Activation of HSP90/HSF1 Signaling as an Adaptive Response to an Electrophilic Metabolite of Morphine.

Morphinone (MO) is an electrophilic metabolite of morphine that covalently binds to protein thiols, resulting in toxicity in vitro and in vivo. We have previously identified a variety of redox signaling pathways that are activated during electrophilic stress. However, the role of MO in such activation remains unknown. In this study, we examined whether MO could activate heat shock protein (HSP) 90/heat shock factor (HSF) 1 signaling in HepG2 cells. MO exposure caused S-modification of HSP90 (determined using biotin-PEAC5-maleimide labeling) and nuclear translocation of transcription factor HSF1, thereby up-regulating its downstream genes encoding B-cell lymphoma 2-associated anthanogene 3 and heat shock 70 kDa protein 1. However, dihydromorphinone, a non-electrophilic metabolite of morphine, had little effect on HSF1 activation or upregulation of these genes, suggesting that covalent modification plays a role in this process and that the HSP90/HSF1 pathway is a redox-signaled adaptive response to morphine metabolism.

Activation of the Keap1/Nrf2 Pathway as an Adaptive Response to an Electrophilic Metabolite of Morphine.

Morphinone (MO) is an electrophilic metabolite of morphine that covalently binds to protein thiols via its α,ß-unsaturated carbonyl group, resulting in toxicity in vitro and in vivo. Our previous studies identified a variety of redox signaling pathways that are activated during electrophilic stress. Here, we examined in vitro activation of a signaling pathway involving Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) in response to MO. Exposure of HepG2 cells to MO caused covalent modification of Keap1 thiols (evaluated using biotin-PEAC5-maleimide labeling) and nuclear translocation of Nrf2, thereby up-regulating downstream genes encoding ATP binding cassette subfamily C member 2, solute carrier family 7 member 11, glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, glutathione S-transferase alpha 1, and heme oxygenase 1. However, dihydromorphinone, a metabolite of morphine lacking the reactive C7-C8 double bond, had little effect on Nrf2 activation. These results suggest that covalent modification is crucial in the Keap1/Nrf2 pathway activation and that this pathway is a redox signaling-associated adaptive response to MO metabolism.

Insulin induces expression of the hepatic vaspin gene.

Visceral adipose tissue-derived serine protease inhibitor (vaspin), initially identified in the visceral adipose tissue, is an adipokine that improves endoplasmic reticulum stress in obesity or insulin sensitivity and glucose tolerance. However, the transcriptional regulation of the hepatic vaspin gene remains elusive. We have previously shown that CCAAT-enhancer-binding protein α, a transcription factor of the basic leucine zipper class, positively regulates the vaspin gene. The present study aimed to investigate the nutritional or hormonal regulators of vaspin expression in the liver. For the fasting and refeeding study, mice in the fasting group were subjected to fasting for 24 h and then sacrificed. Mice in the refeeding group were subjected to fasting for 24 h and then refed with a 50% (w/w) sucrose/MF diet for further 24 h and then sacrificed. For the streptozotocin (STZ) study, STZ (50 mg/kg) was intraperitoneally injected into C57BL/6JJc1 mice for 5 d. Hepatic vaspin was repressed due to fasting for 24 h and was induced upon refeeding with a high-sucrose diet. In studies on liver-specific C/EBPα-deficient mice, C/EBPα was not involved in the induction of hepatic vaspin upon refeeding. In addition, the depletion of insulin by streptozotocin treatment markedly decreased hepatic vaspin expression. Finally, fasting-repressed vaspin expression in the liver was significantly increased by direct injection of insulin into fasting mice. In conclusion, our results suggest that insulin is a positive regulator of hepatic vaspin expression.

Fat-specific protein 27b is regulated by hepatic peroxisome proliferator-activated receptor γ in hepatic steatosis.

