Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
Bioorg Med Chem Lett ; 22(12): 4133-8, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22578458
2.
Bioorg Med Chem Lett ; 19(11): 3059-62, 2009 Jun 01.
Article in English | MEDLINE | ID: mdl-19394221

ABSTRACT

Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone analogs with modified ring structure. In one representative chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates two new carboxylic groups on the molecule. These groups served as reaction sites for further derivatization involving biocatalytic ring closure reactions with structurally diverse bifunctional reagents, including different diols and diamines. As a result, a library of cyclic bislactones and bislactams was created, with modified ring structures covering chemical space and structure activity relationships unattainable by conventional synthetic means.


Subject(s)
Zearalenone/chemistry , Biocatalysis , Drug Design , Enzymes/metabolism , Lipase/metabolism , Structure-Activity Relationship , Zearalenone/biosynthesis
3.
Anal Biochem ; 349(1): 129-35, 2006 Feb 01.
Article in English | MEDLINE | ID: mdl-16325756

ABSTRACT

Current immunoassays for the measurement of leukotriene B(4) (LTB(4)) typically utilize an enzyme-linked immunosorbent assay (ELISA) format that requires multiple incubations and washing steps and often expensive immunoassay kits. We have developed a bead-based, mix and read, indirect fluorescence-linked immunosorbent assay utilizing fluorometric microvolume assay technology (FMAT). The assay employs a monoclonal anti-LTB(4) antibody-coated onto goat antimouse antibody coupled polystyrene beads and an AlexaFluor-647-coupled LTB(4) ligand. Because the FMAT measurement is made only in the portion of the well volume containing the settled beads coated with AF647-LTB(4), the free label in the solution is not measured. Similarly, substances present in plasma that interfere with other immunoassays are largely ignored. The assay is robust (Z=0.8; S/N=250) and can be measured in the presence of relatively high concentrations of dimethyl sulfoxide or serum. It is inexpensive (<0.10 dollars/assay) and amenable to robotics and has a sensitivity comparable to that of the most sensitive ELISA assays; the concentration of LTB(4) giving 50% inhibition (IC(50)) was ca. 55pg/ml. Cross-reactivity in the FMAT assay was comparable to that of the ELISA assay with significant cross-reactivity found only with 20-hydroxy LTB(4) and 12-epi LTB(4). Measurements of LTB(4) determined by FMAT were equivalent to those measured by standard ELISA in samples of ionophore-stimulated human neutrophils or whole blood.


Subject(s)
Fluorometry , Immunoassay , Leukotriene B4/analysis , Humans , Immunoassay/methods , Leukotriene B4/blood , Neutrophils/metabolism
4.
DNA Cell Biol ; 23(8): 519-26, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15307955

ABSTRACT

Polyunsaturated fatty acids (PUFAs) have been previously reported as agonists of peroxisome proliferatoractivated receptor and antagonists of the liver X receptor. The activities on these two nuclear receptors have been attributed to their beneficial effects such as improvement of dyslipidemia and insulin sensitivity and decrease of hepatic lipogenesis. Here we report that PUFAs are ligands of farnesoid X receptor (FXR), a nuclear receptor for bile acids. In a conventional FXR binding assay, arachidonic acid (AA, 20:4), docosahexaenoic acid (DA, 22:6), and linolenic acid (LA, 18:3) had an affinity of 2.6, 1.5, and 3.5 microM, respectively. In a cell-free coactivator association assay, AA, DA, and LA decreased FXR agonist-induced FXR activation with IC(50)s ranging from 0.9 to 4.7 microM. In HepG2 cells, PUFAs regulated the expression of two FXR targets, BSEP and kininogen, in an opposite fashion, although both genes were transactivated by FXR. All three PUFAs dose-dependently enhanced FXR agonist-induced BSEP expression but decreased FXR agonist-induced human kininogen mRNA. Saturated fatty acids such as stearic acid (SA, 18:0) and palmitic acid (PA, 16:0) did not bind to FXR and did not change BSEP or kininogen expression. The pattern of BSEP and kininogen regulation by PUFAs is closely similar to that of the guggulsterone, previously reported as a selective bile acid receptor modulator. Our results suggest that PUFAs may belong to the same class of FXR ligands as guggulsterone, and that the selective regulation of FXR targets may contribute to the beneficial effects of PUFAs in lipid metabolism.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , DNA-Binding Proteins/metabolism , Fatty Acids, Unsaturated/pharmacology , Gene Expression Regulation/drug effects , Kininogens/metabolism , RNA, Messenger/metabolism , Transcription Factors/metabolism , Arachidonic Acid/metabolism , Cell Line, Tumor , DNA Primers , DNA-Binding Proteins/agonists , Docosahexaenoic Acids/metabolism , Fatty Acids, Unsaturated/metabolism , Fluorescence , Humans , Ligands , Polymerase Chain Reaction/methods , Receptors, Cytoplasmic and Nuclear , Transcription Factors/agonists , alpha-Linolenic Acid/metabolism
5.
Arterioscler Thromb Vasc Biol ; 24(10): 1783-8, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15271789

