We 80% have established the expression patterns of the genes encoding ATP-binding cassette (ABC) transporters and cytochromes P450 (CYPs) at the adult regulation human blood-brain barrier (BBB) using isolated brain microvessels and cortex biopsies from patients with epilepsia or glioma. Microves synaptophysin (neurons)in and neuron-glial antigen 2 (NG2)(pericytes). ABCG2 [breast cancer resistance protein (BCRP)] and ABCB1 (MDR1) were the main ABC transporter hydrocarbon genes expressed in microvessels, with 20 times more ABCG2 and 25 times more ABCB1 in microvessels than in the cortex.were The CYP1B1 isoform represented over 80% of all the CYPs genes detected in microvessels. There were 14 times more CYP1B1 (BCRP)] in microvessels than in the cortex, showing that CYP1B1 is mainly expressed at the BBB. p-glycoprotein (ABCB1), BCRP (ABCG2) and mainly CYP1B1 proteins were found in microvessels by western blotting. The expression of genes encoding three transcription factors [pregnane xenobiotic receptor cortex, (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR)] was also investigated. The AhR gene, involved in the regulation of microvessels CYP1B1 expression, was highly expressed in brain microvessels, whereas PXR and CAR genes were almost undetected. This detailed pattern of encoding ABC and CYPs gene expression at the human BBB provides useful information for understanding how their substrates enter the brain.ABCB1

The sequence pregnane X receptor (PXR) plays a crucial role in xenobiotic and drug metabolism, being the major transcriptional regulator of cytochrome pigs P-450 monooxygenase 3A4, which metabolizes more than 50% of all clinically used drugs. Recent pharmacodynamic studies have shown that the and mouse is not an ideal model for predicting human clinical drug study outcomes. Therefore, we characterized the porcine PXR (pPXR)RT-PCR gene to evaluate the utility of the pig as an alternate preclinical animal model. The complete sequence of pPXR mRNA characterized and 11 kb of genomic sequence were obtained. Similar to the human PXR gene, the pPXR gene revealed multiple splice study variants in the ligand-binding domain. All pPXR splice variants (SV) were porcine-specific. The pPXR mRNAs varied in 3'-UTR length due were to differential termination and specific deletions. Northern blot analyses identified high levels of pPXR mRNA expression in the liver, small All intestine, heart, kidney, and colon. RT-PCR amplification detected lower levels of pPXR expression in multiple tissues. Ninety-three pigs representing eight The breeds were analyzed for single nucleotide polymorphisms (SNPs). Only one nonsynonymous SNP (S178L) was found in the pPXR ligand-binding domain.role This characterization of the pPXR gene contributes to the development of a porcine model for human drug metabolic studies.

OBJECTIVE:after The effect of cigarette smoking on CYP2C9 activity is unknown. We conducted a study to evaluate whether there is a extent difference in CYP2C9 activity in smokers versus non-smokers by examining S-warfarin AUC after CYP2C9 inhibition with fluvastatin. In addition, the periods effect of the CYP2C9 inhibitor fluvastatin was evaluated using S-warfarin as a probe. METHODS: A randomized, single dose, two-treatment crossover weak study of warfarin with a washout period of 21 days was performed. Eighteen healthy Caucasian smokers and non-smokers, genotyped as period CYP2C9*1/*1 or CYP2C9*1/*2, received warfarin 10 mg plus vitamin K 10 mg to measure baseline CYP2C9 activity. Warfarin dosing was of repeated after 18 days of fluvastatin 40 mg twice daily to evaluate CYP2C9 activity after inhibition. RESULTS: The S-warfarin [Formula:inhibition. see text] between smokers and non-smokers did not differ by >25% after inhibition. There was no difference in S-warfarin [Formula:did see text] during baseline (p= .45) or inhibition (p= .19) periods for smokers versus non-smokers. Fluvastatin increased the AUC of S-warfarin by K 42+/-29% and 26+/-18% in smokers and nonsmokers, respectively. Linear regression analyses showed significant but weak correlations between peak concentrations (C(at unknown. 1 h)) or (-) 3S,5R-fluvastatin AUC( -12 h) and extent of warfarin inhibition. For (+) 3R,5S-fluvastatin, a weak correlation was found plus between C(at 1 h) and extent of warfarin inhibition. CONCLUSIONS: Cigarette smoking does not affect CYP2C9 activity as evaluated using or S-warfarin as a CYP2C9 probe. Fluvastatin is a weak inhibitor of CYP2C9 activity in both smokers and non-smokers.

