a direct inhibitor for 15-LOX with an IC(50) (50% inhibition concentration) value of 2.42 μM. Furthermore, Δ(9)-THC-11-oic acid, a major and nonpsychoactive metabolite of Δ(9) -THC, but not another Δ(9)-THC metabolite 11-OH-Δ(9)-THC (psychoactive), was revealed to inhibit 15-LOX. Taken together, it is suggested that Δ(9) -THC can abrogate atherosclerosis via direct inhibition of 15-LOX, and that Δ(9)-THC-11-oic acid is shown to be an "active metabolite" of Δ(9) -THC in this case.

This paper describes dynamic positioning system developed for 20 years through research and development by Japan Oil, Gas and Metals National Corporation. It summaries a basic concept of design, simulator, tank test, and real applications. Also, the next generation system of Japan and its basic research is mentioned in the paper.

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC), a major constituent of marijuana, has been shown to stimulate the growth of MCF-7 breast cancer cells through cannabinoid receptor-independent signaling [Takeda, S., Yamaori, S., Motoya, E., Matsunaga, T., Kimura, T., Yamamoto, I., Watanabe, K., 2008. Delta(9)-Tetrahydrocannabinol enhances MCF-7 cell proliferation via cannabinoid receptor-independent signaling. Toxicology 245, 141-146]. Although the growth of MCF-7 cells is known to be stimulated by 17beta-estradiol (E(2)), the interaction of Delta(9)-THC and E(2) in MCF-7 cell growth is not fully clarified so far. In the present study, by using E(2)-sensitive MCF-7 cells that have expressed cyclooxygenase-2 (COX-2) and cytochrome P450 19 (aromatase), we studied whether or not COX-2 and aromatase are involved in Delta(9)-THC-mediated MCF-7 cell proliferation. It was shown that Delta(9)-THC-induced MCF-7 cell growth was inhibited by COX-2 inhibitors and was stimulated by arachidonic acid (a COX substrate). However, the growth of MCF-7 cells induced by Delta(9)-THC was not stimulated by PGE(2), and the expression of aromatase was not affected by COX-2 inhibitors, arachidonic acid, and PGE(2), suggesting that there is a disconnection between COX-2 (PGE(2)) and aromatase in Delta(9)-THC-mediated MCF-7 cell proliferation. On the other hand, Delta(9)-THC-induced MCF-7 cell growth was elevated by two kinds of aromatase inhibitors. Taken together with the evidence that Delta(9)-THC-induced MCF-7 cell proliferation was interfered with testosterone (an aromatase substrate) and exogenously provided E(2), it is suggested that (1) the growth stimulatory effects of Delta(9)-THC are mediated by the product(s) of COX-2 except for PGE(2), (2) the action of Delta(9)-THC is modulated by E(2), and (3) COX-2 and aromatase are individually engaged in the proliferation of MCF-7 cells induced by Delta(9)-THC.

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.

System identification of coupled ship dynamics is problematic with standard least squares methods due to the non-linear, multivariable nature of the system. Neural Networks have therefore been applied to this problem, as they are particularly suitable for approximating non-linear, multivariable functions. In this paper, results of identification with Neural Networks are given for a ship motion simulation based on a standard mathematical model, and for real data collected from a 1/50th scale model of the system. The method is seen to be successful at various operating points, and ideas for extension of the work are discussed.

The purpose of this paper is to describe the feasibility research of marine vehicles with oscillating fin propulsion control system. The oscillating fin propulsion system was designed and constructed to be combined with a ship model. Tank tests using the ship model have confirmed the system's feasibility. As a result, several advantages of the oscillating fin system have been found out. A neural network was successfully applied for an identification of the ship model dynamics with the oscillating fin, and its effectiveness was confirmed. Robotic fish, intended as an amusement attraction for aquariums, using the oscillating fin propulsion system have been developed. Its capability of untethered 3-dimensional movement was confirmed.