ABSTRACT

OBJECTIVE: Leukotriene B4 (LTB4), a product of the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism, has been implicated in atherosclerosis. However, the molecular mechanisms for the atherogenic effect of LTB4 are not well understood. This study is to determine candidate mechanisms. METHOD AND RESULTS: Primary human monocytes were treated with LTB4 and the supernatant was analyzed for cytokine/chemokine production by an immuno-protein array. This analysis revealed a strong increase of the monocyte chemoattractant protein-1 (MCP-1), a proinflammatory cytokine. Follow-up analyses with MCP-1 enzyme-linked immunosorbent assay (for quantitation of MCP-1 protein) and real-time polymerase chain reaction (PCR) (for MCP-1 mRNA) demonstrated that LTB4 strongly induced expression of MCP-1 protein and mRNA in a time-dependent and dose-dependent fashion. This induction was effectively abolished by CP-105,696, an antagonist for the LTB4 receptor BLT1. Selective inhibitors of ERK1/2 or JNK MAPK effectively blocked the LTB4-induced MCP-1 production. Furthermore, LTB4 increased NF-[kappa]B DNA binding activity, which was blocked by CP-105,696. CONCLUSIONS: LTB4 strongly induces MCP-1 production in primary human monocytes. This induction is mediated through the BLT1 pathway increasing MCP-1 transcription. Activation of ERK1/2 or JNK MAPK is essential for this induction. The NF-[kappa]B activation may be involved in LTB4-increased MCP-1 expression. The LTB4-induced MCP-1 in human monocytes may play a critical role in the atherogenicity of LTB4.


Subject(s)
Chemokine CCL2/biosynthesis , Leukotriene B4/pharmacology , Monocytes/drug effects , Benzopyrans/pharmacology , Carboxylic Acids/pharmacology , Cells, Cultured , Chemokine CCL2/genetics , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Monocytes/enzymology , NF-kappa B/metabolism , Receptors, Leukotriene B4/antagonists & inhibitors , Transcription, Genetic/drug effects
6.
J Biol Chem ; 279(10): 8856-61, 2004 Mar 05.
Article in English | MEDLINE | ID: mdl-14684751

ABSTRACT

Farnesoid X receptor (FXR) is a nuclear receptor for bile acids. Ligand activated-FXR regulates transcription of genes to allow feedback control of bile acid synthesis and secretion. There are five major bile acids in humans. We have previously demonstrated that lithocholate acts as an FXR antagonist, and here we show that the other four bile acids, chenodeoxycholate (CDCA), deoxycholate (DCA), cholate (CA), and ursodeoxycholate (UDCA), act as selective FXR agonists in a gene-specific fashion. In an in vitro coactivator association assay, CDCA fully activated FXR, whereas CA partially activated FXR and DCA and UDCA had negligible activities. Similar results were also obtained from a glutathione S-transferase pull-down assay in which only CDCA and the synthetic FXR agonist GW4064 significantly increased the interaction of SRC-1 with FXR. In FXR transactivation assays with a bile salt export pump (BSEP) promoter-driven luciferase construct, bile acids showed distinct abilities to activate the BSEP promoter: CDCA, DCA, CA, and UDCA increased luciferase activity by 25-, 20-, 18-, and 8-fold, respectively. Consistently, CDCA increased BSEP mRNA by 750-fold in HepG2 cells, whereas DCA, CA, and UDCA induced BSEP mRNA by 250-, 75-, and 15-fold, respectively. Despite the partial induction of BSEP mRNA, CA, DCA, and UDCA effectively repressed expression of cholesterol 7alpha-hydroxylase, another FXR target. We further showed that all four bile acids significantly increased FXR protein, suggesting the existence of an auto-regulatory loop in FXR signaling pathways. In conclusion, these results suggest that the binding of each bile acid results in a different FXR conformations, which in turn differentially regulates expression of individual FXR targets.