The fat-specific protein 27 gene (Fsp27) belongs to the cell death-inducing DNA fragmentation factor 45-like effector family. Fsp27 is highly expressed in adipose tissue and fatty liver. In adipocytes, FSP27 localizes to the membrane of lipid droplets and promotes lipid droplet hypertrophy. Recently, FSP27 was shown to consist of two isoforms, FSP27α and FSP27ß. Previously, we demonstrated that Fsp27a is directly regulated by peroxisome proliferator-activated receptor γ (PPARγ) in fatty livers of genetically obese leptin deficient ob/ob mice and that Fsp27b may potentially be regulated by different factors transcriptionally as they both have a different promoter region. Thus, the aim of the present study was to elucidate whether Fsp27b is regulated by PPARγ in fatty liver. Fsp27a and Fsp27b were markedly induced in fatty liver of ob/ob mice compared with those in the normal liver. However, both Fsp27a/b were expressed at markedly lower levels in liver-specific PPARγ knockout mice with an ob/ob background. Further, the PPAR response element (PPRE) for the PPARγ-dependent promotion of Fsp27b promotor activity was revealed at position -1,163/-1,151 from the transcriptional start site (+1). Interestingly, the cis-element responsible for the PPARγ-dependent induction of Fsp27b was the same as that responsible for PPARγ-dependent induction of Fsp27a. These results suggest that PPARγ regulates not only Fsp27a but also Fsp27b in fatty liver of ob/ob mice through a common PPRE.

Insulin Represses Fasting-Induced Expression of Hepatic Fat-Specific Protein 27.

The fat-specific protein 27 (Fsp27) gene belongs to the cell death-inducing DNA fragmentation factor 45-like effector family. Fsp27 is highly expressed in adipose tissue as well as the fatty liver of ob/ob mice. Fsp27 is directly regulated by the peroxisome proliferator-activated receptor γ (PPARγ) in livers of genetically obese leptin deficient ob/ob mice. In the present study, Fsp27 was markedly induced by 24 h fasting in genetically normal mouse livers and repressed by refeeding a high sucrose diet. In contrast with the liver, Fsp27 expression was decreased in adipose tissue by fasting and increased by refeeding. Interestingly, fasting-induced Fsp27 liver expression was independent of PPARγ. Moreover, Fsp27 expression was induced in the insulin-depleted livers of streptozotocin-treated mice. Finally, Fsp27 expression was repressed by direct injection of glucose or insulin in fasting mice. These results suggest that insulin represses Fsp27 expression in the fasting liver.

Cloning and characterization of four rabbit aldo-keto reductases featuring broad substrate specificity for xenobiotic and endogenous carbonyl compounds: relationship with multiple forms of drug ketone reductases.

Multiple forms of reductases for several drug ketones were isolated from rabbit liver, but their interrelationship and physiologic roles remain unknown. We isolated cDNAs for four aldo-keto reductases (AKR1C30, AKR1C31, AKR1C32, and AKR1C33), which share high amino acid sequence identity with the partial sequences of two rabbit naloxone reductases. The four recombinant enzymes reduced a variety of carbonyl compounds, including endogenous α-dicarbonyls (e.g., isatin and diacetyl), aldehydes (e.g., farnesal and 4-oxo-2-nonenal), and ketosteroids. They differed in specificity for drug ketones and ketosteroids. Although daunorubicin and befunolol were common substrates of all of the enzymes, AKR enzymes specifically reduced naloxone (AKR1C30, AKR1C32, and AKR1C33), metyrapone (AKR1C32 and AKR1C33), loxoprofen (AKR1C31 and AKR1C32), ketotifen (AKR1C30), and naltrexone and fenofibric acid (AKR1C33). AKR1C30 reduced only 17-keto-5ß-androstanes, whereas the other enzymes were active toward 3-, 17-, and 20-ketosteroids, and AKR1C33 further reduced 3-keto groups of bile acids and 7α-hydroxy-5ß-cholestanes. In addition, AKR1C30, AKR1C31, AKR1C32, and AKR1C33 were selectively inhibited by carbenoxolone, baccharin, phenolphthalein, and zearalenone, respectively. The mRNAs for the four enzymes were ubiquitously expressed in male rabbit tissues, in which highly expressed tissues were the brain, heart, liver, kidney, intestine, colon, and testis (for AKR1C30 and AKR1C31); brain, heart, liver, kidney, testis, lung, and adrenal gland (for AKR1C32); and liver and intestine (for AKR1C33). Thus, the four enzymes correspond to the multiple drug ketone reductases, and may function in the metabolisms of steroids, isatin and reactive carbonyl compounds, and bile acid synthesis.