A new simulator to make an integrated total simulation of an offshore oil production system has been developed to make the production and transportation plan and the cost examination of the system, prior to construction. The simulator is named SIMOP, which consists of software modules for offshore oil production well, floating structure, production facility, shipment facility, sea vessel, and land equipment considering weather condition. The integrated modelling of each module is developed by discrete event modelling method and control theory. In the paper, down time analysis of an offshore platform were successfully conducted in the North sea and the Southeast Asia sea cases.

For the remotely operated vehicle (ROV) with an umbilical cable, it becomes difficult to guide it to a target as the depth increases. Especially, the vehicle for the working in the deep sea under current disturbance, the control to reach the target is the most important and difficult problem, because the tension of the umbilical cable, which connects the vehicle to the mother ship, becomes extremely large by current force. The author proposes the algorithm for automatic control and guidance to reach the target in the sea under the restriction of actuator's maximum force. The application was successfully conducted to ROV with umbilical cable as a basic control algorithm

Characterization of gastric Na+/I- symporter (NIS) of the rat was carried out. Sequencing of the open reading frame of gastric NIS mRNA showed only three nucleotide changes when compared with FRTL-5 NIS cDNA, and two of these changes led to amino acid changes. The results of Northern blot analysis showed that abundant NIS mRNA was expressed in the stomach when compared with other organs. Western blot analysis using gastric mucosa and FRTL-5 lysates detected the difference in molecular weight between FRTL-5 and gastric mucosa lysates, suggesting abnormal posttranslational modification of gastric NIS protein. Immunohistochemically, gastric NIS protein was located in the cornification layer of the stratified squamous epithelium of the pars proventricularis and in parietal cells and on the apical border of surface epithelial cells of the pars glandularis. Gastric NIS protein was present in tubulovesicular structures and lysosomes in parietal cells by immunoelectron microscopy. Gastric NIS protein exists to trap I- from the gastric lumen, except in parietal cells. Results indicated that a very large amount of gastric NIS mRNA is expressed to be translated, whereas only a small amount of immature gastric NIS protein is detected. This may indicate that immature gastric NIS protein rapidly degrades to peptides.

It is difficult for a ship’s pilot to maintain a stationary position by controlling several actuators in severe environmental conditions. As a result, we have developed a dynamic positioning system based on advanced control theories and applied it to a vessel working on the construction of the Kurushima Bridge. Simulations and experiments have evaluated the effectiveness of the system. Accurate position keeping was achieved in the severe environmental conditions in the Kurushima Straits of Japan.

Anandamide (N-Arachidonoylethanolamine) amidohydrolase catalyzing hydrolysis of anandamide was characterized in mice. The enzymatic activity was highest in the liver, followed by the brain and testis. Negligible activity was found in heart, lung and spleen. The activity in brain and liver was mainly localized in the microsomal fractions. Kinetic experiments demonstrated that Km (μM) and Vmax (nmol/min/mg protein) for the brain microsomes were 9.3 and 2.58, respectively, while those for the hepatic microsomes were 180 and 18.9, respectively. The activity in the microsomes from the liver and brain was markedly inhibited by Cu2+, Hg2+, Se4+, phenylmethylsulfonylfluoride and sodium dodecylsulfate. Brain but not hepatic microsomal enzyme activity was inhibited by Δ9-tetrahydrocannabinol, cannabidiol and cannabinol. Kinetic parameters demonstrated that the inhibition by the cannabinoids was competitive in nature. Relatively high distribution of the enzyme activity in brain suggests an importance of the enzyme in the central nervous system to regulate the neuromodulatory fatty-acid amides.

