Philip J Kingsley

Vanderbilt University, Nashville, MI, United States

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Publications (30)171.1 Total impact

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    ABSTRACT: Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in E. coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts whereas histidine and arginine do not. Nε-Oxopropenyllysine, a lysine-lysine cross-link and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is Nε-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA's ability to bind to a DNA substrate.
    Chemical Research in Toxicology 09/2014; · 3.67 Impact Factor
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    ABSTRACT: Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) utilize arachidonic acid for the synthesis of eicosanoids that have been implicated in carcinogenesis and cardiovascular disease. The ability of celecoxib, a selective COX-2 inhibitor, to redirect arachidonic acid into the 5-LO pathway can potentially reduce its efficacy as a chemopreventive agent and increase the risk of cardiovascular complications. Levels of urinary prostaglandin E metabolite (PGE-M) and leukotriene E4 (LTE4), biomarkers of the COX and 5-LO pathways, are elevated in smokers. Here we investigated the effects of zileuton, a 5-LO inhibitor, vs. zileuton and celecoxib for 6 ± 1 days on urinary PGE-M and LTE4 levels in smokers. Treatment with zileuton led to an 18% decrease in PGE-M levels (P=0.03); the combination of zileuton and celecoxib led to a 62% reduction in PGE-M levels (P<0.001). Levels of LTE4 decreased by 61% in subjects treated with zileuton alone (P<0.001) and were unaffected by the addition of celecoxib. Although zileuton use was associated with a small overall decrease in PGE-M levels, increased PGE-M levels were found in a subset (19/52) of subjects. Notably, the addition of celecoxib to the 5-LO inhibitor protected against the increase in urinary PGE-M levels (P=0.03). In conclusion, zileuton was an effective inhibitor of 5-LO activity resulting in marked suppression of urinary LTE4 levels and possible redirection of arachidonic acid into the COX-2 pathway in a subset of subjects. Combining celecoxib and zileuton was associated with inhibition of both the COX-2 and 5-LO pathways manifested as reduced levels of urinary PGE-M and LTE4.
    Cancer Prevention Research 05/2013; · 4.89 Impact Factor
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    ABSTRACT: Cannabinoid receptor type 1 (CB1)-induced suppression of transient receptor potential vanilloid type 1 (TRPV1) activation provides a therapeutic option to reduce inflammation and pain in different animal disease models through mechanisms involving dampening of TRPV1 activation and signaling events. As we found in both mouse corneal epithelium and human corneal epithelial cells (HCEC) that there is CB1 and TRPV1 expression colocalization based on overlap of coimmunostaining, we determined in mouse corneal wound healing models and in human corneal epithelial cells (HCEC) if they interact with one another to reduce TRPV1-induced inflammatory and scarring responses. Corneal epithelial debridement elicited in vivo a more rapid wound healing response in wildtype (WT) than in CB1(-/-) mice suggesting functional interaction between CB1 and TRPV1. CB1 activation by injury is tenable based on the identification in mouse corneas of 2-arachidonylglycerol (2-AG) with tandem LC-MS/MS, a selective endocannabinoid CB1 ligand. Suppression of corneal TRPV1 activation by CB1 is indicated since following alkali burning, CB1 activation with WIN55,212-2 (WIN) reduced immune cell stromal infiltration and scarring. Western blot analysis of coimmunoprecipitates identified protein-protein interaction between CB1 and TRPV1. Other immunocomplexes were also identified containing transforming growth factor kinase 1 (TAK1), TRPV1 and CB1. CB1 siRNA gene silencing prevented suppression by WIN of TRPV1-induced TAK1-JNK1 signaling. WIN reduced TRPV1-induced Ca(2+) transients in fura2-loaded HCEC whereas pertussis toxin (PTX) preincubation obviated suppression by WIN of such rises caused by capsaicin (CAP). Whole cell patch clamp analysis of HCEC showed that WIN blocked subsequent CAP-induced increases in nonselective outward currents. Taken together, CB1 activation by injury-induced release of endocannabinoids such as 2-AG downregulates TRPV1 mediated inflammation and corneal opacification. Such suppression occurs through protein-protein interaction between TRPV1 and CB1 leading to declines in TRPV1 phosphorylation status. CB1 activation of the GTP binding protein, G(i/o) contributes to CB1 mediated TRPV1 dephosphorylation leading to TRPV1 desensitization, declines in TRPV1-induced increases in currents and pro-inflammatory signaling events.