RATIONALE:In Increases in depressive symptoms during smoking cessation have been associated with risk for relapse. Several studies have linked plasma levels in of cortisol and dehydroepiandrosterone (DHEA) or DHEA-sulfate (DHEAS) to depressive symptoms. OBJECTIVES: To determine whether changes in plasma cortisol, DHEA,8 or DHEAS levels and emergence of depressive symptoms during smoking cessation are associated with smoking relapse. MATERIALS AND METHODS: Subjects ratio were healthy non-medicated men and women, aged 39+/-12 years, who smoked, on average, 22 cigarettes per day. Depressive symptoms, smoking who withdrawal symptoms, and plasma steroid levels were measured before and after 8 days of verified smoking abstinence. Relapse status at and day 15 was then determined. RESULTS: In the full sample (n=63), there was a trend for changes in depressive symptoms a to be associated with relapse. In the subset of 25 subjects with plasma neuroactive steroid data, there was a significant plasma interaction between the change in the plasma DHEA/cortisol ratio from day to day 8 and relapse status at day of 15. This ratio was similar before abstinence, but lower at day 8 in relapsed, compared to abstinent, subjects. Changes in with the DHEA/cortisol ratio tended to predict changes in depressive symptoms in the women only. CONCLUSION: A decrease in the plasma days DHEA/cortisol ratio during 8 days of smoking abstinence was associated with relapse over the following week. Further research is needed to fully characterize sex-specific relationships between abstinence-induced changes in neuroactive steroid levels, depressive or withdrawal symptoms, and relapse. Such research neuroactive may lead to new interventions for refractory smoking dependence.

OBJECTIVE:DNA Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of over 50% of all drugs currently in use. However, CYP3A4 in expression shows a large inter-individual variation that cannot only be explained by genetic polymorphisms identified in this gene. The pregnane were X receptor (PXR) has been identified as a transcriptional regulator of CYP3A4. Single nucleotide polymorphisms (SNPs) in the PXR gene of could influence PXR activity and thereby CYP3A4 expression. This study was therefore aimed at determining the frequencies of known SNPs influence and detecting yet unknown SNPs in the PXR gene in a Dutch population. METHODS: Genomic DNA was isolated from blood the samples obtained from 100 healthy volunteers and subjected to PCR amplification, followed by DNA sequencing. The population, of which the Caucasian, ethnicity was 93% Caucasian, consisted of 79 female individuals and 21 males. RESULTS: A total of 24 SNPs were found which in the PXR gene, eight of which are previously unknown. The allelic frequencies found in this population varied from .5 unknown to 73%. Most of the previously detected SNPs were located in introns. One new SNP, T8555G in exon 8, causes of an amino acid change of C379G and is located in the Ligand Binding Domain of PXR. CONCLUSION: Several SNPs were yet detected in the PXR gene, one of which is located in the ligand binding domain (LBD). These SNPs may influence 24 PXR-mediated CYP3A4 induction.

Niemann-Pick of C disease (NPC) is an irreversible neurodegenerative disorder without current treatment. It is thought to result from deficient intracellular cholesterol every and/or ganglioside trafficking. We have investigated the effects of allopregnanolone treatments on survival, weight loss, motor function, magnetic resonance imaging mice. (MRI), and neuropathology in the mouse model of NPC (Npc1(-/-) mice). We confirmed previous results showing that a single injection survival of 250 mug of allopregnanolone on postnatal day 7 significantly extended the life span of Npc1(-/-) mice. This caused a 7 marked difference in the weight curves of the treated mice but no statistical difference in the Rota-Rod performance. T2-weighted MRI of and diffusion tensor imaging (DTI) of treated mice showed values of signal intensity and fractional anisotropy closer to those of reaction wild-type mice than those of untreated Npc1(-/-) mice. Neuropathology showed that day-7 treatment markedly suppressed astrocyte reaction and significantly reduced that microglial activation. Furthermore, the steroid treatment also increased myelination in brains of Npc1(-/-) mice. Similar effects of allopregnanolone treatment were statistical observed in Npc1(-/-), mdr1a(-/-) double-mutant mice, which have a deficient blood-brain barrier, resulting in increased steroid uptake. The effects on treatment. survival and weight loss of a single injection on day 7 followed by injections every 2 weeks were also evaluated but in Npc1(-/-) mice, and the beneficial effects were found to be greater than with the single injection at day 7.brains We conclude that allopregnanolone treatment significantly ameliorates several symptoms of NPC in Npc1(-/-) mice, presumably by effects on myelination or day-7 neuronal connectivity.(c) 2005 Wiley-Liss, Inc.