Guinea pig hepatic enzyme, microsomal alcohol oxygenase, was able to oxidize both 7 alpha- and 7 beta-hydroxy-delta 8-tetrahydrocannabinol (7 alpha- and 7 beta-hydroxy-delta 8-THC) to 7-oxo-delta 8-THC. A cytochrome P450, named P450GPF-B, which mediates this oxidative metabolism was purified from hepatic microsomes of untreated female guinea pigs. The purified enzyme showed a single protein band of molecular mass 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequence of P450GPF-B is highly homologous with those of several cytochrome P450s belonging to the CYP3A subfamily. 18O derived from atmospheric oxygen was incorporated into 31 and 6%, respectively, of 7-oxo-delta 8-THC formed from 7 alpha- and 7 beta-hydroxy-delta 8-THC when the substrates were incubated with P450GPF-B under 18O2. The antibody against P450GPF-B significantly suppressed the oxidative activities of 7 alpha- and 7 beta-hydroxy-delta 8-THC to 7-oxo-delta 8-THC in hepatic microsomes of guinea pig. These results indicate that P450GPF-B is a major enzyme responsible for the hepatic microsomal alcohol oxygenase activities in the guinea pig.

ES46.5K, a novel esterase, was found in mouse hepatic microsomes. The enzyme catalyzed hydrolysis of 2-acetylaminofluorene and cannabinoid esters. In latter case, the enzyme regioselectively hydrolyzed acetates of 11-hydroxy-delta8-tetrahydrocannabinol. Western immunoblotting analysis demonstrated that none of immuno-reactive proteins against ES46.5K were present in hepatic microsomes from rats, guinea-pigs, monkeys and humans. Rabbit hepatic microsomes contained an immuno-reactive protein, although molecular weight of the protein was rather high (50 kDa) by SDS-PAGE. Esterase activity stained after PAGE demonstrated that ES46.5K retained at origin. Hepatic microsomes of above animal species contained several activity bands on the PAGE, while only mouse hepatic microsomes exhibited significant activity at origin. In mice, liver was only an organ containing ES46.5K by analyzing Western immunoblotting. These results indicate that distribution of ES46.5K is quite different from known carboxylesterases, and suggest that the enzyme has some role in the biotransformation of xenobiotic amide and esters.

Fallout decay simulation experiments were conducted by exposing potted plants of three different clones of Tradescantia heterozygous for flower color to gamma rays, reducing both the dose and dose rate daily (simulated to decrease to a half after 8, 2 or 1 days). Two stable (KU 27 and KU 9) and one mutable (KU 20) clones were used. Daily changes of the induced somatic pink mutation frequency in the stamen hairs observed in such fallout decay simulation experiments were compared with those obtained after single-dose treatments or treatments with simply fractionated doses at fixed dose rates. As compared with single-dose treatments, the treatments with simply fractionated doses resulted in lower peak values and longer peak periods of mutation frequency as expected. In the fallout decay simulation experiments, higher peak values and earlier and shorter peak periods of mutation frequency were observed than after treating with simply fractionated doses also as expected. Although the spontaneous mutation frequencies determined in clone KU 20 were very much higher than those in clones KU 27 and KU 9, gamma-ray-induced mutation frequencies per unit dose in these three clones were comparable, suggesting different mechanisms of initiation and/or repair of spontaneous and gamma-ray-induced mutations.

Scoring of spontaneous somatic pink mutation frequency in the stamen hairs of Tradescantia clone KU 9, a heterozygote for flower color (blue/pink; the blue color being dominant), was carried out for 11 years on plants grown outdoors, during the period of May 11-31 (for 3 weeks) in every year from 1982 to 1992. Weekly and yearly variations of the spontaneous mutation frequency were observed, and such variations could mostly be correlated to the difference in temperature. That is, the mutation frequency was generally higher in the weeks and years when the temperature was relatively low, showing the strongest negative correlation with the average minimum temperature. The variations were also correlated to the diurnal temperature difference, the mutation frequency being higher with larger diurnal temperature difference in general. However, the mutation frequency observed in 1986 was exceptionally higher than that expected from the temperature for this year, and was very significantly higher than for other years. The scoring of mutation frequency was thus continued in 1986 for an additional 4 weeks (June 1-28), and it was confirmed that such higher mutation frequencies lasted for 6 weeks in total. The exceptionally high mutation frequency seemed to be related to the radioactive fallout which occurred in early to mid May of 1986, even in Japan, after the serious nuclear reactor accident at Chernobyl, and also to the biological concentrations of radioactive nuclides which subsequently occurred, although it was difficult to conclude this definitely. The mutation frequency in 1987 was second highest, and was also significantly higher than the lowest mutation frequency observed in 1990.