    Cellular Signalling 11/2012; · 4.47 Impact Factor
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    ABSTRACT: Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA). We recently reported that (R)-profens selectively inhibit endocannabinoid oxygenation but not arachidonic acid oxygenation. In this work, we synthesized achiral derivatives of five profen scaffolds and evaluated them for substrate-selective inhibition using in vitro and cellular assays. The size of the substituents dictated the inhibitory strength of the analogs, with smaller substituents enabling greater potency but less selectivity. Inhibitors based on the flurbiprofen scaffold possessed the greatest potency and selectivity, with desmethylflurbiprofen (3a) exhibiting an IC(50) of 0.11 μM for inhibition of 2-AG oxygenation. The crystal structure of desmethylflurbiprofen complexed to mCOX-2 demonstrated a similar binding mode to other profens. Desmethylflurbiprofen exhibited a half-life in mice comparable to that of ibuprofen. The data presented suggest that achiral profens can act as lead molecules toward in vivo probes of substrate-selective COX-2 inhibition.
    ACS Medicinal Chemistry Letters 09/2012; 3(9):759-763. · 3.31 Impact Factor
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    ABSTRACT: Prostaglandins (PGs) are powerful lipid mediators in many physiological and pathophysiological responses. They are produced by oxidation of arachidonic acid (AA) by cyclooxygenases (COX-1 and COX-2) followed by metabolism of endoperoxide intermediates by terminal PG synthases. PG biosynthesis is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). Specific inhibition of COX-2 has been extensively investigated, but relatively few COX-1-selective inhibitors have been described. Recent reports of a possible contribution of COX-1 in analgesia, neuroinflammation, or carcinogenesis suggest that COX-1 is a potential therapeutic target. We designed, synthesized, and evaluated a series of (E)-2'-des-methyl-sulindac sulfide (E-DMSS) analogues for inhibition of COX-1. Several potent and selective inhibitors were discovered, and the most promising compounds were active against COX-1 in intact ovarian carcinoma cells (OVCAR-3). The compounds inhibited tumor cell proliferation but only at concentrations >100-fold higher than the concentrations that inhibit COX-1 activity. E-DMSS analogues may be useful probes of COX-1 biology in vivo and promising leads for COX-1-targeted therapeutic agents.
    Journal of Medicinal Chemistry 03/2012; 55(5):2287-300. · 5.61 Impact Factor
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    ABSTRACT: Oxidative stress triggers DNA and lipid peroxidation, leading to the formation of electrophiles that react with DNA to form adducts. A product of this pathway, (3-(2'-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-10(3H)-one), or M(1)dG, is mutagenic in bacterial and mammalian cells and is repaired by the nucleotide excision repair pathway. In vivo, M(1)dG is oxidized to a primary metabolite, (3-(2-deoxy-β-d-erythro-pentofuranosyl)-pyrimido[1,2-α]purine-6,10(3H,5H)-dione, or 6-oxo-M(1)dG, which is excreted in urine, bile, and feces. We have developed a specific monoclonal antibody against 6-oxo-M(1)dG and have incorporated this antibody into a procedure for the immunoaffinity isolation of 6-oxo-M(1)dG from biological matrices. The purified analyte is quantified by LC-MS/MS using a stable isotope-labeled analogue ([(15)N(5)]-6-oxo-M(1)dG) as an internal standard. Healthy male Sprague-Dawley rats excreted 6-oxo-M(1)dG at a rate of 350-1893 fmol/kg·d in feces. This is the first report of the presence of the major metabolite of M(1)dG in rodents without exogenous introduction of M(1)dG.
    Chemical Research in Toxicology 01/2012; 25(2):454-61. · 3.67 Impact Factor
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    ABSTRACT: COX-2 is a major contributor to the inflammatory response and cancer progression so it is an important target for prevention and therapy. COX-2 is absent or expressed at low levels in most epithelial cells but is found at high levels in inflammatory lesions, and many premalignant and malignant tumors. Thus, it is an attractive target for molecular imaging. We report a series of novel fluorinated imaging agents, derived from indomethacin or celecoxib that selectively inhibit COX-2. The most promising lead, compound 7, was a fluorinated derivative of celecoxib. Kinetic analysis revealed that this fluorinated compound is a slow, tight-binding inhibitor of COX-2 and exhibits minimal inhibitory activity against COX-1. Efficient incorporation of (18)F into compound 7 by radiochemical synthesis and intravenous injection provided sufficient signal for in vivo positron emission tomography (PET) imaging. Selective uptake of (18)F-7 was observed in inflamed rat paws compared with the noninflamed contralateral paws and uptake was blocked by pretreatment with the COX-2 inhibitor, celecoxib. Uptake of (18)F-7 was not observed when inflammation was induced in COX-2-null mice. In nude mice bearing both a COX-2-expressing human tumor xenograft (1483) and a COX-2-negative xenograft (HCT116), (18)F-7 selectively accumulated in the COX-2-expressing tumor. Accumulation was blocked by pretreatment of the animals with celecoxib. The in vitro and in vivo properties of compound 7 suggest it will be a useful probe for early detection of cancer and for evaluation of the COX-2 status of premalignant and malignant tumors.