Hepatic steatosis in leptin-deficient mice is promoted by the PPARgamma target gene Fsp27.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is induced in leptin-deficient (ob/ob) mouse liver and is critical for the development of hepatic steatosis. The present study shows that fat-specific protein 27 (Fsp27) in ob/ob liver is a direct target gene of PPARgamma and can elevate hepatic triglyceride levels. FSP27 belongs to the CIDE family, composed of CIDE A, CIDE B, and FSP27/CIDE C, all of which contain a conserved CIDE-N domain. FSP27 was recently reported to be a lipid droplet-binding protein and to promote lipid accumulation in adipocytes. The Fsp27 gene was expressed at high levels in ob/ob liver and at markedly lower levels in ob/ob livers lacking PPARgamma. Forced expression of FSP27 by adenovirus in hepatocytes in vitro or in vivo led to increased triglyceride levels. Knockdown by adenovirus expressing FSP27 shRNA resulted in lower accumulation of hepatic triglycerides compared to control adenovirus-infected liver. Taken together, these results indicate that FSP27 is a direct mediator of PPARgamma-dependent hepatic steatosis.

Characterization of rabbit morphine 6-dehydrogenase and two NAD(+)-dependent 3α(17ß)-hydroxysteroid dehydrogenases.

Mammalian morphine 6-dehydrogenase (M6DH)(1) converts morphine into a reactive electrophile, morphinone. M6DH belongs to the aldo-keto reductase (AKR) superfamily, but its endogenous substrates and entire amino acid sequence remain unknown. A recent rabbit genomic sequencing predicts three genes for novel AKRs (1C26, 1C27 and 1C28) that share >87% amino acid sequence identity and are similar to the partial sequence of rabbit liver M6DH. We isolated cDNAs for the three AKRs, and compared the properties of their recombinant enzymes. Like M6DH, only AKR1C26 that shares the highest sequence identity with hepatic M6DH oxidized morphine. The three AKRs showed NAD(+)-dependent dehydrogenase activity towards other non-steroidal alicyclic alcohols and 3α/17ß-hydroxy-C(18)/C(19)/C(21)-steroids, and their mRNAs were ubiquitously expressed in rabbit tissues. The kinetic constants for the substrates suggest that at least AKR1C26 and AKR1C28 act as NAD(+)-dependent 3α/17ß-hydroxysteroid dehydrogenases. AKR1C27 differed from AKR1C28 in its high K(m) values for the substrates and low sensitivity towards competitive inhibitors (ikarisoside A, hinokitiol, hexestrol and zearalenone), despite their 95% sequence identity. The site-directed mutagenesis of Tyr118 and Phe310 in AKR1C27 to the corresponding residues (Phe and Ile, respectively) in AKR1C28 produced an enzyme that was similar to AKR1C28, suggesting their key roles in ligand binding.

Expression of hepatic fat-specific protein 27 depends on the specific etiology of fatty liver.

Fat-specific protein 27 gene (FSP27), isolated by screening for genes specifically expressed in fully differentiated mouse adipocytes, belongs to the cell death-inducing DNA fragmentation factor, alpha subunit-like effector family. FSP27 is induced in not only adipose tissue but also the liver of ob/ob mice, and it promotes the development of fatty liver. The FSP27 gene is expressed in a fatty liver-specific manner and is not detected in the normal mouse liver. FSP27 expression is directly regulated by the induction of the hepatic peroxisome proliferator-activated receptor γ (PPARγ) in ob/ob fatty liver. In the present study, expression of hepatic FSP27 mRNA was determined in non-genetic fatty liver models. The FSP27 gene was markedly induced in the high-fat- or methionine- and choline-deficient (MCD) diet-induced fatty liver, but it was not elevated in alcohol-induced fatty liver. Interestingly, the induction of FSP27 mRNA due to the MCD diet was independent of PPARγ levels and completely absent in the liver from PPARγ-null mice. These results suggest that FSP27 mRNA expression in the liver depends on the etiology of fatty liver.

Non-Gaussian model-based diffusion-weighted imaging of oral squamous cell carcinoma: associations with Ki-67 proliferation status.

