Transcriptome profiling reveals the molecular processes for survival of Lysinibacillus fusiformis strain 15-4 in petroleum environments.

A bacterial strain designated Lysinibacillus fusiformis 15-4 was isolated from oil-free soil on the Qinghai-Tibet Plateau, which can grow well utilizing petroleum hydrocarbons as a carbon source at a lower temperature. To deeply characterize the molecular adaptations and metabolic processes of this strain when grown in a petroleum-containing environment, transcriptome analysis was performed. A total of 4664 genes and the expression of 3969 genes were observed in strain 15-4. When the strain was grown in petroleum-containing medium, 2192 genes were significantly regulated, of which 1312 (60%) were upregulated and 880 (40%) were downregulated. This strain degraded and adapted to petroleum via modulation of diverse molecular processes, including improvements in transporter activity, oxidoreductase/dehydrogenase activity, two-component system/signal transduction, transcriptional regulation, fatty acid catabolism, amino acid metabolism, and environmental stress responses. Many strain-specific genes were involved in the oxidation of hydrocarbon compounds, such as several luciferase family alkane monooxygenase genes, flavin-utilizing monooxygenase family genes, and flavoprotein-like family alkanesulfonate monooxygenase genes. Several cold shock protein genes were also induced suggesting adaptation to cold environments and the potential for petroleum degradation at low temperatures. The results obtained in this study may broaden our understanding of molecular adaptation of bacteria to hydrocarbon-containing environments and may provide valuable data for further study of L. fusiformis.

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

Endocrine sensitivity of estrogen receptor-positive breast cancer is negatively correlated with aspartate-ß-hydroxylase expression.

Although prognostic markers for early estrogen receptor (ER)-positive breast cancer have been extensively developed, predictive markers for adjuvant endocrine therapy are still lacking. Focusing on the mechanisms underlying endocrine resistance, we investigated whether the endocrine sensitivity of ER-positive breast cancer cells was correlated with the expression of aspartate-ß-hydroxylase (ASPH), which is involved in the development of hepatocellular carcinoma. ASPH expression in ER-positive and tamoxifen-resistant breast cancer cells was upregulated by the MAPK and phosphoinositide-3 kinase (PI3K) pathways, which both play pivotal roles in endocrine resistance. In the clinical setting, ASPH expression was negatively correlated with recurrence-free survival of luminal B breast cancer patients that received adjuvant endocrine therapy, but not in patients that did not receive adjuvant endocrine therapy. Luminal B breast cancer is one of the intrinsic molecular subtypes identified by the Prediction Analysis of Microarray 50 (PAM50) multiple gene classifier, and because of its poor response to endocrine therapy, chemotherapy in addition to endocrine therapy is generally required after surgical resection. Our results suggest that the endocrine sensitivity of luminal B breast cancer can be assessed by examining ASPH expression, which promotes the consideration of a prospective study on the association between ASPH expression at the mRNA and protein levels in luminal B breast cancer and subsequent response to endocrine therapy.

Molecular cloning of melatonin 3-hydroxylase and its production of cyclic 3-hydroxymelatonin in rice (Oryza sativa).

Melatonin is metabolized in animals to cyclic 3-hydroxymelatonin (3-OHM) not by an enzymatic pathway, but by interaction with hydroxyl radicals. The production of 3-OHM in animals suggests the possible presence of 3-OHM in plants. Prior to the identification of 3-OHM in plants, we directly cloned the corresponding gene(s) responsible for 3-OHM synthesis using Escherichia coli library strains expressing genes belonging to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily from rice. Three of 35 E. coli library strains supplemented with 1 mmol/L melatonin were found to produce 3-OHM in their extracellular medium, suggestive of three 2-ODD genes involved in 3-OHM production. The purified recombinant 2-ODD 11, 2-ODD 26, and 2-ODD 33 proteins were shown to catalyze the metabolism of melatonin to 3-OHM, with 2-ODD 11 showing the highest melatonin 3-hydroxylase (M3H) catalytic activity. Consistent with the presence of M3H genes, rice leaves supplemented with 5 mmol/L melatonin produced 3-OHM [233 µg/g fresh weight (FW)], 2-hydroxymelatonin (21 µg/g FW), and N1 -acetyl-N2 -formyl-5-methoxykynuramine (5 µg/g FW). Three M3H transcripts were induced upon the treatment of rice leaves with cadmium followed by an increase in M3H enzyme activity. Cloning of M3H genes in plants has paved the way for the studies of melatonin in plants in terms of its multiple physiological roles.

Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification.

The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)-B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5-7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5-2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.