    Cancer Prevention Research 09/2011; 4(10):1536-45. · 4.89 Impact Factor
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    ABSTRACT: The antitumor effects of nonsteroidal anti-inflammatory drugs (NSAID) are assumed to be due to the inhibition of COX activity, but COX-independent mechanisms may also play an important role. NSAID-activated gene (NAG-1/GDF15) is induced by NSAIDs and has antitumorigenic activities. To determine the contribution of COX-2 inhibition and NAG-1/GDF15 expression to the prevention of colon carcinogenesis by NSAIDs, we evaluated several sulindac derivatives [des-methyl (DM)-sulindac sulfide and its prodrug DM-sulindac] that do not inhibit COX-2 activity. Sulindac sulfide and DM-sulindac induced the expression of NAG-1/GDF15 in HCT116 cells as determined by quantitative real-time PCR and Western blot. We fed APC/Min mice with 320 ppm of sulindac and doses of DM-sulindac. Only sulindac significantly inhibited tumor formation inAPC/Min mice. To determine the pharmacokinetic properties of sulindac and DM-sulindac in vivo, wild-type C57/B6 mice were fed with sulindac and DM-sulindac at 80, 160, and 320 ppm. High-performance liquid chromatography analysis revealed that the conversion of DM-sulindac to DM-sulindac sulfide (active form) was less efficient than the conversion of sulindac to sulindac sulfide (active form) in the mice. Lower levels of DM-sulindac sulfide accumulated in intestinal and colon tissues in comparison with sulindac sulfide. In addition, NAG-1/GDF15 was induced in the liver of sulindac-fed mice but not in the DM-sulindac-fed mice. Collectively, our results suggest that the tumor-inhibitory effects of sulindac in APC/Min mice may be due to, in part, NAG-1/GDF15 induction in the liver. Our study also suggests that pharmacologic properties should be carefully evaluated when developing drug candidates.
    Cancer Prevention Research 01/2011; 4(1):150-60. · 4.89 Impact Factor
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    ABSTRACT: Effective diagnosis of inflammation and cancer by molecular imaging is challenging because of interference from nonselective accumulation of the contrast agents in normal tissues. Here, we report a series of novel fluorescence imaging agents that efficiently target cyclooxygenase-2 (COX-2), which is normally absent from cells, but is found at high levels in inflammatory lesions and in many premalignant and malignant tumors. After either i.p. or i.v. injection, these reagents become highly enriched in inflamed or tumor tissue compared with normal tissue and this accumulation provides sufficient signal for in vivo fluorescence imaging. Further, we show that only the intact parent compound is found in the region of interest. COX-2-specific delivery was unambiguously confirmed using animals bearing targeted deletions of COX-2 and by blocking the COX-2 active site with high-affinity inhibitors in both in vitro and in vivo models. Because of their high specificity, contrast, and detectability, these fluorocoxibs are ideal candidates for detection of inflammatory lesions or early-stage COX-2-expressing human cancers, such as those in the esophagus, oropharynx, and colon.
    Cancer Research 05/2010; 70(9):3618-27. · 9.28 Impact Factor
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    Melissa V Turman, Philip J Kingsley, Lawrence J Marnett
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    ABSTRACT: N-(4-Hydroxyphenyl)arachidonoylamide (AM404) is an inhibitor of endocannabinoid inactivation that has been used in cellular and animal studies. AM404 is a derivative of arachidonic acid and has been reported to inhibit arachidonate oxygenation by prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and -2, respectively). While examining the structural requirements for inhibition of PGHS, we discovered that the meta isomer of AM404, N-(3-hydroxyphenyl)arachidonoylamide (3-HPAA), is a substrate for purified PGHS. PGHS-2 efficiently oxygenated 3-HPAA to prostaglandin and hydroxyeicosatetraenoate products. No oxidation of the phenolamide moiety was observed. 3-HPAA appeared to be converted by PGHS-1 in a similar manner; however, conversion was less efficient than that by PGHS-2. PGHS-2 was selectively, dose-dependently, and irreversibly inactivated in the presence of 3-HPAA. Complete inactivation of PGHS-2 was achieved with 10 muM 3-HPAA. Preliminary characterization revealed that 3-HPAA inactivation did not result from covalent modification of PGHS-2 or damage to the heme moiety. These studies provide additional insight into the structural requirements for substrate metabolism and inactivation of PGHS and report the first metabolism-dependent, selective inactivator of PGHS-2.
