Prostaglandin Endoperoxide Research Articles

Nonsteroidal Anti-Inflammatory Drugs and the Heart

Aspirin has been on the market for 115 years. Beginning with the marketing of indomethacin for the treatment of rheumatoid arthritis in 1963, at least 20 other nonsteroidal anti-inflammatory drugs (NSAIDs) with aspirin-like actions have been developed over the past 50 years,1 culminating with the introduction of a new class of selective inhibitors of cyclooxygenase (COX)-2, the coxibs, approximately 15 years ago.2 The NSAIDs represent the single most crowded family of drugs sharing the same therapeutic activities and mechanism of action, perhaps reflecting the unmet therapeutic need in the area of pain management and the large interindividual variability in response to these agents. NSAIDs provide symptomatic relief of pain and inflammation associated with a variety of human disorders, including the rheumatic diseases. Their shared therapeutic actions (ie, analgesic, anti-inflammatory, and antipyretic) are usually accompanied by mechanism-based adverse effects that can, at least in part, be attenuated as a function of individual pharmacokinetic or pharmacodynamic properties.1 Nuances in the tolerability of different NSAIDs had been described in the precoxib era on the basis of clinical trials of a few hundred patients treated for up to a few months with soft end points. More recently, however, important differences in safety have been demonstrated in head-to-head randomized comparisons of individual coxibs and 1 or more traditional NSAIDs that involved tens of thousands of patients treated for up to a few years with hard end points. Even more significantly, the interpretation of the effects of NSAIDs has been greatly enhanced by the availability, for the first time, of large, placebo-controlled trials that aimed to assess the potential for chemoprevention of colorectal cancer by rofecoxib or celecoxib. As a result of these developments, the whole field of NSAIDs has been illuminated during the past 15 years. Although epidemiological studies had previously associated …

In Vivo Chronic and In Vitro Acute Effects of Di(2-Ethylhexyl) Phthalate on Pseudopregnant Rabbit Corpora Lutea: Possible Involvement of Peroxisome Proliferator-Activated Receptor Gamma1

The in vivo chronic and in vitro acute effects of di(2-ethylhexyl) phthalate (DEHP) on the reproductive function of peroxisome proliferator-activated receptor gamma (PPARG) were studied in rabbit corpora lutea (CL) at early stage (Day 4), midstage (Day 9), and late stage (Day 13) of pseudopregnancy. The rabbits were in vivo treated with DEHP for 15 days before induction of pseudopregnancy. Immunohistochemistry provided evidence for the presence of PPARG, prostaglandin endoperoxide synthase 1 (PTGS1), PTGS2, prostaglandin E2-9-ketoreductase (PGE2-9-K), and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in all the luteal cells during pseudopregnancy. DEHP decreased progesterone plasma levels and CL production in all the luteal stages and PPARG protein and gene expressions in early and mid-CL. DEHP in vivo treatment reduced PTGS2 protein expression at the late stage and that of PGE2-9-K at all the stages, whereas PTGS1 and 3beta-HSD were not affected. In in vitro cultured CL, DEHP alone, the PPARG antagonist T0070907 alone, or DEHP plus T0070907 diminished progesterone production and 3beta-HSD activity and increased PGF2alpha and PTGS2 in early and mid-CL, whereas DEHP plus the PPARG agonist 15d-PGJ2 did not affect these hormones and enzymes. All the in vitro treatments did not affect PGE2 secretion as well as PTGS1 and PGE2-9-K enzymatic activities in all the luteal stages. These results provided evidence that DEHP favors functional luteolysis of pseudopregnant rabbit CL, with a mechanism that seems to involve PPARG expression down-regulation, an increase of PTGS2 activity and prostaglandin F2alpha secretion, 3beta-HSD down-regulation, and decrease in progesterone.

Tumour angiogenesis in Epstein-Barr virus-associated post-transplant smooth muscle tumours