This review describes recent advances in the metabolism of cannabinoids. Cannabidiol was metabolized to cannabielsoin, 6 beta-hydroxymethyl-delta 9-tetrahydrocannabinol and an oxepine derivative through epoxide intermediates by hepatic microsomal enzymes containing cytochrome P450 of animals. Cannabidiol inactivated cytochrome P450 UT-2 (CYP2C11) not equal to in male rats and a member of 3A subfamily in mouse liver. These inactivations may be very important because serious drug-drug interactions will occur in the case that cannabidiol is co-administered with drugs which are metabolized mainly by the enzyme system containing these P450 isozymes. A member of cytochrome P450 belonging to 2C subfamily was the major isozymes responsible for the cannabinoid metabolism in many experimental animals and that of 3A subfamily made some contribution to the metabolism of cannabinoids by human hepatic microsomes. Microsomal aldehyde oxygenase, a particular isozyme of cytochrome P450 catalyzing the oxidation of 11-oxo-tetrahydrocannabinol to tetrahydrocannabinol-11-oic acid, was found for the first time by the authors. Cytochrome P450 MUT-2 (CYP2C29) is the major isozyme responsible for the microsomal aldehyde oxygenase activity in mouse hepatic microsomes.

The most abundant metabolite of delta 9-tetrahydrocannabinol (delta 9-THC) formed with hepatic microsomes of monkeys was 11-OH-delta 9-THC, followed by 8 alpha-OH-, 8 beta-OH- and 3'-OH-delta 9-THCs. Two cytochrome P450 isozymes, P450RM-A and P450JM-C, were purified from monkey hepatic microsomes and found to have a molecular weight of 51,000. In the reconstituted system, the activities of P450RM-A towards formation of 11-OH-, 8 alpha-OH-, 8 beta-OH- and 3'-OH-delta 9-THCs were 19-, 40-, 22- and 10-fold higher, respectively, than the corresponding activities of the hepatic microsomes. The activity of P450JM-C towards formation of 3'-OH-delta 9-THC was 10-fold higher than that of P450RM-A, while the activities of both isozymes for 11- and 8 alpha-hydroxylation were not so much different and the 8 beta-hydroxylation activity was 14-fold higher in P450RM-A than in P450JM-C. Antibodies against P450RM-A and P450JM-C markedly inhibited the microsomal formation of 11-OH- and 8 alpha-OH-delta 9-THCs, and 3'-OH-delta 9-THC, respectively. These results suggest that P450RM-A and P450JM-C are major isozymes responsible for the formation of 11-OH- and 8 alpha-OH-delta 9-THCs, and 3'-OH-delta 9-THC, respectively, in monkeys.

Addition of saturated and α, β-unsaturated aliphatic aldehydes (C 8 to C 11 ) significantly increased NADPH oxidation with mouse hepatic microsomes, and the aldehydes themselves were oxidized to the corresponding carboxylic acids. When these aldehyde substrates were incubated similarly under oxygen-18 gas and the carboxylic acids formed were analyzed by GC-MS after methylation, it was indicated that oxygen-18 was significantly incorporated into the carboxylic acids formed from α,β-unsaturated aldehydes, but not significantly into the carboxylic acids formed from saturated aldehydes. These results indicate that enzyme and/or mechanism responsible for the oxidation of these two types of aldehydes is different from each other.