Cloning and characterization of a novel ß-carotene hydroxylase gene from Lycium barbarum and its expression in Escherichia coli.

Lycium barbarum contains high levels of zeaxanthin, which is produced by the conversion of ß-carotene into zeaxanthin. ß-Carotene hydroxylase catalyzes this reaction. We cloned a cDNA (chyb) encoding ß-carotene hydroxylase (Chyb) from the L. barbarum leaf. A 939-bp full-length cDNA sequence was determined with 3'-rapid amplification of cDNA end assay encoding a deduced Chyb protein (34.8 kDa) with a theoretical isoelectric point of 8.36. A bioinformatics analysis showed that the L. barbarum Chyb was located in the chloroplast. Further, to investigate the catalytic activity of the L. barbarum Chyb, a complementation analysis was conducted in Escherichia coli. The results strongly demonstrated that Chyb can catalyze ß-carotene to produce zeaxanthin. Thus, this study suggests that L. barbarum ß-carotene hydroxylase could be a means of zeaxanthin production by genetic manipulation in E. coli.

Subcellular/tissue distribution and responses to oil exposure of the cytochrome P450-dependent monooxygenase system and glutathione S-transferase in freshwater prawns (Macrobrachium malcolmsonii, M. lamarrei lamarrei).

Subcellular fractions (mitochondrial, cytosolic and microsomal) prepared from the tissues (hepatopancreas, muscle and gill) of freshwater prawns Macrobrachium malcolmsonii and Macrobrachium lamarrei lamarrei were scrutinized to investigate the presence of mixed function oxygenase (MFO) and conjugating enzymes (glutathione-S-transferase, GST). Cytochrome P450 (CYP) and other components (cytochrome b(5); NADPH-cytochrome c (CYP) reductase and NADH-cytochrome c-reductase activities) of the MFO system were predominantly present in the hepatic microsomal fraction of M. malcolmsonii and M. lamarrei lamarrei. The results are in agreement with the notion that monooxygenase system is mainly membrane bound in the endoplasmic reticulum, and that the hepatopancreas is the major metabolic tissue for production of biotransformation enzymes in crustaceans. Further, the prawns were exposed to two sublethal (0.9 ppt (parts per thousand) and 2.3 ppt) concentrations of oil effluent. At the end of 30th day, hydrocarbons and detoxifying enzymes were analysed in the hepatopancreas. The accumulations of hydrocarbon in the tissues gradually increased when exposed to sublethal concentrations of oil effluent and were associated with significantly enhanced levels of cytochrome P450 (180.6+/-6.34 pmol mg(-1) protein (P<0.05 versus control, 136.5+/-7.1 pmol mg(-1) protein) for 2.3 ppt and 305.6+/-8.5 pmol mg(-1) protein (P<0.001 versus control, 132.3+/-6.8 pmol mg(-1) protein] for 0.9 ppt of oil exposed M. malcolmsonii; 150+/-6.5 pmol mg(-1 )protein (P<0.01 versus control, 84.6+/-5.2 pmol mg(-1) protein) for 2.3 ppt and 175+/-5.5 pmol mg(-1) protein (P<0.01 versus control, 87.6+/-5.4 pmol mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei), NADPH cytochrome c-reductase activity (14.7+/-0.6 nmol min(-1 )mg(-1) protein (P<0.05 versus control, 6.8+/-0.55 nmol min(-1 )mg(-1) protein) for 2.3 ppt and 12.1+/-0.45 nmol min(-1 )mg(-1) protein (P<0.01 versus control, 6.9+/-0.42 nmol min(-1 )mg(-1) protein) for 0.9 ppt of oil exposed M. malcolmsonii; 12.5+/-0.31 nmol min(-1 )mg(-1) protein (P<0.001 versus control, 4.6+/-0.45 nmol min(-1 )mg(-1) protein) for 2.3 ppt and 9.6+/-0.32 nmol min(-1 )mg(-1) protein (P<0.01 versus control, 4.9+/-0.41 nmol min(-1 )mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei) and cytochrome b(5 )(124.8+/-3.73 pmol mg(-1) protein (P<0.01 versus control, 76.8+/-4.2 pmol mg(-1) protein) for 2.3 ppt and 115.3+/-3.86 pmol mg(-1) protein (P<0.01 versus control, 76.4+/-4.25 pmol mg(-1 )protein) for 0.9 ppt of oil exposed M. malcolmsonii and 110+/-3.11 pmol mg(-1) protein (P<0.01 versus control, 63.7+/-3.24 pmol mg(-1 )protein) for 2.3 ppt and 95.3+/-2.63 pmol mg(-1) protein (P<0.01 versus control, 61.4+/-2.82 pmol mg(-1) protein) for 0.9 ppt of oil exposed M. lamarrei lamarrei). The enhanced levels of biotransformation enzymes in oil-exposed prawns demonstrate a well-established detoxifying mechanism in crustaceans, and the response offers the possibility of use as a biomarker for the early detection of oil pollution.

