Polydatin alleviated alcoholic liver injury in zebrafish larvae through ameliorating lipid metabolism and oxidative stress.

Hepatic steatosis is the early stage of alcoholic liver disease (ALD), may progress to steatohepatitis, fibrosis even cirrhosis. Polydatin, the primary active component of Polygonum cuspidatum Sieb. et Zucc, has been recognized to possess hepatoprotective and anti-inflammatory properties. To investigate whether polydatin alleviates ethanol induced liver injury and to elucidate the underlying molecular mechanisms, zebrafish larvae at 4 days post-fertilization (dpf) were exposed to 350 mmol/L of ethanol for 32 h, then treated with polydatin for 48 h. Oil red O, Nile Red and H&E staining were used to analyze the pathological changes in liver. The mRNA levels were measured by quantitative PCR and the antioxidant capacity was detected using H2O2-specific fluorescent probe. Here, polydatin strongly alleviated hepatic steatosis and decreased the expression levels of alcohol and lipid metabolism-related genes, including CYP2Y3, CYP3A65, HMGCRa, HMGCRb and FASN. Additionally, polydatin inhibited oxidative stress in the liver according to fluorescent probe. Moreover, significantly up-regulated expression of DNA damage-related genes (CHOP, GADD45αa) revealed that polydatin attenuated hepatic apoptosis in larvae. In conclusion, polydatin may improve the liver function of zebrafish with acute alcoholic liver injury through attenuating hepatic fat accumulation, ameliorating lipid and ethanol metabolism and reducing oxidative stress and DNA damage.

Inhibitory action of Epilobium hirsutum extract and its constituent ellagic acid on drug-metabolizing enzymes.

Epilobium hirsutum (EH) is a medicinal plant for treating various diseases. Despite its wide usage, there is no available information about its potential influences on drug metabolism. The present study was undertaken to determine the in vivo effects of EH on hepatic CYP2B, CYP2C, CYP2D, and CYP3A enzymes that are primarily involved in drug metabolism. Male Wistar rats were injected intraperitoneally with EH water extract (EHWE) and ellagic acid (EA) at a daily dose of 37.5 and 20 mg/kg, respectively, for 9 days and hepatic drug-metabolizing enzymes were assessed at activity, protein and mRNA levels. Erythromycin N-demethylase activity was inhibited by 53 and 21 % in EHWE- and EA-treated rats, respectively. Benzphetamine N-demethylase and 7-benzyloxyresorufin-O-debenzylase activities were decreased by 53 and 43 %, and 57 and 57 % in EHWE-and EA-treated rats, respectively. Moreover, protein levels of CYP2B1, CYP2C6, CYP2D2, and CYP3A1 also decreased by 55, 15, 33, and 82 % as a result of EHWE treatment of rats, respectively. Similarly, CYP2B1, CYP2C6, CYP2D2, and CYP3A1 protein levels decreased by 62, 63, 49, and 37 % with EA treatment, respectively. qRT-PCR analyses also showed that mRNA levels of these enzymes were significantly inhibited with bothEHWE and EA treatments. In conclusion, inhibition of drug clearances leading to drug toxicity because of the lowered activity and expression of drug-metabolizing enzymes might be observed in the people who used EH as complementary herbal remedy that might be contributed by its EA content.

Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction.

UNLABELLED: The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a 'gatekeeper' amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60,000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the 'oxyanion hole' in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle. DATABASE: Structural data are available in PDB database under the accession numbers 4PVE, 4PVH, 4PVJ, 4PVK, 4PWB, 4PWC and 4PZF.

Expression of endogenous retroviruses is negatively regulated by the pluripotency marker Rex1/Zfp42.

Rex1/Zfp42 is a Yy1-related zinc-finger protein whose expression is frequently used to identify pluripotent stem cells. We show that depletion of Rex1 levels notably affected self-renewal of mouse embryonic stem (ES) cells in clonal assays, in the absence of evident differences in expression of marker genes for pluripotency or differentiation. By contrast, marked differences in expression of several endogenous retroviral elements (ERVs) were evident upon Rex1 depletion. We demonstrate association of REX1 to specific elements in chromatin-immunoprecipitation assays, most strongly to muERV-L and to a lower extent to IAP and musD elements. Rex1 regulates muERV-L expression in vivo, as we show altered levels upon transient gain-and-loss of Rex1 function in pre-implantation embryos. We also find REX1 can associate with the lysine-demethylase LSD1/KDM1A, suggesting they act in concert. Similar to REX1 binding to retrotransposable elements (REs) in ES cells, we also detected binding of the REX1 related proteins YY1 and YY2 to REs, although the binding preferences of the two proteins were slightly different. Altogether, we show that Rex1 regulates ERV expression in mouse ES cells and during pre-implantation development and suggest that Rex1 and its relatives have evolved as regulators of endogenous retroviral transcription.