A protein (designated as protein-B) was purified from liver microsomes of adult male guinea pigs by an affinity chromatography with omega-aminooctyl Sepharose 4B, followed by HPLC using DEAE-5PW and hydroxyapatite columns which had been used to purify a cytochrome P450 (P450) isozyme (P450-A) from the same subcellular fraction (Narimatsu et al., Biochem Biophys Res Commun 172: 607-613, 1990). Protein-B had a molecular mass of 49 kDa in SDS-PAGE, but did not show absorbance at 417 nm for heme. Further, it did not show any oxidative activities towards aniline (AN), d-benzphetamine (d-BP), p-nitroanisole (p-NA) or delta 9-tetrahydrocannabinol (delta 9-THC) in a reconstituted system including dilauroylphosphatidylcholine, NADPH-P450 reductase, and cytochrome b5. However, antiserum against protein-B raised in rabbits suppressed liver microsomal oxidative activities towards d-BP and p-NA dose-dependently. The antibody decreased delta 9-THC oxidative activity most effectively, but did not decrease AN hydroxylation activity. Antiserum against P450-A suppressed all the activities towards these four substrates, especially towards delta 9-THC, in liver microsomes of male guinea pigs. Moreover, reconstitution with hemin made it possible for protein-B to produce some oxidative activity toward delta 9-THC. These results suggest that protein-B is also a cytochrome P450 isozyme which has lost a heme moiety during purification steps. Both P450-A and protein-B could have a role as cytochrome P450 isozymes in the oxidative metabolism of drugs, especially that of delta 9-THC by the liver microsomes of adult male guinea pigs.

Cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyse the oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) to delta 8-THC-11-oic acid in the presence of NADPH, cytochrome P450 reductase and dilauroylphosphatidylcholine. The catalytic activities (nmol/min/nmol P450) of cytochrome P450s, UT-2 (IIC11), UT-4 (IIA2), UT-5 (IIC13), PB-1, PB-2 (IIC6), PB-4 (IIB1), MC-1 (IA2), MC-5 (IA1) and IF-3 (IIA1), were 0.69, 0.08, 0.07, 0.23, 0.46, 0.02, 0.06, 0.07 and 0.34, respectively, whereas the activities of cytochrome P450s, PB-5 (IIB2) and DM (IIE1), were less than 0.02 nmol/min/nmol P450. Cytochrome P450 IIC11 showed the highest catalytic activity of the cytochromes examined. The mechanism for the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid by cytochrome P450 IIC11 was established as being an oxygenation since one atom of oxygen-18 was exclusively incorporated into the carboxylic acid formed under 18O2. The antibody raised to cytochrome P450 IIC11 inhibited by 60% the hepatic microsomal oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in male rats. These results indicate that cytochrome P450 IIC11 is a major form of the cytochrome to catalyse the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in the hepatic microsomes of male rats and that the oxidation of aldehyde to carboxylic acid is a catalytic activity common to most isozymes of P450.

Pyrolysates of cyanocobalamin, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, and ascorbic acid were tested for mutagenicity in the histidine-requiring mutants Salmonella typhimurium TA98 and TA100. Each vitamin was sealed in a glass tube and heated at 100–600°C in a muffle furnace. Methanol-chloroform extracts of the pyrolysate of each vitamin tested did not show any mutagenicity in either TA98 or TA100 without rat liver 9000 × mg supernatant fraction (S9) added. In the presence of S9, the B-group vitamins (cyanocobalamin, thiamine hydrochloride, riboflavin, and pyridoxine hydrochloride) were all mutagenic in TA98 and TA100, with the highest activity among the vitamins tested found in the pyrolysate of cyanocobalamin. The pyrolysate of 0.25 μmole cyanocobalamin produced 3200 revertants, while the pyrolysates of 0.25 μmole thiamine hydrochloride and riboflavin produced only 910 revertants, and the pyrolysate of pyridoxine hydrochloride did not show any mutagenicity at that amount. The mutagenicity was generally more active to TA98 than to TA100, indicating that frameshift-type mutagens were contained in the pyrolysates. The pyrolysate of ascorbic acid did not show any mutagenic activity in either TA98 or TA100 under the present experimental conditions.