OBJECTIVES: To investigate possible associations between diffusion-weighted imaging (DWI) parameters derived from a non-Gaussian model fitting and Ki-67 status in patients with oral squamous cell carcinoma (OSCC). METHODS: Twenty-four patients with newly diagnosed OSCC were prospectively recruited. DWI was performed using six b-values (0-2500). The diffusion-related parameters of kurtosis value (K), kurtosis-corrected diffusion coefficient (DK), diffusion heterogeneity (α), distributed diffusion coefficient (DDC), slow diffusion coefficient (Dslow), and apparent diffusion coefficient (ADC) were calculated from four diffusion fitting models. Ki-67 status was categorized as low (Ki-67 percentage score < 20%), middle (20-50%), or high (> 50%). Kruskal-Wallis tests were performed between each non-Gaussian diffusion model parameters and Ki-67 grade. RESULTS: The Kruskal-Wallis tests revealed that multiple parameters (K, ADC, Dk, DDC and Dslow) showed statistically significant differences between the three levels of Ki-67 status (K: p = 0.020, ADC: p = 0.012, Dk: p = 0.027, DDC: p = 0.007 and Dslow: p = 0.026). CONCLUSIONS: Several non-Gaussian diffusion model parameters and ADC values were significantly associated with Ki-67 status and have potential as promising prognostic biomarkers in patients with OSCC.

Vaspin is a novel target gene of hepatic CCAAT-enhancer-binding protein.

Vaspin, initially identified in visceral adipose tissue, is an adipokine, and administration of recombinant vaspin leads to lowering of the endoplasmic reticulum stress which is elevated in obesity or enhancement of insulin sensitivity. CCAAT/enhancer binding protein (C/EBP), as a basic leucine zipper transcription factor, plays a critical role in adipocyte development and glucose and lipid metabolisms in liver. The present study aimed to investigate the effect of C/EBPα on vaspin gene expression. The expression of hepatic vaspin was markedly decreased in liver-specific C/EBPα knockout mice. A reporter assay indicated that two C/EBP-responsive elements (CEBPREs) are necessary for C/EBPα-dependent induction of vaspin promoter activities. Furthermore, electrophoretic mobility shift assay showed that C/EBPα in mouse liver is capable of directly binding the two CEBPREs. These results suggest that C/EBPα positively regulates hepatic vaspin expression through two functional CEBPREs. Thus, vaspin is a novel C/EBPα target gene in the liver.

Redox cycling of 9,10-phenanthraquinone to cause oxidative stress is terminated through its monoglucuronide conjugation in human pulmonary epithelial A549 cells.

9,10-Phenanthraquinone (PQ), a component of airborne particulate matter, causes marked cellular protein oxidation and cytotoxicity through a two-electron reduction to 9,10-dihydroxyphenanthrene (PQH2), which is associated with the propagation of reactive oxygen species (K. Taguchi et al., Free Radic. Biol. Med. 43:789-799, 2007). In the present study, we explored a biotransformation pathway for the detoxification of PQ. Exposure of human pulmonary epithelial A549 cells to PQ resulted in a time-dependent appearance of an unknown metabolite in the medium that was identified as the monoglucuronide of PQH2 (PQHG). Whereas a variety of isozymes of uridine 5'-diphosphate glucuronosyltransferase (UGTs) are responsible for PQHG formation, UGT1A10 and UGT1A6 were particularly effective catalysts for glucuronide conjugation. In cell-free systems, PQ exhibited a rapid thiol oxidation and subsequent oxygen consumption in the presence of dithiothreitol, whereas PQHG did not. Unlike the parent compound, PQHG completely lost the ability to oxidize cellular proteins and cause cell death in A549 cells. In addition, deletion of the transcription factor Nrf2 decreased PQHG formation and increased PQ-mediated toxicity of mouse primary hepatocytes. Thus, we conclude that PQHG is a metabolite of PQ, generated through PQH2, that terminates its redox cycling and transports it to extracellular space.

Oxidation of 5-methoxy-N,N-diisopropyltryptamine in rat liver microsomes and recombinant cytochrome P450 enzymes.

The oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), a tryptamine-type designer drug, was studied using rat liver microsomal fractions and recombinant cytochrome P450 (CYP) enzymes. 5-MeO-DIPT was biotransformed mainly into a side-chain N-deisopropylated metabolite and partially into an aromatic ring O-demethylated metabolite in liver microsomal fractions from untreated rats of both sexes. This metabolic profile is different from our previous findings in human liver microsomal fractions, in which the aromatic ring O-demethylation was the major pathway whereas the side-chain N-deisopropylation was minor [Narimatsu S, Yonemoto R, Saito K, Takaya K, Kumamoto T, Ishikawa T, et al. Oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes. Biochem Pharmacol 2006;71:1377-85]. Kinetic and inhibition studies indicated that the side-chain N-dealkylation is mediated by CYP2C11 and CYP3A2, whereas the aromatic ring O-demethylation is mediated by CYP2D2 and CYP2C6 in untreated male rats. Pretreatment of male rats with beta-naphthoflavone (BNF) produced an aromatic ring 6-hydroxylated metabolite. Recombinant rat and human CYP1A1 efficiently catalyzed 5-MeO-DIPT 6-hydroxylation under the conditions used. These results provide valuable information on the metabolic fate of 5-MeO-DIPT in rats that can be used in the toxicological study of this designer drug.