Time-dependent induction of midazolam-1-hydroxylation enzymes in rats treated with St. John's wort.

Time-dependent effects of St. John's wort (SJW) on midazolam 1-hydroxylation were investigated in Wistar rats. Wistar rats treated with SJW (1000 mg/kg/d) for 1, 3, and 7 d were administered midazolam orally at a dose of 10 mg/kg. Oral clearance of midazolam in the SJW treated rats increased time dependently, and was significant after 7 d of treatment with SJW. The midazoram-1-hydroxylation activity in liver microsomes obtained from the SJW treated rats was significantly higher than in the control group. Linear correlation was observed between oral clearance and midazolam-1-hydroxylation activity in the liver microsomes, suggesting that CYP3A induction in liver mainly decreased the midazolam concentration in plasma. Immunoblotting revealed that the protein amount of CYP3A was induced within 3 d of SJW treatment. Since the midazolam-1-hydroxylation activity continuously increased for at least 7 d, the induction of CYP3A by SJW continued to cause interactions with drugs metabolized by CYP3A. It is important for persons receiving SJW for an extended time to consider its interactions with prescription drugs.

Fatal seizures due to potential herb-drug interactions with Ginkgo biloba.

Alternative therapy including herbal drugs and complementary medicine is becoming increasingly popular. However, the rise in the incidence of herb-drug interactions is causing concern, especially in the absence of warning labels addressing potential adverse effects. We present the case of a 55-year-old male who suffered a fatal breakthrough seizure, with no evidence of non-compliance with his anticonvulsant medications. The autopsy report revealed subtherapeutic serum levels for both anticonvulsants Depakote and Dilantin. Concomitant with his prescribed medications, the decedent was also self-medicating with a cornucopia of herbal supplements and nutraceuticals, prominent among which was Ginkgo biloba. Ginkgo, an herbal extract from the leaves of the Ginkgo biloba tree, has been used medicinally for centuries and has been touted as a cure for a variety of medical conditions. The induction of Cytochrome P450 enzymes by components of herbal drugs has been known to affect the metabolism of various drugs. Dilantin is primarily metabolized by CYP2C9, and secondarily metabolized by CYP2C19. Valproate metabolism is also modulated in part by CYP2C9 and CYP2C19. A recent study revealed significant inductive effect of ginkgo on CYP2C19 activity. CYP2C19 induction by ginkgo could be a plausible explanation for the subtherapeutic levels of Dilantin and Depakote. Additionally, ginkgo nuts contain a potent neurotoxin, which is known to induce seizure activity. Evidence of other herbal drugs diminishing the efficacy of anticonvulsant medication does exist; however, there has been only one other documented instance of ginkgo potentiating seizure activity in the presence of anticonvulsant therapy. Highlighting the potential adverse effects and drug interactions of ginkgo on the packaging of the drug may help prevent inadvertent use in vulnerable individuals.

CYP2U1, a novel human thymus- and brain-specific cytochrome P450, catalyzes omega- and (omega-1)-hydroxylation of fatty acids.

Long chain fatty acids have recently emerged as critical signaling molecules in neuronal, cardiovascular, and renal processes, yet little is presently known about the precise mechanisms controlling their tissue distribution and bioactivation. We have identified a novel cytochrome P450, CYP2U1, which may play an important role in modulating the arachidonic acid signaling pathway. Northern blot and real-time PCR analysis demonstrated that CYP2U1 transcripts were most abundant in the thymus and the brain (cerebellum), indicating a specific physiological role for CYP2U1 in these tissues. Recombinant human CYP2U1 protein, expressed in baculovirus-infected Sf9 insect cells, was found to metabolize arachidonic acid exclusively to two region-specific products as determined by liquid chromatography-mass spectrometry. These metabolites were identified as 19- and 20-hydroxy-modified arachidonic acids by liquid chromatography-tandem mass spectrometry analysis. In addition to omega/omega-1 hydroxylation of arachidonic acid, CYP2U1 protein also catalyzed the hydroxylation of structurally related long chain fatty acid (docosahexaenoic acid) but not fatty acids such as lauric acid or linoleic acid. This is the first report of the cloning and functional expression of a new human member of P450 family 2, CYP2U1, which metabolizes long chain fatty acids. Based on the ability of CYP2U1 to generate bioactive eicosanoid derivatives, we postulate that CYP2U1 plays an important physiological role in fatty acid signaling processes in both cerebellum and thymus.