Hepatic microsomal oxygenation of aldehydes to carboxylic acids was investigated. Aldehydes (veratrum aldehyde, cinnamic aldehyde, myrtenal, cuminaldehyde, 3-phenylpropionaldehyde, perillaldehyde and 9-anthraldehyde) were incubated with hepatic microsomes of mice in the presence of an NADPH-generating system under 18O2 (97 atom%). The incorporation of oxygen-18 into carboxylic acids formed was determined by gas chromatography-mass spectrometry. Oxygen-18 was incorporated into the carboxylic acids formed from all aldehyde substrates examined. Hepatic microsomal formation of 3,4-dimethoxybenzoic acid and cumic acid from veratrum aldehyde and cuminaldehyde, respectively, was inhibited by CO and SKF 525-A. These results indicate that the oxygenation of aldehydes which may be catalyzed by cytochrome P450 is a common reaction in the biotransformation of xenobiotic aldehydes.

A cDNA clone encoding a cytochrome P-450 (P-450) isozyme was isolated from a mouse liver cDNA library. This P-450, designated CYP2C29, the first member of the mouse CYP2C subfamily to be reported, contained the complete coding region of 490 amino acid residues. The deduced amino acid sequence of CYP2C29 exhibited 83% identity with that of the rat CYP2C7 and had an N-terminal sequence identical to that of P-450 MUT-2, a microsomal aldehyde oxygenase. Two peptides, derived from BrCN cleavage of P-450 MUT-2, were also identical to the cDNA deduced protein of CYP2C29. These results indicate that CYP2C29 cDNA encodes P-450 MUT-2.

The oxygenation of an aldehyde, 11-oxo-delta 8-tetrahydrocannabinol to a carboxylic acid, delta 8-tetrahydrocannabinol-11-oic acid was catalyzed by cytochrome P-450 MUT-2 purified from hepatic microsomes of male ddN mice. The oxygenation mechanism was confirmed by the incorporation of oxygen-18 from molecular oxygen into the carboxylic acid formed. An aldehyde form but not a hydrated form of 11-oxo-delta 8-tetrahydrocannabinol may be a substrate for the cytochrome P-450. The oxygenation of aldehyde catalyzed by cytochrome P-450 might be a common metabolic reaction in biological systems, and should be considered as an additional role of cytochrome P-450 in biotransformation of endogenous compounds and xenobiotics.

Thyroid peroxidase (TPO) plays a central role in thyroid gland function. The enzyme catalyzes two important reactions of thyroid hormone synthesis, i.e., the iodination of tyrosine residues in thyroglobulin and phenoxy-ester formation between pairs of iodinated tyrosines to generate the thyroid hormones, thyroxine and triiodothyronine. Previously, we isolated the cDNAs encoding human and mouse TPOs and assigned the human TPO gene to the short arm of chromosome 2 by somatic cell hybrid mapping. By a similar analysis of DNA from somatic cell hybrids, the human TPO gene was mapped to 2pter-p12. The mouse TPO gene was localized to chromosome 12 using a rat TPO cDNA as a probe to hybridize with mouse-hamster somatic cell hybrids. In this study, we used fluorescence in situ hybridization (FISH) to confirm the localization of human and mouse TPO genes to human chromosome 2 and mouse chromosome 12 and to assign them regionally to 2p25 and 12C, respectively. 7 refs., 1 fig.

