ABSTRACTPBREM, the phenobarbital-responsive enhancer module of cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with finding that anti-retinoid X (RXR) could supershift NR1-nuclear protein complex, DNA affinity chromatography oligonucleotides enriched orphan RXR from hepatic extracts phenobarbital-treated mice. In addition to RXR, CAR was present in same fraction. mice, both rapidly increased before induction CYP2B10 mRNA....

The endogenous CYP2B6 gene becomes phenobarbital (PB) inducible in androstenol-treated HepG2 cells either transiently or stably transfected with a nuclear receptor CAR expression vector. PB induction mediated by is regulated conserved 51-base pair element called PB-responsive enhancer module (PBREM) that has now been located between −1733 and −1683 bp the gene's 5′-flanking region. An vitro translated acting as retinoid X α heterodimer binds directly to two sites NR1 NR2 within PBREM. In...

The constitutively active receptor (CAR) transactivates a distal enhancer called the phenobarbital (PB)-responsive module (PBREM) found in PB-inducible CYP2B genes. CAR dramatically increases its binding to PBREM livers of PB-treated mice. We have investigated cellular mechanism PB-induced increase binding. Western blot analyses mouse revealed an extensive nuclear accumulation following PB treatment. Nuclear contents perfectly correlate with PBREM. PB-elicited appears be general step...

Phenobarbital (PB) induces various gene encoding drug/steroid-metabolizing enzymes such as cytochromes P450 (P450s) and transferases. Although the nuclear orphan constitutive active receptor (CAR) has been identified a key transcription factor that regulates induction of CYP2B, full scope CAR-regulated genes still remains major question. To this end, reverse transcriptase-polymerase chain reaction cDNA microarray techniques were employed to examine expression in wild-type CAR-null mice. The...

The UDP-glucuronosyltransferase, UGT1A1, is the critical enzyme responsible for detoxification of potentially neurotoxic bilirubin by conjugating it with glucuronic acid. For decades, phenobarbital (PB) treatment hyperbilirubinemia has been known to increase expression UGT1A1 gene in liver. We have now delineated PB response activity a 290-bp distal enhancer sequence (-3483/-3194) gene. contains 3 putative nuclear receptor motifs, and was activated orphan receptor, human constitutive active...

Both the human pregnane X receptor (hPXR) and constitutive androstane (hCAR) are capable of regulating <i>CYP3A4</i> <i>CYP2B6</i> gene expression. However, majority currently identified CYP3A4 CYP2B6 inducers confirmed activators hPXR but not hCAR. To compare these receptors with respect to their chemical selectivities, 16 drugs known induce and/or CYP2B expression were evaluated for relative activation versus Because high basal low chemical-induced hCAR in immortalized cells, alternative...

The nuclear constitutive active receptor (CAR) is a key transcription factor regulating phenobarbital (PB)-inducible of various hepatic genes that encode xenobiotic/steroid-metabolizing enzymes. CAR retained in the cytoplasm noninduced livers and translocates into nucleus after PB induction. HepG2 cells lack capability retaining cytoplasm; thus, spontaneously accumulates nucleus. We have now cloned characterized tetratricopeptide repeat (TPR) protein, designated cytoplasmic retention protein...

Upon drug activation, the nuclear pregnane X receptor (PXR) regulates not only hepatic but also energy metabolism. Using Pxr(-/-) mice, we have now investigated PXR-mediated repression of lipid metabolism in fasting livers. Treatment with PXR activator pregnenolone 16alpha-carbonitrile (PCN) down-regulated mRNA levels carnitine palmitoyltransferase 1A (in beta-oxidation) and mitochondrial 3-hydroxy-3-methylglutarate-CoA synthase 2 ketogenesis) wild-type (Pxr(+/+)) mice only. In contrast,...

The pregnane X receptor (PXR) plays an important role in the response to xenobiotics and endogenous toxins. We have used a specific anti-PXR antibody Western blotting of mouse liver nuclear extracts show that PXR is accumulated nucleus after treatment with 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN), followed by increase Cyp3a11 mRNA. Expression wild type various mutants as green fluorescent fusion proteins livers showed was retained cytoplasm from where PCN translocated into nucleus....

The epidermal growth factor receptor is an unexpected target of the barbiturate phenobarbital.