An investigation of the bioactivation potential and metabolism profile of Zebrafish versus human.

The zebrafish model has been increasingly explored as an alternative model for toxicity screening of pharmaceutical drugs. However, little is understood about the bioactivation of drug to reactive metabolite and phase I and II metabolism of chemical in zebrafish as compared with human. The primary aim of our study was to establish the bioactivation potential of zebrafish using acetaminophen as a probe substrate. Our secondary aim was to perform metabolite profiling experiments on testosterone, a CYP3A probe substrate, in zebrafish and compare the metabolite profiles with that of human. The glutathione trapping assay of N-acetyl-p-benzoquinone imine demonstrated that zebrafish generates the same reactive metabolite as humans from the bioactivation of acetaminophen. Zebrafish possesses functional CYP3A4/5-like and UDP-glucuronosyltransferase metabolic activities on testosterone. Differential testosterone metabolism was observed among the two species. In silico docking studies suggested that the zebrafish CYP3A65 was responsible for the bioactivation of acetaminophen and phase I hydroxylation of testosterone. Our findings reinforce the need to further characterize the drug metabolism phenotype of zebrafish before the model can fully achieve its potential as an alternative toxicity screening model in drug research.

Cytochrome P450-dependent drug oxidation activity of liver microsomes from Microminipigs, a possible new animal model for humans in non-clinical studies.

SUMMARY: Small minipigs (Bland name, Micromini Pig; registered as a novel variety of pig in the Japanese Ministry of Agriculture, Forestry and Fisheries) were developed with the aim of non-clinical pharmacological/toxicological use. They were principally mated with<10 kg body weight at 7 months-old resulting in good handling. Cytochrome P450 (P450)-and flavin-containing monooxygenases (FMO)-dependent drug oxidation activity of liver microsomes prepared from male Microminipigs (8 months-old) was compared with that for pooled dogs, monkeys, and humans. High P450 2D-dependent bufuralol 1'-hydroxylation and FMO-dependent benzydamine N-oxygenation activity was observed in liver microsomes from Microminipigs. Typical P450 1A, 2B, 2C, 2E, and 3A-dependent drug oxidation activity was also seen in Microminipigs. However, occasional differences might give undetected low P450 2A-dependent coumarin 7-hydroxylation in Microminipigs at 8-months-old, in contrast to liver microsomes from one 10-days-old Microminipis and commercially available pooled minipigs which had low but detectable coumarin 7-hydroxylation activity. The present results suggest that there is some overlap in Microminipig and human P450 substrate specificity. These findings should provide important information for greater understanding of drug metabolism in Microminipigs, as an experimental animal model for non-clinical use.

Inhibition of human CYP2B6-catalyzed bupropion hydroxylation by Ginkgo biloba extract: effect of terpene trilactones and flavonols.

Cytochrome P450 2B6 (CYP2B6) is expressed predominantly in human liver. It catalyzes the oxidative biotransformation of various drugs, including bupropion, which is an antidepressant and a tobacco use cessation agent. Serious adverse effects of high dosages of bupropion have been reported, including the onset of seizure. As Ginkgo biloba extract may be consumed with bupropion or another CYP2B6 drug substrate, potential exists for an herb-drug interaction. Therefore, we investigated the effect of G. biloba extract and some of its chemical constituents (terpene trilactones and flavonols) on the in vitro catalytic activity of CYP2B6 as assessed by the bupropion hydroxylation assay with recombinant enzyme and hepatic microsomes. The amount of hydroxybupropion was quantified by a novel and validated ultraperformance liquid chromatography/mass spectrometry method. Enzyme kinetic analysis indicated that G. biloba extract competitively inhibited hepatic microsomal CYP2B6-catalyzed bupropion hydroxylation (apparent K(i) was 162 +/- 14 microg/ml). Bilobalide and ginkgolides A, B, C, and J were not responsible for the inhibition of CYP2B6 catalytic activity by the extract. Whereas the 3-O-glucoside and 3-O-rutinoside of quercetin, kaempferol, and isorhamnetin had no effect, the corresponding aglycones (10 and 50 microg/ml) decreased hepatic microsomal bupropion hydroxylation. The inhibition of CYP2B6 by kaempferol was competitive (apparent K(i) was 10 +/- 1 microg/ml). In summary, G. biloba extract and its flavonol aglycones are naturally occurring inhibitors of in vitro CYP2B6 catalytic activity and bupropion hydroxylation. Future studies are needed to investigate whether G. biloba extract interacts in vivo with bupropion or other clinically important CYP2B6 drug substrates.