The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2.

We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.

Fat-specific protein 27 is a novel target gene of liver X receptor α.

Fat-specific protein 27 (FSP27) is highly expressed in the fatty liver of genetically obese ob/ob mice and promotes hepatic triglyceride (TG) accumulation. The nuclear hormone receptor liver X receptor α (LXRα) also plays a critical role in the control of TG levels in the liver. The present study demonstrated transcriptional regulation of Fsp27a and Fsp27b genes by LXRα. Treatment with the LXR ligand T0901317 markedly increased Fsp27a and Fsp27b mRNAs in wild-type C57BL/6J and ob/ob mouse livers. A reporter assay indicated that two LXR-responsive elements (LXREs) are necessary for LXRα-dependent induction of Fsp27a and Fsp27b promoter activities. Furthermore, the LXRα/retinoid X receptor α complex is capable of directly binding to the two LXREs both in vitro and in vivo. These results suggest that LXRα positively regulates Fsp27a and Fsp27b expression through two functional LXREs. Fsp27a/b are novel LXR target genes in the ob/ob fatty liver.

An approach to evaluate two-electron reduction of 9,10-phenanthraquinone and redox activity of the hydroquinone associated with oxidative stress.

Quinones are widely used as medicines or redox agents. The chemical properties are based on the reactions against an electron donor. 9,10-Phenanthraquinone (PQ), which is a quinone contaminated in airborne particulate matters, forms redox cycling, not Michael addition, with electron donors. Redox cycling of PQ contributes to its toxicity, following generation of reactive oxygen species (ROS). Detoxification of quinones is generally thought to be two-electron reduction forming hydroquinones. However, a hydroquinone of PQ, 9,10-dihydroxyphenanthrene (PQH(2)), has been never detected itself, because it is quite unstable. In this paper, we succeeded in detecting PQH(2) as its stable derivative, 9,10-diacetoxyphenanthrene (DAP). However, higher concentrations of PQ (>4 microM) form disproportionately with PQH(2), producing the 9,10-phenanthraquinone radical (PQ(-)) which is a one-electron reducing product of PQ. In cellular experiments using DAP as a precursor of PQH(2), it was shown that PQH(2) plays a critical role in the oxidative protein damage and cellular toxicity of PQ, showing that two-electron reduction of PQ can also initiate redox cycling to cause oxidative stress-dependent cytotoxicity.

Identification of N-ethylglycine in urine of cancer patients with metastatic bone disease.

BACKGROUND: Previously, a HPLC method for the determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine with fluorescence detection after pre-column derivatization with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride (DMS-Cl) [Inoue H, Iguchi H, Kono A, Tsuruta Y. Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride. J Chromatogr 1999;724:221-230] was developed to study the relation between those analytes and bone diseases. When the urinary analytes were measured, a large peak due to an unknown substance was recognized in the chromatograms of cancer patients with metastatic bone disease, although it was scarcely present in normal subjects. In this study, we identified the unknown substance. METHODS: The fluorescent fraction based on the unknown substance was collected using HPLC and the structure of the fluorescence product was analyzed with MS, (1)H NMR and (13)C NMR. RESULTS: The fluorescence product based on the unknown substance was established to be a DMS-derivative of N-ethylglycine. CONCLUSIONS: Excretion of N-ethylglycine in the urine of cancer patients with metastatic bone disease is recognized, although N-ethylglycine is scarcely excreted in the urine of normal subjects.

Inflammatory response to acute myocardial infarction augments neointimal hyperplasia after vascular injury in a remote artery.