Metabolism of human cytochrome P450 marker substrates in mouse: a strain and gender comparison.

The aim was to characterize mouse gender and strain differences in the metabolism of commonly used human cytochrome (CYP) P450 probe substrates. Thirteen human CYP probe substrates (phenacetin, coumarin, 7-ethoxy-4-trifluoromethyl coumarin, amiodarone, paclitaxel, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, chlorzoxazone, p-nitrophenol, testosterone and lauric acid) were used in activity measurements. The metabolism of the probe substrates was compared in liver microsomes from male and female NMRI, CBA, C57bl/6, 129/SvJ and CD1 strains. The expression of proteins identified on Western blots with commonly available antibodies selective for specific human and rat CYP enzymes were compared in the different mouse strains. Males had higher metabolism than corresponding females for phenacetin O-deethylation (human marker for CYP1A2 activity), and a high correlation was found between phenacetin activity and immunoreactivity in Western blots produced with rat CYP1A2 antibodies. Protein detected by antibodies cross-reacting with human CYP2B6 and rat CYP2B1/2 antibodies was female specific except for the 129/SvJ strain, where it was absent in both genders. Females generally had a higher metabolism of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation (human markers for CYP2D activity). Bufuralol 1'-hydroxylation correlated with a female-dominant mouse CYP, which was detected with antibodies against rat CYP2D4. p-Nitrophenol 2-hydroxylation correlated better than chlorzoxazone 6-hydroxylation with the protein detected with antibodies against rat CYP2E1, indicating that p-nitrophenol is a more specific substrate for mouse CYP2E1.

cDNAs for the synthesis of cyclic carotenoids in petals of Gentiana lutea and their regulation during flower development.

cDNAs encoding lycopene epsilon -cyclase, lycopene beta-cyclase, beta-carotene hydroxylase and zeaxanthin epoxidase were isolated from a Gentiana lutea petal cDNA library. The function of all cDNAs was analyzed by complementation in Escherichia coli. Transcript levels during different stages of flower development of G. lutea were determined and compared to the carotenoid composition. Expression of all genes increased by a factor of up to 2, with the exception of the lycopene epsilon -cyclase gene. The transcript amount of the latter was strongly decreased. These results indicate that during flower development, carotenoid formation is enhanced. Moreover, metabolites are shifted away from the biosynthetic branch to lutein and are channeled into beta-carotene and derivatives.

Induction of liver monooxygenases by annatto and bixin in female rats.

Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'). Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and beta-Apo-8'-carotenal) are known to be potent inducers of CYP1A1, a property not shared by others (beta-carotene, lycopene and lutein). Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28% bixin) and bixin (95% pure) on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight), bixin (250 mg/kg body weight) or the vehicle only (corn oil, 3.75 g/kg body weight) for 5 consecutive days, or were not treated (untreated control). The activities of aniline-4-hydroxylase (A4H), ethoxycoumarin-O-deethylase (ECOD), ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD) and benzyloxy- (BROD) resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg) induced EROD (3.8x), MROD (4.2x), BROD (3.3x) and PROD (2.4x). Bixin (250 mg/kg) was a weaker inducer of EROD (2.7x), MROD (2.3x) and BROD (1.9x) and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings.

Induction of liver monooxygenases by annatto and bixin in female rats

Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'). Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and á-Apo-8'-carotenal) are known to be potent inducers of CYP1A1, a property not shared by others (á-carotene, lycopene and lutein). Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28 percent bixin) and bixin (95 percent pure) on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight), bixin (250 mg/kg body weight) or the vehicle only (corn oil, 3.75 g/kg body weight) for 5 consecutive days, or were not treated (untreated control). The activities of aniline-4-hydroxylase (A4H), ethoxycoumarin-O-deethylase (ECOD), ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD) and benzyloxy- (BROD) resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg) induced EROD (3.8x), MROD (4.2x), BROD (3.3x) and PROD (2.4x). Bixin (250 mg/kg) was a weaker inducer of EROD (2.7x), MROD (2.3x) and BROD (1.9x) and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings

Alkane biodegradation in Pseudomonas aeruginosa strains isolated from a polluted zone: identification of alkB and alkB-related genes.