Sequence-independent construction of ordered combinatorial libraries with predefined crossover points.

Combinatorial libraries coding for mosaic enzymes with predefined crossover points constitute useful tools to address and model structure-function relationships and for functional optimization of enzymes based on multivariate statistics. The presented method, called sequence-independent generation of a chimera-ordered library (SIGNAL), allows easy shuffling of any predefined amino acid segment between two or more proteins. This method is particularly well adapted to the exchange of protein structural modules. The procedure could also be well suited to generate ordered combinatorial libraries independent of sequence similarities in a robotized manner. Sequence segments to be recombined are first extracted by PCR from a single-stranded template coding for an enzyme of interest using a biotin-avidin-based method. This technique allows the reduction of parental template contamination in the final library. Specific PCR primers allow amplification of two complementary mosaic DNA fragments, overlapping in the region to be exchanged. Fragments are finally reassembled using a fusion PCR. The process is illustrated via the construction of a set of mosaic CYP2B enzymes using this highly modular approach.

The phytoestrogen coumestrol is a naturally occurring antagonist of the human pregnane X receptor.

Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.

Effect of Ginkgo biloba extract on oxidative metabolism of valproic acid in hepatic microsomes from donors with the CYP2C9*1/*1 genotype.

We investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the oxidative metabolism of valproic acid (VPA) in hepatic microsomes from donors with the CYP2C9*1/*1 genotype. G. biloba extract decreased 4-ene-VPA, 3-OH-VPA, 4-OH-VPA, and 5-OH-VPA formation with mean (+/- SE) IC50 values of 340 +/- 40 microg/mL, 370 +/- 100 microg/mL, 180 +/- 30 microg/mL, and 210 +/- 20 microg/mL, respectively. This was associated with inhibition of not only CYP2C9*1, but also CYP2A6 and CYP2B6. Bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, and isorhamnetin-3-O-rutinoside were not responsible for the inhibition of VPA metabolism by the extract. When analyzed as the sum of the aglycone and total glycosides present in the extract, quercetin decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 76%, 51%, and 70%, respectively, kaempferol decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 65%, 46%, and 49%, respectively, and isorhamnetin decreased 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation by 29%, 26%, and 31%, respectively. The 3 aglycones did not affect 3-OH-VPA formation. In summary, G. biloba extract decreased hepatic microsomal formation of 4-ene-VPA, 4-OH-VPA, 5-OH-VPA, and 3-OH-VPA, but the effect was not due to the terpene trilactones or flavonol glycosides investigated in our study.

Characterization of histone lysine-specific demethylase in relation to thyroid hormone-regulated anuran metamorphosis.

The thyroid hormone receptor (THR) is a member of the nuclear transcription factor and plays a central role in regulating anuran metamorphosis. Previous studies with mammalian cells have suggested that THR is involved in chromatin remodeling through histone methylation. In the present study, we cloned cDNA of lysine-specific demethylase gene, xLSD1, from Xenopus laevis and examined its expression in relation to metamorphosis. Overexpression of xLSD1 in A6 cells, a Xenopus laevis cell line, resulted in the decrease of methylation status of lysine residues of histone H3, indicating that the protein of cloned xLSD1 was functionally active. The expression of LSD1 at mRNA levels was up-regulated in the body skin and the intestine during natural and thyroid hormone-induced metamorphosis. Larval epidermal basal cells and intestinal epithelial cells at the premetamorphic stage were identified as the xLSD1-expressing cells. At the metamorphic climax stage the progenitor cells of adult epidermal basal cells also expressed xLSD1, whereas those of the adult intestinal epithelial cells did not. We propose that LSD1 participates in the regulation of metamorphosis through THR- or another transcriptional factor-induced chromatin remodeling.

Stereoselective block of hERG channel by (S)-methadone and QT interval prolongation in CYP2B6 slow metabolizers.