The pharmacological effect in mice of N 3 -benzylthymidine (N 3 -ByTd) was examined by two routes of administration; intravenous (i.v.) and intracerebroventricular (i.c.v.), and compared with the effect of administration of N 3 -benzyluridine (N 3 -ByUd) previously reported. Hypnotic activity, pentobarbital (PB)-induced sleeping time, motor incoordination and spontaneous activity were used as indices of pharmacological effects. N 3 -ByTd (0.5–2.0 μmol/mouse, (i.c.v.) and N 3 -ByUd (1.5–3.0 μmol/mouse, i.c.v.) were found to possess dose-dependent hypnotic activity, and N 3 -ByTd had more potent hypnotic activity than N 3 -ByUD. Both N 3 -ByTd and N 3 -ByUd (0.5 and 1.0 μmol/mouse, i.c.v., respectively) showed a synergistic effect on PB-induced sleep, although their parent compounds, thymidine (Td) and uridine (Ud), did not potentiate the activity at each dose. In motor incoordination, the effect on N 3 -ByTd (0.5 μmol/mouse) continued for 6 hr after i.c.v. injection. All compounds decreased the spontaneous activity of mice by i.c.v. administration. Furthermore, both N 3 -ByTd and N 3 -ByUd decreased the activity, when they were administered by i.v. These results reveal that both N 3 -benzylpyramidine nucleosides have more direct depressant effects on the central nervous system (CNS) than the parent compounds. Among the pyrimidine nucleoside derivatives tested, N 3 -ByTd was found to be the most potent hypnotic substance.

N,N′-Diallylpentobarbital (DAPB) antagonized barbital (B)-induced sleep in mice and rats. DAPB [80 mg/kg, intraperitoneal (i.p.)] reduced the barbital (350 mg/kg, i.p.)-induced sleeping time to 40% of the control in mice. Twenty, 40 and 80 mg/kg, i.p. of DAPB reduced barbital-induced sleeping time when administered 60 min prior to injection of barbital. DAPB (80 mg/kg, i.p.) also shortened barbital (250 mg/kg, i.p.)-induced sleeping time to about 70% of the control in rats. The results indicates that DAPB is an antagonist against hypnotic activity of barbital.

Rabbit liver microsomes were found to catalyze oxidation of 11-hydroxy-Δ 8 -tetrahydrocannabinol to 11-oxo-Δ 8 -tetrahydrocannabinol. This enzyme reaction required NADPH and molecular oxygen, and it was partially inhibited by CO. Pyrazole, potassium cyanide and sodium azide showed no effect on this oxidation, but SKF-525 A caused a significant inhibition. Thus, it is concluded that this enzymatic reaction is mediated by a mixed function oxidase involving cytochrome P-450.

The effects of delta 8-tetrahydrocannabinol (delta 8-THC) and its major and active metabolite, 11-hydroxy-delta 8-tetrahydrocannabinol (11-OH-delta 8-THC), on the hepatic microsomal drug-metabolizing enzyme system were studied in mice. The repeated administration of 11-OH-delta 8-THC (5 mg/kg/day, i.v.) for 3 or 7 days increased significantly the activities of aniline hydroxylase and p-nitroanisole O-demethylase. By the same treatment, cytochrome P-450 content (3 days) or NADPH-cytochrome c reductase activity (7 days) was also increased significantly. The treatment with delta 8-THC for 7 days (5 mg/kg/day, i.v.) significantly increased aniline hydroxylase only. 11-OH-delta 8-THC increased the Vmax, but not the Km, values for both drug-metabolizing enzymes, whereas delta 8-THC decreases significantly the Km value (270 microM) for p-nitroanisole O-demethylase as compared with the control (398 microM). Repeated administration of these cannabinoids for 7 days also increased the metabolism of delta 8-THC by hepatic microsomes; this was attributed to an enhanced formation of 11-OH-delta 8-THC. In contrast, microsomal formation of 7 alpha-OH-delta 8-THC was decreased significantly by treatment with delta 8-THC. 11-OH-delta 8-THC, but not delta 8-THC, treatment increased the metabolism of 11-OH-delta 8-THC by hepatic microsomes. These findings indicate that delta 8-THC and 11-OH-delta 8-THC treatment can induce hepatic microsomal drug-metabolizing enzymes and affect differently the catalytic properties of the enzymes.