OBJECTIVE: Percutaneous coronary intervention (PCI) is currently the most widely accepted treatment for acute myocardial infarction (AMI). It remains unclear, however, whether post-AMI conditions might exacerbate neointimal hyperplasia and restenosis following PCI. Given that both a medial smooth muscle cell lineage and a bone marrow (BM)-derived hematopoietic stem cell lineage are now thought to contribute to neointima formation, the primary aims of the present study were to determine whether AMI augments neointimal hyperplasia at sites of arterial injury, and whether BM-derived cells contribute to that process. METHODS AND RESULTS: We simultaneously generated models of AMI and arterial injury in the same mice, some of which had received BM transplantation. We found that AMI augments neointimal hyperplasia at sites of femoral artery injury by approximately 35% (P<0.05), but that while BM-derived cells contributed to neointimal hyperplasia, they did not contribute to the AMI-related augmentation. Expression of interleukin (IL)-6 mRNA was approximately 7-fold higher in the neointimas of mice subjected to both AMI and arterial injury than in those of mice subjected to arterial injury alone. In addition, we observed increased synthesis of tumor necrosis factor (TNF)-alpha within infarcted hearts and TNF-alpha receptor type 1 (TNFR1) within injured arteries. Chronic treatment with pentoxifylline, which mainly inhibits TNF-alpha synthesis, reduced levels of circulating TNF-alpha and attenuated neointimal hyperplasia after AMI. CONCLUSIONS: Conditions after AMI could exacerbate postangioplasty restenosis, not by increasing mobilization of BM-derived cells, but by stimulating signaling via TNF-alpha, TNFR1 and IL-6.

Cloning of a cDNA encoding a novel marmoset CYP2C enzyme, expression in yeast cells and characterization of its enzymatic functions.

We cloned a cDNA encoding a novel CYP2C enzyme, called P450 M-2C, from a marmoset liver. The deduced amino acid sequence showed high identities to those of human CYP2C8 (87%), CYP2C9 (78%) and CYP2C19 (77%). The P450 M-2C enzyme expressed in yeast cells catalyzed p-methylhydroxylation of only tolbutamide among four substrates tested, paclitaxel as a CYP2C8 substrate, diclofenac and tolbutamide as CYP2C9 substrates and S-mephenytoin as a CYP2C19 substrate. p-Methylhydroxylation of tolbutamide by marmoset liver microsomes showed monophasic kinetics, and the apparent K(m) value (1.2 mM) for the substrate was similar to that of the recombinant P450 M-2C (1.8 mM). Although all of the recombinant human CYP2C8, CYP2C9 and CYP2C19 expressed in yeast cells catalyzed tolbutamide p-methylhydroxylation, the kinetic profile of CYP2C8 was most similar to that of P450 M-2C. Tolbutamide oxidation by the marmoset liver microsomes and the recombinant P450 M-2C was inhibited most effectively by quercetin, a CYP2C8 inhibitor, followed by omeprazole, a CYP2C19 inhibitor, whereas sulfaphenazole, a CYP2C9 inhibitor, was less potent under the conditions used. These results indicate that P450 M-2C is the major tolbutamide p-methylhydroxylase in the marmoset liver.

Ligand-activated PPARbeta efficiently represses the induction of LXR-dependent promoter activity through competition with RXR.

Angiopoietin-like protein 3 (angptl3), a member of the vascular endothelial growth factor family, was shown to play an important role in regulating lipid metabolism. To elucidate the mechanism by which PPARbeta represses angptl3 promoter activity, reporter constructs were prepared and transfection analysis carried out. PPARbeta repressed angptl3-Luc promoter activity and activation of PPARbeta by L-165041, a PPARbeta-specific ligand, increased the extent of repression. The repression by L-165041 was lost in angptl3-Luc plasmids having a deleted or mutated LXRalpha binding site (DR4). PPARbetaL405R, deficient in RXRalpha binding, had no effect on angptl3-Luc promoter activity. PPARbeta did not repress the activity of GAL4-LXRalpha which activates of GAL4DBD TK-Luc independent of RXR. Addition of RXRalpha completely abolished the repression of angptl3-Luc activity by PPARbeta. Mammalian two-hybrid analysis revealed that PPARbeta ligand binding enhanced the dissociation of the LXRalpha-RXRalpha heterodimer. Gel shift assays also indicated that PPARbeta ligand binding increased dissociation of LXRalpha/RXRalpha binding to a DR4 oligonucleotide probe; addition of RXRalpha restored the binding lost by addition of PPARbeta. Collectively, these results suggest that the binding of PPARbeta-specific ligand enhances the affinity between RXRalpha and activated PPARbeta and thus may regulate angptl3 gene expression through a DR4 element by competing with LXRalpha for RXRalpha.