Pseudomonas aeruginosa strains that grow on crude oil as the sole source of carbon and energy were isolated from an environment in Morocco polluted by petroleum refinery effluents. The twenty isolates grew on saturated alkanes from C12 to C22. Three of the isolates were also able to grow on low molecular weight C6 to C10 n-alkanes, but the other 17 strains were not. The strains were tested for alkB and a/kB-related genes encoding alkane-1-monooxygenase (alkane hydroxylase). Oligonucleotide primers specific for the alkB gene of strain P. putida (GPo1 ) and for the alkB1 and alkB2 genes of P. aeruginosa strain PAO1 allowed amplification from the P. aeruginosa isolates of fragments similar to alkB1 and alkB2 genes of strain PAO1. Only 3 strains carried an alkB gene very similar to that of strain GPo1, and these strains were the same ones that could utilise C6 to C10 n-alkanes.

Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy.

California poppy (Eschscholzia californica Cham.) cell cultures produce several benzophenanthridine alkaloids, such as sanguinarine, chelirubine, and macarpine, with potent pharmacological activity. Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3'-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures. Transformed cell lines expressing antisense BBE or antisense CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared with control cultures transformed with a beta-glucuronidase gene. Pathway intermediates were not detected in any of the transformed cell lines. The suppression of benzophenanthridine alkaloid biosynthesis using BBE or CYP80B1 antisense RNA constructs also reduced the growth rate of the cultures. Two-dimensional (1)H-nuclear magnetic resonance and in vivo (15)N-nuclear magnetic resonance spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic cell lines. However, transformed cells with reduced benzophenanthridine alkaloid levels contained larger cellular pools of several amino acids including alanine, leucine, phenylalanine, threonine, and valine compared with controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was less than 2-fold higher in antisense-suppressed cells relative to controls. These results show that alterations in the metabolic flux through benzophenanthridine alkaloid biosynthesis can affect the regulation of amino acid pools. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways.

Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells.

The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated. Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1-2 microM) in plastic flasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow. Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 microM 1alpha,25-dihydroxyvitamin D(3) to promote cellular CYP3A4 expression. Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213 +/- 54.35%) compared to that of parent cells (100 +/- 6.05%). Furthermore, they retained significantly less [(3)H]vincristine sulphate (p < 0.05), a P-gp substrate, after efflux (272.89 +/- 11.86 fmol/mg protein) than the parent cells (381.39 +/- 61.82 fmol/mg protein). The expression of CYP3A4 in parental cells after 1alpha,25-dihydroxyvitamin D(3) treatment was quantified to be 76.2 +/- 7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0 +/- 20.0 pmol/mg protein). Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting. Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0 +/- 20.0 to 1057.0 +/- 46.0 pmol/min/mg protein. Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells. The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics.

Cost-effective whole-cell assay for laboratory evolution of hydroxylases in Escherichia coli.

Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and longchain fatty acids at the omega-1, omega-2, and omega-3 positions. A continuous spectrophotometric assay for P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycarboxylic acids and the chromophore p-nitrophenolate was reported recently. However, this pNCA assay procedure contained steps that limited its application in high throughput screening, including expression of P450 BM-3 variant F87A in 4-ml cultures, centrifugation, resuspension of the cell pellet, and cell lysis. We have shown that permeabilization of the outer membrane of Escherichia coli DH5alpha with polymyxin B sulfate, EDTA, polyethylenimine, or sodium hexametaphosphate results in rapid conversion of 12-pNCA. A NADPH-generating system consisting of NADP(+), D/L-isocitric acid, and the D/L-isocitrate dehydrogenase of E. coli DH5alpha reduced the cofactor expense more than 10-fold. By avoiding cell lysis, resuspension, and centrifugation, the high throughput protocol allows screening of thousands of samples per day.

Regulation of phenobarbital-mediated induction of CYP102 (cytochrome P450(BM-3)) in Bacillus megaterium by phytochemicals from soy and green tea.

Cytochrome P450 102 (CYP102 or Cytochrome P450(BM)(-)(3)) is induced in Bacillus megaterium by barbiturates, perioxisome proliferators, estrogen, and nonsteroidal antiinflammatory drugs. We have previously demonstrated that a CYP102 construct (BMC 143) coupled with a luciferase reporter gene can be used to identify the inducers of CYP102. We now describe the effect of added phytochemicals on the induction of CYP102 by phenobarbital (PB) in B. megaterium. The isoflavones genistein, biochanin A, coumestrol, and equol, the green tea flavanoid epicatechin, and the fungal toxin zearalenone inhibit the induction of CYP102 by PB in a dose-dependent manner. However, the isoflavone daidzein, the phytoalexin glyceollin, and catechin, an epimer of epicatechin, failed to exhibit a similar inhibitory effect on PB-mediated CYP102 induction.