BACKGROUND:the The identification of promoter regions that are regulated by a given transcription factor has traditionally relied upon the identification and strategy. distributions of binding sites recognized by the factor. In this study, we have developed a tandem machine learning approach for method the identification of regulatory target genes based on these parameters and on the corresponding binding site information contents that measure regions) the affinities of the factor for these cognate elements. RESULTS: This method has been validated using models of DNA binding of sites recognized by the xenobiotic-sensitive nuclear receptor, PXR/RXRalpha, for target genes within the human genome. An information theory-based weight matrix has was first derived and refined from known PXR/RXRalpha binding sites. The promoter region of candidate genes was scanned with the classification weight matrix. A novel information density-based clustering algorithm was then used to identify clusters of information rich sites. Finally, transformed clustering data representing metrics of location, strength and clustering of binding sites were used for classification of promoter regions using an refined ensemble approach involving neural networks, decision trees and Naïve Bayesian classification. The method was evaluated on a set of 24 transcription known target genes and 288 genes known not to be regulated by PXR/RXRalpha. We report an average accuracy (proportion of derived correctly classified promoter regions) of 71%, sensitivity of 73%, and specificity of 70%, based on multiple cross-validation and the leave-one-out Bayesian strategy. The performance on a test set of 13 genes showed that 10 were correctly classified. CONCLUSION: We have developed binding a machine learning approach for the successful detection of gene targets for transcription factors with high accuracy. The method has to been validated for the transcription factor PXR/RXRalpha and has the potential to be extended to other transcription factors.

The base constitutive androstane receptor (CAR) NR1I3 is a transcription factor that upon activation by xenobiotics induces transcription of drug-metabolizing and drug a transporter genes. Our goal was to identify whether alternative splicing of CAR makes an important contribution to the generation of site) novel CAR proteins. The wild-type CAR mRNA (CAR.1) and splice variants (SVs) were amplified from human liver cDNAs and in Transcripts a panel of cDNAs from 36 human tissues, using exon 1- and 3'-untranslated region primers, cloned and sequenced. Twenty-two unique exon hCAR splice variants (CAR-SVs) containing different combinations of splicing (deletion of exons 2, 4, 5, 7, partial deletion of exon 36 9, or insertion of 12 or 15 base pairs from introns 6 or 7) were identified. CAR mRNAs were expressed prostate in small intestine, kidney, testis, adrenal, and brain caudate nucleus. Intestine expressed only CAR.1 mRNA, whereas spleen, heart, and prostate CAR.1 expressed only CAR-SVs. In vitro transcription and translation of CAR-SVs lacking exon 2 (missing ATG start site) generated CAR proteins 5, that differed in M(r) from CAR.1. These CAR-SVs used a translation initiation site in exon 1, generating CAR with a upon unique amino-terminal sequence. Transcripts lacking part of exon 9 altered the CAR reading frame generating CAR proteins with a unique 2, carboxy-terminal end. CAR-SVs demonstrated compromised binding to CYP2B6 NR elements and transcriptional activation of a CYP2B6 luciferase reporter. The expression (missing of CAR in additional human cell types increases the range of tissue specific transcriptional responses regulated by this receptor, suggesting mRNA, additional biological roles for CAR and CAR-SV proteins in these tissues.

CYP2B6 (SNPs) metabolizes many drugs, and its expression varies greatly. CYP2B6 genotype-phenotype associations were determined using human livers that were biochemically phenotyped was for CYP2B6 (mRNA, protein, and CYP2B6 activity), and genotyped for CYP2B6 coding and 5'-flanking regions. CYP2B6 expression differed significantly between an sexes. Females had higher amounts of CYP2B6 mRNA (3.9-fold, P < .001), protein (1.7-fold, P < .009), and activity (1.6-fold,varied P < .05) than did male subjects. Furthermore, 7.1% of females and 20% of males were poor CYP2B6 metabolizers. Striking of differences among different ethnic groups were observed: CYP2B6 activity was 3.6- and 5. -fold higher in Hispanic females than in Caucasian 7.1% (P < .022) or African-American females (P < .038). Ten single nucleotide polymorphisms (SNPs) in the CYP2B6 promoter and seven most in the coding region were found, including a newly identified 13072A>G substitution that resulted in an Lys139Glu change. Many CYP2B6 splice splice variants (SV) were observed, and the most common variant lacked exons 4 to 6. A nonsynonymous SNP in exon Caucasian 4 (15631G>T), which disrupted an exonic splicing enhancer, and a SNP 15582C>T in an intron-3 branch site were correlated with associations this SV. The extent to which CYP2B6 variation was a predictor of CYP2B6 activity varied according to sex and ethnicity.in The 1459C>T SNP, which resulted in the Arg487Cys substitution, was associated with the lowest level of CYP2B6 activity in livers (15631G>T), of females. The intron-3 15582C>T SNP (in significant linkage disequilibrium with a SNP in a putative hepatic nuclear factor 4 (SV) (HNF4) binding site) was correlated with lower CYP2B6 expression in females. In conclusion, we found several common SNPs that are found, associated with polymorphic CYP2B6 expression.

The of ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 and levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory line. mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required Based to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators protect increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in compensatory Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile expression acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated increased pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.