Subject(s)
Gene Expression Regulation , Receptors, Cytoplasmic and Nuclear/genetics , Signal Transduction/genetics , Cell Line , Chenodeoxycholic Acid/metabolism , Chenodeoxycholic Acid/pharmacology , Cholates/metabolism , Cholates/pharmacology , DNA-Binding Proteins , Deoxycholic Acid/metabolism , Deoxycholic Acid/pharmacology , Humans , Promoter Regions, Genetic , Protein Conformation/drug effects , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/chemistry , Signal Transduction/drug effects , Transcription Factors , Ursodeoxycholic Acid/metabolism , Ursodeoxycholic Acid/pharmacology
7.
J Biol Chem ; 278(51): 51085-90, 2003 Dec 19.
Article in English | MEDLINE | ID: mdl-14527955

ABSTRACT

The human multidrug resistance gene MDR3 encodes a P-glycoprotein that belongs to the ATP-binding cassette transporter family (ABCB4). MDR3 is a critical trans-locator for phospholipids across canalicular membranes of hepatocytes, evidenced by the fact that human MDR3 deficiencies result in progressive familial intrahepatic cholestasis type III. It has been reported previously that MDR3 expression is modulated by hormones, cellular stress, and xenobiotics. Here we show that the MDR3 gene is trans-activated by the farnesoid X receptor (FXR) via a direct binding of FXR/retinoid X receptor alpha heterodimers to a highly conserved inverted repeat element (a FXR response element) at the distal promoter (-1970 to -1958). In FXR trans-activation assays, both the endogenous FXR agonist chenodeoxycholate and the synthetic agonist GW4064 activated the MDR3 promoter. Deletion or mutation of this inverted repeat element abolished FXR-mediated MDR3 promoter activation. Consistent with these data, MDR3 mRNA was significantly induced by both chenodeoxycholate and GW4064 in primary human hepatocytes in time- and dose-dependent fashions. In conclusion, we demonstrate that MDR3 expression is directly up-regulated by FXR. These results, together with the previous report that the bile salt export pump is a direct FXR target, suggest that FXR coordinately controls secretion of bile salts and phospholipids. Results of this study further support the notion that FXR is a master regulator of lipid metabolism.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/biosynthesis , ATP-Binding Cassette Transporters/biosynthesis , Phospholipid Transfer Proteins , Receptors, Cytoplasmic and Nuclear/physiology , Transcriptional Activation , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP-Binding Cassette Transporters/genetics , Base Sequence , Carrier Proteins , Cell Line, Tumor , DNA-Binding Proteins , Hepatocytes , Humans , Ligands , Membrane Proteins , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors , Transcription Factors/metabolism
8.
J Biol Chem ; 278(31): 28765-70, 2003 Aug 01.
Article in English | MEDLINE | ID: mdl-12761213

ABSTRACT

Human kininogen belongs to the plasma kallikreinkinin system. High molecular weight kininogen is the precursor for two-chain kinin-free kininogen and bradykinin. It has been shown that the two-chain kinin-free kininogen has the properties of anti-adhesion, anti-platelet aggregation, and anti-thrombosis, whereas bradykinin is a potent vasodilator and mediator of inflammation. In this study we show that the human kininogen gene is strongly up-regulated by agonists of the farnesoid X receptor (FXR), a nuclear receptor for bile acids. In primary human hepatocytes, both the endogenous FXR agonist chenodeoxycholate and synthetic FXR agonist GW4064 increased kininogen mRNA with a maximum induction of 8-10-fold. A more robust induction of kininogen expression was observed in HepG2 cells, where kininogen mRNA was increased by chenodeoxycholate or GW4064 up to 130-140-fold as shown by real time PCR. Northern blot analysis confirmed the up-regulation of kininogen expression by FXR agonists. To determine whether kininogen is a direct target of FXR, we examined the sequence of the kininogen promoter and identified a highly conserved FXR response element (inverted repeat, IR-1) in the proximity of the kininogen promoter (-66/-54). FXR/RXRalpha heterodimers specifically bind to this IR-1. A construct of a minimal promoter with the luciferase reporter containing this IR-1 was transactivated by FXR. Deletion or mutation of this IR-1 abolished FXR-mediated promoter activation, indicating that this IR-1 element is responsible for the promoter transactivation by FXR. We conclude that kininogen is a novel and direct target of FXR, and bile acids may play a role in the vasodilation and anti-coagulation processes.


Subject(s)
DNA-Binding Proteins/physiology , Kininogens/genetics , Transcription Factors/physiology , Transcriptional Activation , Binding Sites , Blotting, Northern , Carcinoma, Hepatocellular/metabolism , Chenodeoxycholic Acid/pharmacology , DNA/metabolism , DNA-Binding Proteins/agonists , Gene Deletion , Gene Expression Regulation/drug effects , Hepatocytes/metabolism , Humans , Isoxazoles/pharmacology , Liver Neoplasms/metabolism , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , RNA, Messenger/analysis , Receptors, Cytoplasmic and Nuclear , Receptors, Retinoic Acid/physiology , Repetitive Sequences, Nucleic Acid , Retinoid X Receptors , Transcription Factors/agonists , Transfection , Tumor Cells, Cultured
9.
J Biol Chem ; 278(12): 10214-20, 2003 Mar 21.
Article in English | MEDLINE | ID: mdl-12525500

