Oxidation of 1,8-cineole, the monoterpene cyclic ether originated from eucalyptus polybractea, by cytochrome P450 3A enzymes in rat and human liver microsomes.
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1,8-Cineole, the monoterpene cyclic ether known as eucalyptol, is one of the components in essential oils from Eucalyptus polybractea. We investigated the metabolism of 1,8-cineole by liver microsomes of rats and humans and by recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human P450 and NADPH-P450 reductase cDNAs had been introduced. 1,8-Cineole was found to be oxidized at high rates to 2-exo-hydroxy-1,8-cineole by rat and human liver microsomal P450 enzymes. In rats, pregenolone-16alpha-carbonitrile (PCN) and phenobarbital induced the 1,8-cineole 2-hydroxylation activities by liver microsomes. Several lines of evidence suggested that CYP3A4 is a major enzyme involved in the oxidation of 1,8-cineole by human liver microsomes: (1), 1,8-cineole 2-hydroxylation activities by liver microsomes were inhibited very significantly by ketoconazole, a CYP3A inhibitor, and anti-CYP3A4 immunoglobulin G; (2), there was a good correlation between CYP3A4 contents and 1,8-cineole 2-hydroxylation activities in liver microsomes of eighteen human samples; and (3), of various recombinant human P450 enzymes examined, CYP3A4 had the highest activities for 1,8-cineole 2-hydroxylation; the rate catalyzed by CYP3A5 was about one-fourth of that catalyzed by CYP3A4. Kinetic analysis showed that K(m) and V(max) values for the oxidation of 1,8-cineole by liver microsomes of human sample HL-104 and rats treated with PCN were 50 microM and 91 nmol/min/nmol P450 and 20 microM and 12 nmol/min/nmol P450, respectively. The rates observed using human liver microsomes and recombinant CYP3A4 were very high among other CYP3A4 substrates reported so far. These results suggest that 1,8-cineole, a monoterpenoid present in nature, is one of the effective substrates for CYP3A enzymes in rat and human liver microsomes. (+info)
Requirement of the Lec35 gene for all known classes of monosaccharide-P-dolichol-dependent glycosyltransferase reactions in mammals.
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The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-beta-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate. (+info)
In vitro inhibition of cytochrome P450 enzymes in human liver microsomes by a potent CYP2A6 inhibitor, trans-2-phenylcyclopropylamine (tranylcypromine), and its nonamine analog, cyclopropylbenzene.
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Currently, there are no selective, well characterized inhibitors for CYP2A6. Therefore, the effects of trans-(+/-)-2-phenylcyclopropylamine (tranylcypromine), a potent CYP2A6 inhibitor, on human liver microsomal cytochromes P450 (CYP) were studied to elucidate its selectivity. The IC50 value of tranylcypromine in coumarin 7-hydroxylation (CYP2A6 model activity) was 0.42 +/- 0.07 microM and in chlorzoxazone 6-hydroxylation (CYP2E1 model activity) 3.0 +/- 1.1 microM. The IC50 values for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activities were >10 microM. Potency and selectivity of tranylcypromine were strongly dependent on the amine group, because its nonamine analog cyclopropylbenzene was much less potent inhibitor of CYP1A, CYP2A6, CYP2C19, and CYP2E1 activities and did not inhibit at all CYP2C9, CYP2D6, or CYP3A4 activities. In human liver microsomes tranylcypromine induced type II and cyclopropylbenzene type I difference spectrum. According to the double reciprocal analysis of these spectral responses both tranylcypromine and cyclopropylbenzene may have at least two P450-related binding sites in liver microsomes. The K(a) values of tranylcypromine varied from 4.5 to 15.1 microM and -34.3 to 167 microM in microsomes derived from three different livers and of cyclopropylbenzene from -1.6 to 10.1 microM and -34.6 and 75.2 microM in the same liver microsomes. Based on these results, tranylcypromine seems an adequately selective CYP2A6 inhibitor for in vitro use. (+info)
Simple synthesis of 5,9-dimethylated long-chain alkanes, the sex pheromones of leaf miner moths.
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Each stereoisomeric mixture of 5,9-dimethylpentadecane and 5,9-dimethylhexadecane, the major and the minor sex pheromone components of Perileucoptera coffeella, respectively, was synthesized in about 25% overall yield through 6 steps from beta-citronellol. 5,9-Dimethylheptadecane, the major sex pheromone component of Leucoptera malifoliella, was also synthesized analogously as a stereoisomeric mixture in a 22% overall yield. (+info)
Identification of a compound in Chamaecyparis taiwanensis inhibiting the ice-nucleating activity of Pseudomonas fluorescens KUIN-1.