The other commonly prescribed antiepileptic drug phenytoin has a narrow therapeutic range and wide inter-individual variability in clearance explained partially by CYP2C9 haplotype. and CYP2C19 coding variants. After finding a paradoxically low urinary phenytoin metabolite (S)/(R) ratio in subjects on phenytoin maintenance therapy had with a CYP2C9*1/*1 & CYP2C19*1/*2 genotype, we hypothesized that CYP2C9 regulatory polymorphisms (rPMs), G-3089A and -2663delTG, in linkage disequilibrium with CYP2C9 CYP2C19*2 were responsible. These rPMs explained as much as 10% of the variation in phenytoin maintenance dose in epileptic patients,-2663delTG, but were not correlated with other patients' warfarin dose requirements or with phenytoin metabolite ratio in human liver microsomes. We polymorphisms hypothesized the rPMs affected CYP2C9 induction by phenytoin, a PXR and CAR activator. Transfection studies showed CYP2C9 reporters with wild-type and versus variant alleles had similar basal activity but significantly greater PHT induction by co-transfected PXR, CAR and Nrf2 and less induction YY1 repression. Phenytoin induction of CYP2C9 was greater in human hepatocytes with the CYP2C9 wild-type vs. variant haplotype. Therefore, CYP2C9 phenytoin rPMs affect phenytoin-dependent induction of CYP2C9 and phenytoin metabolism in humans, with an effect size comparable to that for CYP2C9*2 narrow and 2C9*3. These findings may also be relevant to the clinical use of other PXR, CAR and Nrf2 activators.

The and aim of this study was to identify the extent of genetic variability in breast cancer resistance protein (BCRP) in humans.intestinal We first analysed the sequence of BCRP cDNA from human livers and from human intestines phenotyped for expression of intestinal small BCRP. We then determined the frequency of all known coding single nucleotide polymorphisms (cSNPs) using DNA from individuals representing 11 of different ethnic populations. Nine SNPs including four non-synonymous and three synonymous cSNPs and two intronic SNPs were identified. Of the and missense mutations, exon 2 SNP (G34A) resulted in a V12M change; exon 5 SNP (C421A) resulted in a Q141K substitution;non-synonymous exon 6 SNP (A616C) resulted in an I206L amino acid substitution; and exon 15 SNP (A1768T) resulted in a N590Y marked change in the BCRP protein. The two most frequent polymorphisms identified in the human population studied were the G34A and G34A C421A transitions. There was marked variation in BCRP genotypes and allele frequencies in the different populations. BCRP mRNA was phenotyped Q141K in human small bowel intestinal samples by real-time polymerase chain reaction and BCRP protein was analysed on immunoblots of tissue genetic from the same individuals. There was a 78-fold variation in expression of BCRP mRNA and significant variation in BCRP protein in expression in human intestine. Expression of intestinal BCRP mRNA and protein was not different between persons expressing the common Gln141 phenotyped allele compared to the Lys141 allele. Thus, common natural allelic variants of BCRP have been identified, and did not influence and interindividual variation in expression of BCRP mRNA in human intestine, but remain to be tested for their effect on BCRP BCRP function.

Marked mutations interindividual variability in expression of CYP3A4 influences the disposition of many endo- and xenobiotics, including the metabolism of steroids, environmental the toxins and therapeutically useful drugs. The present study was designed to determine the genetic basis of CYP3A4 variability. We analysed in DNA from 82 individuals with known CYP3A4 phenotype including 53 Caucasians and 21 African-American liver donors, seven individuals who were polymorphisms outliers in CYP3A4 metabolism and five individuals in a family of a poor nifedipine metabolizer. In addition, we analysed DNA five from the eight person DNA Polymorphism Discovery Resource subset (Coriell Institute) and 89 individuals representing nine ethnic groups. Five non-synonymous outliers mutations in the coding region of CYP3A4 were observed. CYP3A4*14 (T44C) in exon 1 resulted in an L15P change; CYP3A4*15 CYP3A4*10 (G14387A) in exon 6 resulted in a R162Q substitution; CYP3A4*10 (G14422C) in exon 6 resulted in a D174H substitution; CYP3A4*16 resulted (C15721G) in exon 7 resulted in a T185S amino acid substitution; and CYP3A4*12 (C22002T) in exon 11 resulted in a subset L373F change in the CYP3A4 protein. An additional six single nucleotide polymorphisms (SNPs) in the 5'-UTR, 13 SNPs in the of introns and three SNPs in the 3'-UTR were observed. Extensive population differences were observed in the frequencies of various CYP3A4 Discovery alleles. None of the 28 CYP3A4 SNPs identified in CYP3A4 phenotyped persons (most individuals being heterozygous for any CYP3A4 variant)exon was associated with low hepatic CYP3A4 protein expression or low CYP3A4 activity in vivo.