    Biochemistry 11/2009; 48(51):12233-41. · 3.38 Impact Factor
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    ABSTRACT: Psychosocial stress is a risk factor for development and exacerbation of neuropsychiatric illness. Repeated stress causes biochemical adaptations in endocannabinoid (eCB) signaling that contribute to stress-response habituation, however, the synaptic correlates of these adaptations have not been examined. Here, we show that the synthetic enzyme for the eCB 2-arachidonoylglycerol (2-AG), diacylglycerol (DAG) lipase alpha, is heterogeneously expressed in the amygdala, and that levels of 2-AG and precursor DAGs are increased in the basolateral amygdala (BLA) after 10 days, but not 1 day, of restraint stress. In contrast, arachidonic acid was decreased after both 1 and 10 days of restraint stress. To examine the synaptic correlates of these alterations in 2-AG metabolism, we used whole-cell electrophysiology to determine the effects of restraint stress on depolarization-induced suppression of inhibition (DSI) in the BLA. A single restraint stress exposure did not alter DSI compared with control mice. However, after 10 days of restraint stress, DSI duration, but not magnitude, was significantly prolonged. Inhibition of 2-AG degradation with MAFP also prolonged DSI duration; the effects of repeated restraint stress and MAFP were mutually occlusive. These data indicate that exposure to repeated, but not acute, stress produces neuroadaptations that confer BLA neurons with an enhanced capacity to elevate 2-AG content and engage in 2-AG-mediated short-term retrograde synaptic signaling. We suggest stress-induced enhancement of eCB-mediated suppression of inhibitory transmission in the BLA could contribute to affective dysregulation associated with chronic stress.
    Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 09/2009; 34(13):2699-709. · 8.68 Impact Factor
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    ABSTRACT: A novel series of iodinated indomethacin derivatives was synthesized, and evaluated as selective inhibitors of COX-2. Two candidate compounds N-(p-iodobenzyl)-2-(1-(p-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamide (3) and 1-(p-iodobenzyl)-5-methoxy-2-methyl-3-indoleacetic acid (9) possessed optimum properties suitable for potential in vivo imaging. Arylstannane precursors for radioiododestannylation were synthesized in 70–85% yield from the iodo compounds by reaction with hexabutylditin and tetrakis(triphenylphosphine)palladium(0) in refluxing dioxane. Radioiododestannylation was conducted by reaction with carrier-added Na[123I] in the presence of Chloramine-T in an EtOAc/H2O binary system under acidic conditions (pH 3.5), allowing direct isolation of the labeled products by separation of the organic phase. Radioiodinated products [123I]3 and [123I]9 were recovered in a decay-corrected radiochemical yield of 86–87% and radiochemical purity of 98–99%. Copyright © 2009 John Wiley & Sons, Ltd.
    Journal of Labelled Compounds 07/2009; 52(9):387 - 393.
  • Philip J Kingsley, Lawrence J Marnett
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    ABSTRACT: Since the discovery of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) in the early 1990s, the endocannabinoid system has been implicated in a wide array of physiological processes, such as control of food intake and energy balance, fertility and obesity. As the importance of this system becomes apparent, there is a tremendous need for robust, sensitive and efficient analytical methodology for the examination of the endocannabinoids, their congeners and putative metabolites. This review will summarize quantitative analytical methodology as reported in the literature from 1992 to present for the analysis of endocannabinoids and related compounds.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 06/2009; 877(26):2746-54. · 2.78 Impact Factor
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    ABSTRACT: Sulindac sulfide is a benzylidene-indene that is a potent, time-dependent inhibitor of cyclooxygenases-1 and -2. Removal of the 2'-methyl group from the indene ring dramatically reduces time-dependent inhibition of both enzymes but also changes the geometry of the benzylidene double bond from Z to E. Herein, we explore the importance of double bond geometry on cyclooxygenase inhibition. The Z-isomer of 2'-des-methyl sulindac sulfide was synthesized by reduction of a bromoindene precursor or by photoisomerization of the E-isomer. The Z-isomer inhibited both cyclooxygenases, but with diminished potency compared to sulindac sulfide. Thus, although the 2'-methyl group is a major determinant of time-dependent cyclooxygenase inhibition, the geometry of the benzylidene double bond plays a role as well.