Cannabidiol induced a carbon monoxide-like complex with mouse hepatic microsomal cytochrome P-450 during NADPH-dependent metabolism in vitro on a spectral basis. The reduction by dithionite was required for the maximal development of a spectrum. The complex showed a peak at 450 nm which shifted to 419 or 423 nm, respectively, by further addition of hemoglobin or myoglobin. Cannabidiol-induced complex formation required molecular oxygen, and was decreased by the addition of inhibitors of cytochrome P-450-dependent monooxygenase. Pretreatment of mice with phenobarbital (80 or 100 mg/kg, i.p. for 3 days) but not 3-methylcholanthrene (80 mg/kg, i.p.) increased the complex formation. In contrast, pretreatment with cobaltous chloride (40 mg/kg, i.p. for 3 days) decreased the complex formation. 8,9-Dihydro- and 1,2,8,9-tetrahydrocannabidiols also induced the same spectrum as that of above complex, whereas cannabidiol monomethyl- and dimethylethers reduced this ability. In addition, both cannabidivarin and cannabigerol induced the complex formation, although Δ9-tetrahydrocannabinol, cannabinol and cannabielsoin did not. Olivetol but not d-limonene induced the spectrum of the complex to some extent. These results indicate that cannabidiol induces a carbon monoxide-like complex with cytochrome P-450 during hepatic microsomal metabolism, and suggest that phenobarbital-inducible cytochrome P-450s mediate at least one of the metabolic steps of CBD to form the complex, as well as the importance of the resorcinol moiety of CBD for the complex formation.

Brain microsomes of mice, rats, guinea pigs and rabbits catalyzed the oxidation of delta 8- and delta 9-tetrahydrocannabinol to their monohydroxylated metabolites. The most prominent metabolite was the 4'-hydroxylated metabolite on the pentyl side chain of the cannabinoids in all species tested, except that the 5'-hydroxylation of delta 9-tetrahydrocannabinol was most abundant in the guinea pig. These results are quite different from the metabolic profile of the cannabinoids with hepatic microsomes.

delta 8-Tetrahydrocannabinol (THC)- and 8 beta, 9 beta-epoxyhexahydrocannabinol (EHHC)-tolerant mice were tolerant to the hypothermia produced by morphine while 8 alpha, 9 alpha-EHHC-tolerant mice were not. Morphine-tolerant mice acquired partial tolerance to the hypothermic effect of 8 alpha,9 alpha-EHHC, but not to the effect of delta 8-THC and 8 beta,9 beta-EHHC. Cataleptogenic effects of all cannabinoids were enhanced in morphine-tolerant mice as compared to non-tolerant animals. It seems that morphine-tolerant mice exhibited hypersensitivity to the cataleptogenic effect of the cannabinoids without potentiation of the hypothermia. These results suggest that the morphine-tolerant mouse may acquire 'latent' cross-tolerance to the hypothermic effect of the cannabinoids.

8α,9α-Epoxyhexahydrocannabinol ( EHHC ) was formed from Δ 8 -tetrahydrocannabinol ( THC ) by mouse liver microsomes. The reaction required O 2 , and partially inhibited by CO. The optimal pH for the epoxide formation was from 7.4 to 8.0. EDTA did not affect the epoxide formation, but SKF 525-A, α-naphthoflavone and CCl 4 caused a significant inhibition. In addition, the rate of epoxidation increased significantly after treatment. with phenobarbital and 3-methylcholanthrene, but decreased after CoCl 2 treatment. 8β,9β-EHHC, a stereoisomer of 8α,9α-EHHC, was not found under all the conditions used in this study. These results indicate that 8α,9α-EHHC formation is mediated by monooxygenase system involving cytochrome P-450.

In this paper, an attempt is made to estimate the air pollutant concentration at an arbitrary site from observed concentrations at several fixed monitoring stations. The Group Method of Data Handling (GMDH) is applied to this problem. A numerical example based on actual data is presented.