The transduced pregnane X receptor, PXR (PXR.1) can be activated by structurally diverse lipophilic ligands. PXR.2, an alternatively spliced form of PXR,bound lacks 111 nucleotides encoding 37 amino acids in the ligand binding domain. PXR.2 bound a classic CYP3A4 PXRE in electrophoretic that mobility shift assays, but transfected PXR.2 failed to transactivate a CYP3A4-promoter-luciferase reporter plasmid in HepG2 cells treated with various PXR assays ligands. Co-transfection experiments showed that PXR.2 behaved as a dominant negative, interfering with PXR.1/rifampin activation of CYP3A4-PXRE-LUC. In HepG2 and showed LS180 cells stably transduced with PXR.1, PXR target genes (CYP3A4, MDR1, CYP2B6, UGT1A1) were higher than mock transduced cells in various the absence of ligand and were further induced in the presence of rifampin. In contrast, PXR.2 stably introduced into the induce same host cells failed to induce target genes over levels in mock transfected cells following drug treatment. Our homology modeling same suggests that ligands bind PXR.1 more favorably likely because of the presence of a key disordered loop region, which is PXR.1, missing in PXR.2. Yeast two hybrid assays revealed that, even in the presence of ligand, the co-repressors remain tightly bound structurally to PXR.2 and co-activators are unable to bind at helix 12. In summary, PXR.2 can bind to PXREs but fails transduced to transactivate target genes because ligands do not bind the ligand binding domain of PXR.2 productively, co-repressors remain tightly bound,homology and co-activators are not recruited to PXR.2.

We (> have investigated several in silico and in vitro methods in order to improve our ability to predict potential drug interactions methods of antibiotics. Our focus was to identify those antibiotics that activate PXR and induce CYP3A4 in human hepatocytes and intestinal was cells. Human PXR activation was screened using reporter assays in HepG2 cells, kinetic measurements of PXR activation were made in for DPX-2 cells, and induction of CYP3A4 expression and activity was verified by quantitative PCR, immunoblotting and testosterone 6â-hydroxylation in primary by human hepatocytes and LS180 cells. We found that in HepG2 cells CYP3A4 transcription was activated strongly (>10-fold) by rifampin and expression troleandomycin; moderately (> 7-fold) by dicloxacillin, tetracycline, clindamycin, griseofulvin and (> 4-fold) by erythromycin; weakly (>2.4-fold) by nafcillin, cefaclor and expression sulfisoxazole; and (>2-fold) by cefadroxil and penicillin V. Similar though not identical results were obtained in DPX-2 cells. CYP3A4 mRNA DPX-2 and protein expression were induced by these antibiotics to differing extents in both liver and intestinal cells. CYP3A4 activity was (>10-fold) significantly increased by rifampin (9.7-fold), nafcillin and dicloxacillin (5.9-fold), and weakly induced (2-fold) by tetracycline, sufisoxazole, troleandomycin and clindamycin. Multiple order pharmacophore models and docking indicated a good fit for dicloxacillin and nafcillin in PXR. These results suggest that in vitro strongly and in silico methods can help to prioritize and identify antibiotics that are most likely to reduce exposures of medications cells. (such as oral contraceptive agents) which interact with enzymes and transporters regulated by PXR. In summary, nafcillin, dicloxacillin, cephradine, tetracycline,cells. sulfixoxazole, erythromycin, clindamycin, and griseofulvin exhibit a clear propensity to induce CYP3A4 and warrant further clinical investigation.

The ERBT CYP3A5*1 allele has been associated with differences in the metabolism of some CYP3A substrates. CYP3A polymorphism may also influence susceptibility 2.14%, for certain drug interactions. We have previously noted a correlation between basal CYP3A activity and the inductive effects of dexamethasone increased using the erythromycin breath test (ERBT). To determine if CYP3A5 polymorphism influences induction of CYP3A activity, we examined the effect while of an anti-emetic regimen of dexamethasone, and the prototypical inducer rifampin, on the ERBT in a African American volunteers prospectively determine stratified by CYP3A5*1 allele carrier-status. Mean basal ERBTs were significantly higher in CYP3A5*1 carriers (2.71%+/- .53%) versus non-carriers (2.12%test +/- .37%, P = .006). Rifampin increased ERBTs in CYP3A5*1 carriers (4.68% vs 2.60%, P = .0008) and non-carriers (3.55%(2.71% vs 2.11%, P = .0017), while dexamethasone increased ERBTs only in CYP3A5*1 non-carriers (3.03% vs 2.14%, P = .031). CYP3A5 higher polymorphism appears to influence susceptibility to induction-type drug interactions for some inducers, and CYP3A5*1 non-carriers may be more susceptible to of the inductive effects of dexamethasone due to lower basal CYP3A activity.