    Bioorganic & medicinal chemistry letters 05/2009; 19(12):3271-4. · 2.65 Impact Factor
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    ABSTRACT: Marijuana is the most commonly used illicit drug. Although there is some indication that reproductive functions in males are impaired in chronic marijuana users, the genetic evidence and underlying causes remain largely unknown. Herein we show that genetic loss of Faah, which encodes fatty acid amide hydrolase (FAAH), results in elevated levels of anandamide, an endocannabinoid, in the male reproductive system, leading to compromised fertilizing capacity of sperm. This defect is rescued by superimposing deletion of cannabinoid receptor 1 (Cnr1). Retention of Faah(-/-) sperm on the egg zona pellucida provides evidence that the capacity of sperm to penetrate the zona barrier is hampered by elevated anandamide levels. Collectively, the results show that aberrant endocannabinoid signaling via CNR1 impairs normal sperm function. Besides unveiling a new regulatory mechanism of sperm function, this study has clinical significance in male fertility.
    Biology of Reproduction 12/2008; 80(2):235-42. · 4.03 Impact Factor
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    ABSTRACT: Pharmacological inhibition or genetic deletion of cyclooxygenase (COX)-2, but not COX-1, has been shown to increase susceptibility to kainic acid (KA)-induced excitotoxicity. However, it is unclear if susceptibility to excitotoxins that act through other neurotransmitter receptors is altered by COX-2 inhibition. To further understand the involvement of COX-2 in regulating susceptibility to excitotoxicity, we investigated the effect of COX-2 deletion on excitotoxicity induced by peripheral injection of N-methyl-d-aspartate (NMDA, a specific agonist of the NMDA receptors) or lindane (a GABA(A) receptor antagonist). COX-2(-/-) mice injected intraperitoneally with NMDA (50-100mg/kg) exhibited significantly increased median seizure intensity when compared to COX-2(+/+) mice. Further, COX-2(-/-) mice exposed to NMDA showed neuronal damage, detected by Fluoro Jade B (FJB) staining, in the CA3 region of the hippocampus. There was no FJB staining nor any significant difference in median or maximal seizure intensity in COX-2(+/+) and COX-2(-/-) mice exposed to lindane. LC-MS/MS analysis of brain prostaglandin profile in COX-2(-/-) mice demonstrated a significant increase in PGF(2alpha), TXB(2), PGE(2) and PGD(2) expression 1h after administration of an excitotoxic dose of KA, but not of NMDA. Our findings demonstrate that COX-2 regulates susceptibility to KA and NMDA excitotoxicity, which directly activate glutamatergic neurotransmission, but not to lindane, which indirectly alters glutamatergic neurotransmission. Furthermore, increased levels of prostaglandins after seizures are associated with consistent manifestation of neuronal damage.
    NeuroToxicology 10/2008; 29(6):1114-20. · 2.65 Impact Factor
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    ABSTRACT: A novel class of lipids, N-acyltaurines, was recently discovered in fatty acid amide hydrolase knockout mice. In some peripheral tissues, such as liver and kidney, N-acyltaurines with long, polyunsaturated acyl chains are most prevalent. Polyunsaturated fatty acids are converted to a variety of signaling molecules by cyclooxygenases (COXs) and lipoxygenases (LOXs). The ability of COXs and LOXs to oxygenate arachidonoyltaurine was evaluated to gain insight into the potential metabolic fate of N-acyltaurines. Although arachidonoyltaurine was a poor substrate for COXs, mammalian 12 S- and 15 S-LOXs oxygenated arachidonoyltaurine with similar or better efficiency than arachidonic acid. Products of arachidonoyltaurine oxygenation were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The positional specificity of single oxygenation was retained for 15 S-LOXs. However, platelet-type 12 S-LOX produced 12- and 15-hydroxyeicosatetraenoyltaurines (HETE-Ts). Furthermore, LOXs generated dihydroxyeicosatetraenoyltaurines (diHETE-Ts). Metabolism of arachidonoyltaurine by murine resident peritoneal macrophages (RPMs) was also profiled. Arachidonoyltaurine was rapidly taken up and converted primarily to 12-HETE-T. Over prolonged incubations, RPMs also generated small amounts of diHETE-T. Oxidative metabolism of polyunsaturated N-acyltaurines may represent a pathway for the generation or termination of novel signaling molecules.