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Inactivation of the ice-nucleating activity of Pseudomonas fluorescens KUIN-1 by compounds in the leaves from coniferous trees were investigated, and the inactivated material was identified. Intact cells of the strain KUIN-1 and the acetone or methanol extracts of leaves of various coniferous trees were allowed to react for 30 min at 18 degrees C. Antinucleation compounds were obtained from Chamaecyparis taiwanensis. When the acetone extract from the leaves of coniferous trees was added to the cell suspension (about 10(6) cells/ml) in 50 mM potassium phosphate buffer (pH 7.0), the ice nucleating temperature, T50, was significantly decreased (T50 < -5 degrees C). This inhibitor was isolated by using TLC, then identified as hinokitiol based on UV-VIS, IR, and mass spectral data. When intact cells of the strain KUIN-1 were incubated with hinokitiol, limonene, and alpha-pinene of the principal constituent of the leaves of coniferous trees in 50 mM potassium phosphate buffer (pH 7.0), the ice-nucleating activity decreased, but not in alpha-terpinene. Furthermore, the ice-nucleating activities from other ice-nucleating bacteria also decreased in the presence of hinokitiol. This inhibition was proportional to the concentration of hinokitinol. The pH and thermal stabilities of the ice-nucleating activity of the cells were changed by the addition of hinokitiol (10 mM). (+info)
Plant-derived oils reduce pathogens and gaseous emissions from stored cattle waste.
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Carvacrol and thymol in combination at 6.7 mM each completely inhibited the production of short-chain volatile fatty acids and lactate from cattle waste in anoxic flasks over 23 days. Fecal coliforms were reduced from 4.6 x 10(6) to 2.0 x 10(3) cells per ml 2 days after treatment and were nondetectable within 4 days. Total anaerobic bacteria were reduced from 8.4 x 10(10) to 1.5 x 10(7) cells per ml after 2 days and continued to be suppressed to that level after 14 days. If the concentration of carvacrol or thymol were doubled (13.3 mM), either could be used to obtain the same inhibitory fermentation effect. We conclude that carvacrol or thymol may be useful as an antimicrobial chemical to control pathogens and odor in stored livestock waste. (+info)
Defensive function of herbivore-induced plant volatile emissions in nature.
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Herbivore attack is known to increase the emission of volatiles, which attract predators to herbivore-damaged plants in the laboratory and agricultural systems. We quantified volatile emissions from Nicotiana attenuata plants growing in natural populations during attack by three species of leaf-feeding herbivores and mimicked the release of five commonly emitted volatiles individually. Three compounds (cis-3-hexen-1-ol, linalool, and cis-alpha-bergamotene) increased egg predation rates by a generalist predator; linalool and the complete blend decreased lepidopteran oviposition rates. As a consequence, a plant could reduce the number of herbivores by more than 90% by releasing volatiles. These results confirm that indirect defenses can operate in nature. (+info)
Cytotoxicity of the hinokitiol-related compounds, gamma-thujaplicin and beta-dolabrin.
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Gamma-thujaplicin and beta-dolabrin, the constituents of the wood of Thujopsis dolabrata Sieb. et Zucc. var. hondai showed strong in vitro cytotoxic effects against the human stomach cancer cell lines KATO-III and Ehrlich's ascites carcinoma. The cytotoxic effects of the two compounds against both tumor cell lines were clear when cell growth was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Gamma-thujaplicin and beta-dolabrin at 0.32 microg/ml inhibited cell growth of human stomach cancer KATO-III by 85 and 67%, and Ehrlich's ascites carcinoma by 91 and 75%, respectively. There is no large difference in cytotoxicity between these compounds, but the activity of gamma-thujaplicin was slightly more potent than that of beta-dolabrin. On the other hand, hinokitiol acetate did not show a cytotoxic effect, suggesting that at least a part of the mechanism of the cytotoxic effect of hinokitiol-related compounds is due to metal chelation between the carbonyl group at C-1 and the hydroxyl group at C-2 in the tropolone skeleton of these molecules. The acute toxicities [50% lethal dose (LD50) value: intraperitoneal injection, Van der Waedem] of gamma-thujaplicin and beta-dolabrin in mice were 277 mg/kg and 232 mg/kg, respectively. (+info)