Methadone inhibits the cardiac potassium channel hERG and can cause a prolonged QT interval. Methadone is chiral but its therapeutic activity is mainly due to (R)-methadone. Whole-cell patch-clamp experiments using cells expressing hERG showed that (S)-methadone blocked the hERG current 3.5-fold more potently than (R)-methadone (IC50s (half-maximal inhibitory concentrations) at 37 degrees C: 2 and 7 microM). As CYP2B6 slow metabolizer (SM) status results in a reduced ability to metabolize (S)-methadone, electrocardiograms, CYP2B6 genotypes, and (R)- and (S)-methadone plasma concentrations were obtained for 179 patients receiving (R,S)-methadone. The mean heart-rate-corrected QT (QTc) was higher in CYP2B6 SMs (*6/*6 genotype; 439+/-25 ms; n=11) than in extensive metabolizers (non *6/*6; 421+/-25 ms; n=168; P=0.017). CYP2B6 SM status was associated with an increased risk of prolonged QTc (odds ratio=4.5, 95% confidence interval=1.2-17.7; P=0.03). This study reports the first genetic factor implicated in methadone metabolism that may increase the risk of cardiac arrhythmias and sudden death. This risk could be reduced by the administration of (R)-methadone.

Identification of histone demethylases in Saccharomyces cerevisiae.

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.

The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases.

Histone methylation regulates chromatin structure and transcription. The recently identified histone demethylase lysine-specific demethylase 1 (LSD1) is chemically restricted to demethylation of only mono- and di- but not trimethylated histone H3 lysine 4 (H3K4me3). We show that the X-linked mental retardation (XLMR) gene SMCX (JARID1C), which encodes a JmjC-domain protein, reversed H3K4me3 to di- and mono- but not unmethylated products. Other SMCX family members, including SMCY, RBP2, and PLU-1, also demethylated H3K4me3. SMCX bound H3K9me3 via its N-terminal PHD (plant homeodomain) finger, which may help coordinate H3K4 demethylation and H3K9 methylation in transcriptional repression. Significantly, several XLMR-patient point mutations reduced SMCX demethylase activity and binding to H3K9me3 peptides, respectively. Importantly, studies in zebrafish and primary mammalian neurons demonstrated a role for SMCX in neuronal survival and dendritic development and a link to the demethylase activity. Our findings thus identify a family of H3K4me3 demethylases and uncover a critical link between histone modifications and XLMR.

Contribution of CYP2C9, CYP2A6, and CYP2B6 to valproic acid metabolism in hepatic microsomes from individuals with the CYP2C9*1/*1 genotype.

The present study investigated the role of specific human cytochrome P450 (CYP) enzymes in the in vitro metabolism of valproic acid (VPA) by a complementary approach that used individual cDNA-expressed CYP enzymes, chemical inhibitors of specific CYP enzymes, CYP-specific inhibitory monoclonal antibodies (MAbs), individual human hepatic microsomes, and correlational analysis. cDNA-expressed CYP2C9*1, CYP2A6, and CYP2B6 were the most active catalysts of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation. The extent of 4-OH-VPA and 5-OH-VPA formation by CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B was only 1-8% of the levels by CYP2C9*1. CYP2A6 was the most active in catalyzing VPA 3-hydroxylation, whereas CYP1A1, CYP2B6, CYP4F2, and CYP4F3B were less active. Correlational analyses of VPA metabolism with CYP enzyme-selective activities suggested a potential role for hepatic microsomal CYP2A6 and CYP2C9. Chemical inhibition experiments with coumarin (CYP2A6 inhibitor), triethylenethiophosphoramide (CYP2B6 inhibitor), and sulfaphenazole (CYP2C9 inhibitor) and immunoinhibition experiments (including combinatorial analysis) with MAb-2A6, MAb-2B6, and MAb-2C9 indicated that the CYP2C9 inhibitors reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by 75-80% in a panel of hepatic microsomes from donors with the CYP2C9*1/*1 genotype, whereas the CYP2A6 and CYP2B6 inhibitors had a small effect. Only the CYP2A6 inhibitors reduced VPA 3-hydroxylation (by approximately 50%). The extent of inhibition correlated with the catalytic capacity of these enzymes in each microsome sample. Overall, our novel findings indicate that in human hepatic microsomes, CYP2C9*1 is the predominant catalyst in the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA, whereas CYP2A6 contributes partially to 3-OH-VPA formation.

Rat PXR reporter-gene activity correlates with the induction of CYP3A in rat precision-cut liver slices.