The significantly hypothesis was tested that sequence diversity in BCRP's cis-regulatory region is a significant determinant of BCRP expression. The BCRP promoter imbalance and intron 1 were resequenced in lymphoblast DNA from the polymorphism discovery resource (PDR) 44 subset. BCRP SNPs were genotyped 2. in donor human livers, intestines and lymphoblasts quantitatively phenotyped for BCRP mRNA expression. Carriers of the -15622C>T SNP had lower was BCRP expression in multiple tissues. The intron 1 SNP 16702C>T was associated with high expression in livers; 1143G>A was associated intron with low expression in intestine; 12283T>C was associated with higher expression in the PDR44 and White livers. The -15994C>T promoter in SNP was significantly associated with higher BCRP expression in multiple tissues. Patients with the -15994C>T genotype had substantially higher clearance alternative of oral imatinib. We next determined if BCRP expression was related to polymorphic alternative splicing or alternative promoter use. Liver BCRP polymorphically expressed an alternatively spliced mRNA (SV1) skipping exon 2. Although SV1+ livers did not uniformly carry the ex2 G34A expression allele, 90% of G34A livers expressed SV1 (vs. 4% of 34GG livers) and BCRP mRNA was significantly lower among Hispanic region livers with the G34A variant genotype. Analysis of allele expression imbalance (AEI) showed that PDR44 samples with AEI had lower with BCRP mRNA expression, however, no linked cis-polymorphisms were identified. BCRP utilized multiple promoters, and livers differentially using alternative exon 1b (SV1) had lower BCRP. Conclusion: BCRP expression in lymphoblasts, liver and intestine is associated with novel promoter and intron 1 SNPs.expression

The genes Protocadherin 11X/Y (PCDH11X/Y) gene pair has been proposed as a carrier of the variation relating to cerebral asymmetry and psychosis (Tx1-fetal on the ground that the Y gene was generated by duplication at 6 million years (close to the chimpanzee-human separation)of and there is a case for an X/Y determinant of cerebral asymmetry. The present article investigated the patterns of alternative expression splicing and expression of the PCDH11X/Y genes. Twelve alternative transcripts of PCDH11X/Y genes were presently identified by in silico analysis.an To investigate the biological roles of alternative transcripts of PCDH11X/Y genes, the transcripts were analyzed by real-time reverse transcription-polymerase chain a reaction amplification. A total of 31 normal tissues including 11 different regions of human brain were used to investigate a were wide spectrum of expression profiles. Dominant expression patterns were identified in several tissues (Tx1-fetal liver; Tx3-adult brain; Tx4-adult brain and PCDH11X/Y kidney; Tx5-bone marrow; Ty1-fetal brain; Ty2-adrenal gland). Tx4 transcripts showed specific expression patterns in olfactory tissues. The findings can guide of functional investigation of neuropsychiatric disorders.(c) 2009 Wiley-Liss, Inc.

The In pregnane X receptor belongs to the nuclear hormone receptor superfamily and is involved in the transcriptional control of numerous genes.propose It was originally thought that it was a xenobiotic sensor controlling detoxification pathways. Recent studies have shown an increasingly important for role in inflammation and cancer, supporting its function in abrogating tissue damage. PXR orthologs and PXR-like pathways have been identified reported, in several non-mammalian species which corroborate a conserved role for PXR in cellular detoxification. In summary, PXR has a multiplicity in of roles in vivo and is being revealed as behaving like a "Jekyll and Hyde" nuclear hormone receptor. The importance supporting of this review is to elucidate the need for discovery of antagonists of PXR to further probe its biology and hormone therapeutic applications. Although several PXR agonists are already reported, virtually nothing is known about PXR antagonists. Here, we propose the "Jekyll development of PXR antagonists through chemical, genetic and molecular modeling approaches. Based on this review it will be clear that corroborate antagonists of PXR and PXR-like pathways will have widespread utility in PXR biology and therapeutics.

Ketohexokinase hepatocytes (KHK, also known as fructokinase) initiates the pathway through which most dietary fructose is metabolized. Very little is known about isoform the cellular localization of this enzyme. Alternatively spliced KHK-C and KHK-A mRNAs are known, but the existence of the KHK-A blotting, protein isoform has not been demonstrated in vivo. Using antibodies to KHK for immunohistochemistry and western blotting of rodent tissues,protein including those from mouse knockouts, coupled with reverse transcription PCR assays, we determined the distribution of the splice variants. The blotting highly expressed KHK-C isoform localised to hepatocytes in the liver and to the straight segment of the proximal renal tubule.for In both tissues, cytoplasmic and nuclear staining was observed. The KHK-A mRNA isoform was observed exclusively in a range of mRNA other tissues, and by western blotting, the presence of endogenous immunoreactive KHK-A protein was shown for the first time, proving was that the KHK-A mRNA is translated into KHK-A protein in vivo, and supporting the suggestion that this evolutionarily conserved isoform of is physiologically functional. However, the low levels of KHK-A expression prevented its immunohistochemical localization within these tissues. Our results highlight through that the use of in vivo biological controls (tissues from knockout animals) is required to distinguish genuine KHK immunoreactivity from distribution experimental artifact.