ABSTRACT

Guggulipid is an extract of the guggul tree Commiphora mukul and has been widely used to treat hyperlipidemia in humans. The plant sterol guggulsterone (GS) is the active agent in this extract. Recent studies have shown that GS can act as an antagonist ligand for farnesoid X receptor (FXR) and decrease expression of bile acid-activated genes. Here we show that GS, although an FXR antagonist in coactivator association assays, enhances FXR agonist-induced transcription of bile salt export pump (BSEP), a major hepatic bile acid transporter. In HepG2 cells, in the presence of an FXR agonist such as chenodeoxycholate or GW4064, GS enhanced endogenous BSEP expression with a maximum induction of 400-500% that induced by an FXR agonist alone. This enhancement was also readily observed in FXR-dependent BSEP promoter activation using a luciferase reporter construct. In addition, GS alone slightly increased BSEP promoter activation in the absence of an FXR agonist. Consistent with the results in HepG2, guggulipid treatment in Fisher rats increased BSEP mRNA. Interestingly, in these animals expression of the orphan nuclear receptor SHP (small heterodimer partner), a known FXR target, was also significantly increased, whereas expression of other FXR targets including cholesterol 7alpha-hydroxylase (Cyp 7a1), sterol 12alpha-hydroxylase (Cyp 8b1), and the intestinal bile acid-binding protein (I-BABP), remained unchanged. Thus, we propose that GS is a selective bile acid receptor modulator that regulates expression of a subset of FXR targets. Guggulipid treatment in rats lowered serum triglyceride and raised serum high density lipoprotein levels. Taken together, these data suggest that guggulsterone defines a novel class of FXR ligands characterized by antagonist activities in coactivator association assays but with the ability to enhance the action of agonists on BSEP expression in vivo.


Subject(s)
ATP-Binding Cassette Transporters/genetics , DNA-Binding Proteins/antagonists & inhibitors , Pregnenediones/pharmacology , Transcription Factors/antagonists & inhibitors , Transcription, Genetic/drug effects , ATP Binding Cassette Transporter, Subfamily B, Member 11 , Animals , Bile Acids and Salts/metabolism , Cholesterol, HDL/blood , DNA-Binding Proteins/analysis , Humans , Male , Promoter Regions, Genetic , Protein Isoforms , Rats , Rats, Inbred F344 , Receptors, Cytoplasmic and Nuclear , Transcription Factors/analysis , Triglycerides/blood , Tumor Cells, Cultured
10.
J Org Chem ; 67(14): 5001-4, 2002 Jul 12.
Article in English | MEDLINE | ID: mdl-12098324

ABSTRACT

Neuropeptide Y (NPY) is a polypeptide found in the peripheral and central nervous system and is involved in the regulation of feeding. Antagonists of NPY receptor activation could therefore have potential for development as antiobesity drugs. Fermentation of an isolate of Xylaria persicaria yielded two novel eremophilane sesquiterpenoids xylarenals A (1) and B (2). These compounds are selective for the NPY Y5 receptor but have only modest affinity. The isolation, structure elucidation, and biological activities of these compounds are described.


Subject(s)
Ascomycota/chemistry , Neuropeptide Y/metabolism , Receptors, Neuropeptide Y/antagonists & inhibitors , Receptors, Neuropeptide Y/drug effects , Sesquiterpenes/isolation & purification , Animals , Anti-Obesity Agents , Chromatography, High Pressure Liquid , Inhibitory Concentration 50 , Mice , Molecular Structure , Neurotransmitter Agents , New Jersey , Nuclear Magnetic Resonance, Biomolecular , Sesquiterpenes/chemistry , Sesquiterpenes/pharmacology
11.
J Biol Chem ; 277(35): 31441-7, 2002 Aug 30.
Article in English | MEDLINE | ID: mdl-12052824

ABSTRACT

Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.


Subject(s)
ATP-Binding Cassette Transporters/genetics , DNA-Binding Proteins/antagonists & inhibitors , Gene Expression Regulation, Neoplastic/drug effects , Isoxazoles/pharmacology , Lithocholic Acid/pharmacology , Transcription Factors/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 11 , ATP-Binding Cassette Transporters/metabolism , Cloning, Molecular , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Escherichia coli/genetics , Glutathione Transferase/metabolism , Humans , Mixed Function Oxygenases/genetics , Polymerase Chain Reaction , RNA, Messenger/genetics , Receptors, Cytoplasmic and Nuclear , Recombinant Fusion Proteins/metabolism , Rifampin/pharmacology , Transcription, Genetic/drug effects , Transfection , Tumor Cells, Cultured
SELECTION OF CITATIONS
SEARCH DETAIL
...