Recent studies have suggested that both constitutive androstane receptor (CAR) and pregnane X-receptor (PXR) are involved in the induction of rat liver microsomal cytochrome P-450 (CYP) 2B and 3A through a mechanism called cross-talk. In this study we intend to determine if a PXR-reporter gene assay could be used for the prediction of CYP3A and/or CYP2B induction in rats. The induction of rat CYP2B and CYP3A by nineteen structurally diverse compounds was evaluated by using rat precision-cut liver slices and a rat PXR reporter-gene system. Induction of CYP2B and CYP3A mRNAs in rat liver slices was quantified by real-time polymerase chain reaction. Rat PXR activation was measured by induction of luciferase activity in rat PXR reporter-gene system. Linear regression analysis of the fold of induction of mRNA in liver slices and the fold of luciferase activity in rat PXR reporter-gene system shows that a reasonable correlation (r2 = 0.6) exists between the CYP3A induction and the rat PXR activation. A much lower correlation was observed between CYP2B induction and the rat PXR activation (r2 = 0.1). The results from this study suggest that the PXR may play a major role in the induction of rat CYP3A, but not CYP2B. Therefore, the PXR-reporter gene assay may be useful in a high-throughput screening to predict CYP3A induction in rats.

Analysis of the inhibitory potential of Ginkgo biloba, Echinacea purpurea, and Serenoa repens on the metabolic activity of cytochrome P450 3A4, 2D6, and 2C9.

OBJECTIVE: To study the potential of three top-selling herbal products, Ginkgo biloba, Echinacea purpurea, and Serenoa repens to inhibit the in vitro enzymatic activity of three of the most important drug metabolizing enzymes, cytochrome P450 (CYP) 3A4, 2D6, and 2C9. METHODS: High throughput CYP inhibition screening was used to test the inhibitory capacity of extracts of commercially available herbal medications on the metabolism of CYP enzyme substrates. RESULTS: S. repens showed potent inhibition of the metabolic activity of all three CYPs tested. The effects of G. biloba and E. purpurea varied. E. purpurea demonstrated mild inhibition of CYP3A4 activity with 7- benzyloxy-4-trifluoromethylcoumarin (BFC) as the model substrate, but mild inducing effects in the presence of the model substrate resorufin benzyl ether (BzRes). Little effect on CYP2D6 and moderate inhibition of CYP2C9 was seen with both E. purpurea and G. biloba. G. biloba also showed mild-to-moderate inhibition of CYP3A4 depending on the model substrate. CONCLUSIONS: The inhibitory capacity of herbal products varies depending on the concentrations of the model substrate and the herbal extract, as well as the identity of the model substrate, as demonstrated by the varied effects of CYP3A4 enzymatic activity with different model substrates. The potential for strong adverse interactions exists for Serenoa repens, which was a potent inhibitor of all three CYPs examined. Physicians are encouraged to advise patients of the risks of combining herbal products with prescription medications.

Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment.

Three key benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), were localized in cultured opium poppy (Papaver somniferum) cells by sucrose density gradient fractionation and immunogold labeling. CYP80B1 catalyzes the second to last step in the formation of (S)-reticuline, the last common intermediate in sanguinarine and morphine biosynthesis. BBE converts (S)-reticuline to (S)-scoulerine as the first committed step in sanguinarine biosynthesis, and COR catalyzes the penultimate step in the branch pathway leading to morphine. Sanguinarine is an antimicrobial alkaloid that accumulates in the vacuoles of cultured opium poppy cells in response to elicitor treatment, whereas the narcotic analgesic morphine, which is abundant in opium poppy plants, is not produced in cultured cells. CYP80B1 and BBE were rapidly induced to high levels in response to elicitor treatment. By contrast, COR levels were constitutive in the cell cultures, but remained low and were not induced by addition of the elicitor. Western blots performed on protein homogenates from elicitor-treated cells fractionated on a sucrose density gradient showed the cosedimentation of CYP80B1, BBE, and sanguinarine with calreticulin, and COR with glutathione S-transferase. Calreticulin and glutathione S-transferase are markers for the endoplasmic reticulum (ER) and the cytosol, respectively. In response to elicitor treatment, large dilated vesicles rapidly developed from the lamellar ER of control cells and fused with the central vacuole. Immunogold localization supported the association of CYP80B1 and BBE with ER vesicles, and COR with the cytosol in elicitor-treated cells. Our results show that benzylisoquinoline biosynthesis and transport to the vacuole are associated with the ER, which undergoes major ultrastructural modification in response to the elicitor treatment of cultured opium poppy cells.

Impact of African herbal medicines on antiretroviral metabolism.

We examined the effects of two African herbal medicines recommended for HIV/AIDS patients on antiretroviral metabolism. Extracts from Hypoxis and Sutherlandia showed significant effects on cytochrome P450 3A4 metabolism and activated the pregnane X receptor approximately twofold. P-glycoprotein expression was inhibited, with Hypoxis showing 42-51% and Sutherlandia showing 19-31% of activity compared with verapamil. Initiating policies to provide herbal medicines with antiretroviral agents may put patients at risk of treatment failure, viral resistance or drug toxicity.