The be induction of dog CYP3A12 and CYP3A26 mRNA levels was evaluated in liver slices after treatment with 22 xenobiotics. Eleven of the the 22 xenobiotics increased 3A12 mRNA by more than four-fold, while nine did the same for 3A26 mRNA. A four-fold 3A12 increase in the mRNA level was used as the cut-off for indication of induction based on the noise level of pregnane the real time-PCR. A good correlation was found between the mRNA levels for 3A12 and 3A26 after treatment with compounds,the suggesting that these two CYPs may be co-induced. Induction of CYP3A4 in human hepatocytes was evaluated after treatment with the of same 22 compounds. Thirteen out of the 22 compounds increased the 3A4 mRNA levels by more than four-fold. When the 3A4 mRNA levels of 3A4 and 3A12 were compared after treatment with compounds, no correlation was found. The regulation of CYP3A 22 expression has been demonstrated to be controlled by pregnane X receptor (PXR). Upon examination of the sequence homology and the 3A26 three-dimensional structures of human PXR and a dog PXR model, only two different amino acids (met323/val and arg410/lys) were found was in the ligand-binding domain. This finding suggests that these two amino acids may play a role in the binding specificity and of ligands.

Pregnane form, X receptor (PXR) is an important component of the body's adaptive defense system responsible for the elimination of various toxic induced xenobiotics. PXR activation by endogenous and exogenous chemicals, including steroids, antibiotics, bile acids, and herbal compounds, results in induction of human drug metabolism. We investigated the ability of the isoflavones genistein, daidzein, and the daidzein metabolite equol to activate human and Treatment mouse PXR in vitro using cell-based transient transfection studies and primary hepatocytes and in vivo in a mouse model. In vitro transient transfection assays, the isoflavones genistein and daidzein activate full-length, wild-type mouse PXR, but not a mutant form, with genistein and being the most potent. In contrast, equol was a more potent activator of human PXR than genistein or daidzein. In recruitment a mammalian 2-hybrid assay, isoflavones induced recruitment of the coactivator steroid receptor coactivator 1 to PXR. When tested against the a native human Cytochrome P450 3A4 (CYP3A4) promoter, equol was the more potent activator and treatment of human hepatocytes with equol genistein increased CYP3A4 mRNA and immunoreactive protein expression. Treatment of wild-type, but not PXR(-/-), mouse hepatocytes showed that genistein and daidzein body's induced the expression of Cytochrome P450 3A11 (Cyp3A11) mRNA, whereas equol had no effect. Cyp3A11 mRNA was also induced in isoflavones vivo in mice fed a soy protein-containing diet. The results presented herein demonstrate that there is a species-specific difference in the the activation of PXR by isoflavones and equol.

The the hypothesis was tested that sequence diversity in PXR cis-regulatory regions are significant determinants of variation in inducible and constitutive CYP3A4 alleles. expression. A combination of comparative genomics and computational algorithms were used to select regions of the human PXR promoter and 63396C>T intron 1 that were resequenced in the polymorphism discovery resource 24 DNA subset. PXR SNPs were then genotyped in donor alleles human livers phenotyped for CYP3A4 and MDR1 mRNAs and primary human hepatocytes phenotyped for basal and rifampin inducible CYP3A4 activity.CYP3A4 The human PXR promoter (16.9 kb) was significantly larger than in rodents (2.9 kb). Eighty-nine SNPs were identified in the human promoter and intron 1 of PXR. The SNPs most consistently associated with CYP3A4 phenotypic measures were a 44477T>C (-1359) promoter deletion SNP (in LD with SNP 463170, a 6 bp deletion in intron 1a, and SNP 46551, a C nucleotide insertion with in intron 1b); SNP 63396C>T in intron 1 (in LD with SNPs 63704A>G, 63813(CAAA)(CA) variable repeat, and 65104T>C); and SNPs significantly 56348C>A; 69789A>G and 66034T>C. Donor livers with the variant PXR alleles had altered hepatic expression of PXR targets compared to regions livers with PXR wild-type alleles. These results identified PXR promoter and intron 1 SNPs associated with PXR target gene expression was (CYP3A4) in donor livers and cultured hepatocytes and that a striking number of the linked intron1 SNPs will affect putative 1b); binding sites for hepatic HNF3beta (FOXA2), a transcription factor linked with PXR expression.