    Biochemistry 04/2008; 47(12):3917-25. · 3.38 Impact Factor
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    ABSTRACT: Although mainly expressed in neuronal cells, lipocalin-type PGD synthase (L-PGDS) is detected in the macrophages infiltrated to atherosclerotic plaques. However, the regulation and significance of L-PGDS expression in macrophages are unknown. Here, we found that treatment of macrophages with bacterial endotoxin (LPS) or Pseudomonas induced L-PGDS expression. Epigenetic suppression of L-PGDS expression in macrophages blunted a majority of PGD(2) produced after LPS treatment. Chromatin immunoprecipitation assays show that L-PGDS induction was regulated positively by AP-1, but negatively by p53. L-PGDS expression was detected in whole lung and alveolar macrophages treated with LPS or Pseudomonas. L-PGDS overexpressing transgenic mice improved clearance of Pseudomonas from the lung compared with nontransgenic mice. Similarly, intratracheal instillation of PGD(2) enhanced removal of Pseudomonas from the lung in mice. In contrast, L-PGDS knockout mice were impaired in their ability to remove Pseudomonas from the lung. Together, our results identify induction of L-PGDS expression by inflammatory stimuli or bacterial infection, the regulatory mechanism of L-PGDS induction, and the protective role of L-PGDS expression in host immune response. Our study suggests a potential therapeutic usage of L-PGDS or PGD(2) against Pseudomonas pneumonia.
    The Journal of Immunology 09/2007; 179(4):2565-75. · 5.52 Impact Factor
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    ABSTRACT: Cyclooxygenases (COX) have been implicated in the etiology of a number of diseases, but defining the precise contribution of COXs to these diseases is challenging. Potent COX inhibitors exist, but they display off-target effects. 2'-Desmethyl derivatives of indomethacin and sulindac sulfide were synthesized that demonstrated reduced COX inhibitory activity but were inducers of peroxisome proliferator-activated receptor gamma-dependent transcription, adipocyte differentiation, or apoptosis of colon cancer cell lines. 2'-Desmethylindomethacin demonstrated gastrointestinal toxicity lower than that of indomethacin in C57BL6 mice, highlighting the importance of COX activity in maintaining gastrointestinal homeostasis and establishing that COX inhibition contributes to gastrointestinal toxicity by nonsteroidal antiinflammatory drugs. These compounds serve as useful probes of COX-dependent biology and may represent leads for antidiabetic and anticancer drugs.
    ACS Chemical Biology 08/2007; 2(7):479-83. · 5.44 Impact Factor
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    ABSTRACT: Preimplantation embryo development to the blastocyst stage and uterine differentiation to the receptive state are prerequisites for embryo implantation. Burgeoning evidence suggests that endocannabinoid signaling is critical to early pregnancy events. Anandamide (N-arachidonoylethanolamine) and 2-AG (2-arachidonoylglycerol) are two major endocannabinoids that bind to and activate G-protein coupled cannabinoid receptors CB1 and CB2. We have previously shown that a physiological tone of anandamide is critical to preimplantation events in mice, since either silencing or amplification of anandamide signaling causes retarded development and oviductal retention of embryos via CB1, leading to deferred implantation and compromised pregnancy outcome. Whether 2-AG, which also influences many biological functions, has any effects on early pregnancy remains unknown. Furthermore, mechanisms by which differential uterine endocannabinoid gradients are established under changing pregnancy state is not clearly understood. We show here that 2-AG is present at levels one order of magnitude higher than those of anandamide in the mouse uterus, but with similar patterns as anandamide, i.e. lower levels at implantation sites and higher at interimplantation sites. We also provide evidence that region- and stage-specific uterine expression of N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), and sn-1-diacylglycerol (DAG) lipase alpha (DAGLalpha) and monoacylglycerol lipase (MAGL) for synthesis and hydrolysis of anandamide and 2-AG, respectively, creates endocannabinoid gradients conducive to implantation. Our genetic evidence suggests that FAAH is the major degrading enzyme for anandamide, whereas COX-2, MAGL and to some extent COX-1 participate in metabolizing 2-AG in the pregnant uterus. The results suggest that aberrant functioning of these pathways impacting uterine anandamide and/or 2-AG levels would compromise pregnancy outcome.
    Prostaglandins & other lipid mediators 03/2007; 83(1-2):62-74. · 2.42 Impact Factor