Accumulated evidence suggests that cross-talk between the pregnane X receptor (PXR) and constitutive androstane (CAR) results in shared transcriptional activation of <i>CYP2B</i> <i>CYP3A</i> genes. Although most data imply symmetrical cross-regulation these genes by rodent PXR CAR, actual selectivities corresponding human receptors are unknown. The objective this study was to evaluate symmetry (h) hCAR comparing for CYP2B6 CYP3A4. Human hepatocyte studies revealed nonselective induction...

In response to phenobarbital (PB) and other PB-type inducers, the nuclear receptor CAR translocates mouse liver nucleus (T. Kawamoto et al., Mol. Cell. Biol. 19:6318-6322, 1999). To define translocation mechanism, fluorescent protein-tagged human (hCAR) was expressed in livers using situ DNA injection gene delivery systems. As wild-type hCAR, truncated lacking C-terminal 10 residues (i.e., AF2 domain) translocated nucleus, indicating that PB-inducible is independent. Deletion of 30 abolished...

Heparan sulfateN-deacetylase/N-sulfotransferase (HSNST) catalyzes the first and obligatory step in biosynthesis of heparan sulfates heparin. The crystal structure sulfotransferase domain (NST1) human HSNST-1 has been determined at 2.3-Å resolution a binary complex with 3′-phosphoadenosine 5′-phosphate (PAP). NST1 is approximately spherical an open cleft, consists single α/β fold central five-stranded parallel β-sheet three-stranded anti-parallel bearing interstrand disulfide bond. structural...

UDP-glucuronosyltransferase (UGT) 1A1 glucuronidates endogenous metabolites, such as bilirubin, and exogenous substances, plays a critical role in their detoxification excretion. In previous article, we described the phenobarbital response activity to 290-base pair (bp) distal enhancer sequence (–3499/–3210) of human <i>UGT1A1</i> gene that is activated by constitutive androstane receptor (CAR). Here, show dexamethasone at submicromolar concentrations enhances pregnane X (PXR)...

Compared with its rodent orthologs, little is known about the chemical specificity of human constitutive androstane receptor (hCAR) and regulation hepatic enzyme expression. Phenytoin (PHY), a widely used antiepileptic drug, potent inducer CYP2B6 in primary hepatocytes, but does not activate pregnane X (PXR) significantly cell-based transfection assays at same concentrations associated induction CYP2B6. Based on this observation, we hypothesized that PHY may be selective activator hCAR. In...

Kininogen Thiol‐proteinase inhibitor Papain Cathepsin B H L

Upon activation by therapeutics, the nuclear xenobiotic/ constitutive active/androstane receptor (CAR) regulates various liver functions ranging from drug metabolism and excretion to energy metabolism. CAR can also be a risk factor for developing diseases such as hepatocellular carcinoma. Here we have characterized conserved threonine 38 of human primary residue that translocation CAR. Protein kinase C phosphorylates located on α-helix spanning residues 29–42 constitutes part first zinc...

As a promiscuous xenobiotic sensor, the constitutive androstane receptor (CAR; NR1I3) regulates expression of multiple drug-metabolizing enzymes and transporters in liver. The constitutively activated nature CAR cell-based transfection assays has hindered its use as predictor metabolism-based drug-drug interactions. Here, we have identified 1-(2-chlorophenylmethylpropyl)-3-isoquinoline-carboxamide (PK11195), typical peripheral benzodiazepine (PBR) ligand, selective potent inhibitor human (h)...

The solute carrier family 13 member 5 (SLC13A5) is a sodium-coupled transporter that mediates cellular uptake of citrate, which plays important roles in the synthesis fatty acids and cholesterol. Recently, pregnane X receptor (PXR, NR1I2), initially characterized as xenobiotic sensor, has been functionally linked to regulation various physiologic processes are associated with lipid metabolism energy homeostasis. Here, we show <i>SLC13A5</i> gene novel transcriptional target PXR, altered...

A new trisaccharide sugar chain was identified in bovine blood coagulation factors VII and IX. pentapeptide isolated from factor contained Ser-52, which could not be with a gas-phase sequencer, suggesting an unknown substituent on the serine residue (Takeya, H. et al. (1988) J. Biol. Chem., press). The same results were obtained for containing Ser-53 of Component analysis revealed that peptide 1 mol glucose 2 xylose. This component also confirmed by high-resolution fast atom bombardment mass...