The sequence pregnane X receptor (PXR) plays a crucial role in xenobiotic and drug metabolism, being the major transcriptional regulator of cytochrome pigs P-450 monooxygenase 3A4, which metabolizes more than 50% of all clinically used drugs. Recent pharmacodynamic studies have shown that the and mouse is not an ideal model for predicting human clinical drug study outcomes. Therefore, we characterized the porcine PXR (pPXR)RT-PCR gene to evaluate the utility of the pig as an alternate preclinical animal model. The complete sequence of pPXR mRNA characterized and 11 kb of genomic sequence were obtained. Similar to the human PXR gene, the pPXR gene revealed multiple splice study variants in the ligand-binding domain. All pPXR splice variants (SV) were porcine-specific. The pPXR mRNAs varied in 3'-UTR length due were to differential termination and specific deletions. Northern blot analyses identified high levels of pPXR mRNA expression in the liver, small All intestine, heart, kidney, and colon. RT-PCR amplification detected lower levels of pPXR expression in multiple tissues. Ninety-three pigs representing eight The breeds were analyzed for single nucleotide polymorphisms (SNPs). Only one nonsynonymous SNP (S178L) was found in the pPXR ligand-binding domain.role This characterization of the pPXR gene contributes to the development of a porcine model for human drug metabolic studies.

OBJECTIVE:and The nuclear receptor NR1I2 (also called PXR or SXR) is primarily expressed in mouse and human liver and intestines. Direct .04 activation of NR1I2 occurs in response to a range of xenobiotics, which causes the formation of a heterodimer with the were RXR receptor. This heterodimer binds to the nuclear receptor response elements of downstream genes such as ABCB1, CYP2C, and CYP3A.significantly This study determined the extent of NR1I2 variation in three world populations. METHODS: Variation in NR1I2 was identified by pooled This resequencing in African, Asian, and European populations. Validation was performed in European and African populations using PCR and Pyrosequencing technology.as RNA expression of NR1I2, ABCB1 and CYP3A4 was assessed using real-time PCR. RESULTS: Of 36 single nucleotide polymorphisms (SNPs) identified,identified, 24 were in the untranslated region, 8 were intronic, and 4 exonic. Thirty-six percent were unique to the African population.36 In comparison with previously published data, we identified 13 novel polymorphisms. The NR1I2 -566A > C polymorphism was significantly associated in with ABCB1 and CYP3A4 RNA expression in colon tumor (P = .04 in both cases), however, this polymorphism was not SXR) associated with NR1I2 expression. CONCLUSION: With NR1I2 playing such a large role in the regulation of genes involved in drug pooled metabolism and transport, genetic variation contributing to altered NR1I2 function may have an important clinical impact.

The analyzed pregnane X receptor (PXR, NR1I2) and the estrogen receptors (ERalpha, NR3A1 and ERbeta, NR3A2) bind a large number of compounds,were including environmental pollutants and drugs, which exhibit remarkably diverse structural features. This prompted us to investigate if ER ligands could checked be PXR activators. We focused our attention on known estrogens from various chemical classes: physiological and synthetic estrogens and antiestrogens,(54%) plant and fungus estrogens, and other man-made chemicals belonging to phthalate plasticizers, surfactant-derived alkylphenols and cosmetics. Altogether, nearly 50 compounds classes: were thus analyzed for their ability to activate human PXR in stably transfected cells, HGPXR cells, derived from HeLa cells estrogens and expressing luciferase under the control of a chimeric hPXR. Some of the newly identified hPXR activators were also checked of for their ability to induce cytochrome P450 3A4 and 2B6 expressions in a primary culture of human hepatocytes. A significant luciferase proportion (54%) of compounds with estrogenic activity or able to bind ER were found to be hPXR activators: in particular,surfactant-derived antiestrogens, mycoestrogens and phthalates. An even greater proportion is observed if estrogenic pesticides are included. Altogether, these results raise the estrogen question of the meaning and consequences of compounds with double PXR/ER activation ability.

The A human nuclear pregnane X receptor (PXR) responds to a wide variety of xenobiotic and endobiotic compounds, including pregnanes, progesterones, corticosterones,steroid lithocholic acids, and 17beta-estradiol. In response to these ligands, the receptor controls the expression of genes central to the metabolism it and excretion of potentially harmful chemicals from both exogenous and endogenous sources. While the structural basis of PXR's interaction with receptor, small and large xenobiotics has been examined, the detailed nature of its binding to endobiotics, including steroid-like ligands, remains unclear.the We report the crystal structure of the human PXR ligand binding domain (LBD) in complex with 17beta-estradiol, a representative steroid large ligand, at 2.65 A resolution. Estradiol is found to occupy only one region of PXR's expansive ligand binding pocket, leaving residues a notable 1,000 A(3) of space unoccupied, and to bridge between the key polar residues Ser-247 and Arg-410 in the to PXR LBD. Positioning the steroid scaffold in this way allows it to make several direct contacts to alphaAF of the domain receptor's AF-2 region. The PXR-estradiol complex was compared to that of other nuclear receptors, including the estrogen receptor, in complexes wide with analogous ligands. It was found that PXR's placement of the steroid is remarkably distinct relative to other members of ligand the nuclear receptor superfamily. Using the PXR-estradiol complex as a guide, the binding of other steroid- and cholesterol-like molecules was this then considered. The results provide detailed insights into the manner in which human PXR responds to a wide range of bridge endobiotic compounds.