Epstein-Barr virus (EBV)-associated post-transplant smooth muscle tumours (PTSMT), are rare complications following organ/stem cell transplantation. Despite the mainly benign behaviour of PTSMT, alternative therapies are needed for those patients with progressive tumours. In tumours not approachable by surgery or reduction of immunosuppression, the angiogenic microenvironment might be a potential target of therapy, an approach that is well utilised in other soft tissue neoplasms. In a previous study, we evaluated the expression of EBV-related genes and the microRNA profile in PTSMT, but so far the characteristics of angiogenesis in PTSMT are not known. Therefore, the aim of this study was to evaluate the expression pattern of angiogenesis-related genes in PTSMT, in order to identify potential target molecules for anti-angiogenic therapy.PTSMT (n = 5 tumours) were compared with uterine leiomyomas (n = 7). Analyses included real-time PCR of 45 angiogenesis-associated genes, immunohistochemistry (CD31, prostaglandin endoperoxide synthase 1/PTGS1) and assessment of tumour vascularisation by conventional histopathology.PTSMT showed similar or fewer vessels than leiomyomas. Of the genes under investigation, 23 were down-deregulated (pro-angiogenic and some anti-angiogenic factors) and five were up-regulated (e.g. PTGS1 which is expressed at very low levels in leiomyomas but moderately higher levels in PTSMT).In summary, no particular target molecule could be identified, because tumour angiogenesis in PTSMT is characterised by low levels of major pro-angiogenic factors and there is no prominent increase in tumour vascularisation. EBV can induce angiogenesis via its viral late membrane protein 1 (LMP1) but PTSMT frequently do not express LMP1, which could be an explanation why, despite EBV infection, PTSMT show no exaggerated tumour angiogenesis.

An Asymmetric Pattern in Binding of Prostaglandin Endoperoxide H Synthases to their Inhibitors and its Implications for Enzyme Catalysis and Allosteric Regulation

Proteins experience some conformational changes upon ligand bindings and their effects on protein functions are displayed in various ways. Crystallographic data of protein/ligand complexes are essential in exploring this issue; structural determinations of the complexes of interest, however, are often challenging and docking procedure can be carefully designed as an adequate substitute. Prostaglandin endoperoxide H synthases (PGHSs), also known as cyclooxygenases, have been investigated recently in an attempt to understand their half-of-sites enzyme activity and consequent biological significance. PGHSs are heme-containing proteins exist in solution as sequence homodimer, but once their catalytic cycle of cyclooxygenase/peroxidase is initiated, they behave functionally as a heterodimeric manner. Biochemical approach to solve this puzzle has been intensively attempted and suggested their interchangeability of structurally distinct two monomers. This paper presents an analysis of PGHS-ligand interactions upon binding either experimentally or virtually and predicts its biological implications.

Expression of HLA Class I Antigen, Aspirin Use, and Survival After a Diagnosis of Colon Cancer

Use of aspirin (which inhibits platelet function) after a colon cancer diagnosis is associated with improved overall survival. Identifying predictive biomarkers of this effect could individualize therapy and decrease toxic effects. To demonstrate that survival benefit associated with low-dose aspirin use after a diagnosis of colorectal cancer might depend on HLA class I antigen expression. A cohort study with tumor blocks from 999 patients with colon cancer (surgically resected between 2002 and 2008), analyzed for HLA class I antigen and prostaglandin endoperoxide synthase 2 (PTGS2) expression using a tissue microarray. Mutation analysis of PIK3CA was also performed. Data on aspirin use after diagnosis were obtained from a prescription database. Parametric survival models with exponential (Poisson) distribution were used to model the survival. Overall survival. The overall survival benefit associated with aspirin use after a diagnosis of colon cancer had an adjusted rate ratio (RR) of 0.53 (95% CI, 0.38-0.74; P < .001) when tumors expressed HLA class I antigen compared with an RR of 1.03 (0.66-1.61; P = .91) when HLA antigen expression was lost. The benefit of aspirin was similar for tumors with strong PTGS2 expression (0.68; 0.48-0.97; P = .03), weak PTGS2 expression (0.59; 0.38-0.97; P = .02), and wild-type PIK3CA tumors (0.55; 0.40-0.75; P < .001). No association was observed with mutated PIK3CA tumors (0.73; 0.33-1.63; P = .44). Contrary to the original hypothesis, aspirin use after colon cancer diagnosis was associated with improved survival if tumors expressed HLA class I antigen. Increased PTGS2 expression or the presence of mutated PIK3CA did not predict benefit from aspirin. HLA class I antigen might serve as a predictive biomarker for adjuvant aspirin therapy in colon cancer.

Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer

Prostaglandin endoperoxide H synthase-2 (PGHS-2), also known as cyclooxygenase-2 (COX-2), is a sequence homodimer. However, the enzyme exhibits half-site heme and inhibitor binding and functions as a conformational heterodimer having a catalytic subunit (Ecat) with heme bound and an allosteric subunit (Eallo) lacking heme. Some recombinant heterodimers composed of a COX-deficient mutant subunit and a native subunit (i.e. Mutant/Native PGHS-2) have COX activities similar to native PGHS-2. This suggests that the presence of heme plus substrate leads to the subunits becoming lodged in a semi-stable Eallo-mutant/Ecat-Native∼heme form during catalysis. We examined this concept using human PGHS-2 dimers composed of combinations of Y385F, R120Q, R120A, and S530A mutant or native subunits. With some heterodimers (e.g. Y385F/Native PGHS-2), heme binds with significantly higher affinity to the native subunit. This correlates with near native COX activity for the heterodimer. With other heterodimers (e.g. S530A/Native PGHS-2), heme binds with similar affinities to both subunits, and the COX activity approximates that expected for an enzyme in which each monomer contributes equally to the net COX activity. With or without heme, aspirin acetylates one-half of the subunits of the native PGHS-2 dimer, the Ecat subunits. Subunits having an S530A mutation are refractory to acetylation. Curiously, aspirin acetylates only one-quarter of the monomers of S530A/Native PGHS-2 with or without heme. This implies that there are comparable amounts of two noninterchangeable species of apoenzymes, Eallo-S530A/Ecat-Native and Eallo-Native/Ecat-S530A. These results suggest that native PGHS-2 assumes a reasonably stable, asymmetric Eallo/Ecat form during its folding and processing.

Nitro-fatty acids: novel anti-inflammatory lipid mediators

Nitro-fatty acids are formed and detected in human plasma, cell membranes, and tissue, modulating metabolic as well as inflammatory signaling pathways. Here we discuss the mechanisms of nitro-fatty acid formation as well as their key chemical and biochemical properties. The electrophilic properties of nitro-fatty acids to activate anti-inflammatory signaling pathways are discussed in detail. A critical issue is the influence of nitroarachidonic acid on prostaglandin endoperoxide H synthases, redirecting arachidonic acid metabolism and signaling. We also analyze in vivo data supporting nitro-fatty acids as promising pharmacological tools to prevent inflammatory diseases.

A Meta-Analysis of PTGS1 and PTGS2 Polymorphisms and NSAID Intake on the Risk of Developing Cancer

BackgroundSeveral studies have investigated whether the polymorphisms in the prostaglandin endoperoxide synthase 1 (PTGS1) and PTGS2 genes and nonsteroidal anti-inflammatory drug (NSAID) use are associated with cancer risk; however, those studies have produced mixed results. Therefore, we performed a meta-analysis to evaluate the association between the PTGS1 and PTGS2 polymorphisms and the effect of NSAID use on the risk of developing cancer.MethodsWe conducted a comprehensive search in PubMed through March 2012. The odds ratios (ORs) with the corresponding 95% confidence intervals (CIs) were calculated using the fixed-effect model or the random-effect model.ResultsThe database search generated 13 studies that met the inclusion criteria. For PTGS1 rs3842787, NSAID users homozygous for the major allele (CC) had a significantly decreased cancer risk compared with non-NSAID users (OR = 0.73, 95% CI = 0.59–0.89). For PTGS2 rs5275 and rs20417, there were no significant differences between the gene polymorphism and NSAID use on cancer risk among the 8 and 7 studies, respectively. However, in the stratified analysis by the type of cancer or ethnicity population, NSAID users homozygous for the major allele (TT) in rs5275 demonstrated significantly decreased cancer risk compared with non-NSAID users in cancer type not involving colorectal adenoma (OR = 0.70, 95% CI = 0.59–0.83) and among the USA population (OR = 0.67, 95% CI = 0.56–0.82). NSAID users homozygous for the major allele (GG) in rs20417 displayed a significantly decreased cancer risk than non-NSAID users among the US population (OR = 0.72, 95% CI = 0.58–0.88). For the PTGS2 rs689466 and rs2745557 SNPs, there were no significant differences.ConclusionThis meta-analysis suggests that the associations between PTGS polymorphisms and NSAID use on cancer risk may differ with regard to the type of cancer and nationality.

Effect of hCG and eCG Treatments on Prostaglandins Synthesis in the Porcine Oviduct

The oviduct plays a crucial role in fertilization, gamete maturation and embryo transport. Prostaglandins are some of the main factors determining its roles. The present study investigated the influence of oestrus synchronization and superovulation on prostaglandins synthesis in the porcine oviduct. Mature cross-bred gilts after exhibiting oestrous cycles were divided into four groups: I, cyclic; II, inseminated; III, synchronized and inseminated; and IV, superovulated and inseminated. Oviducts were collected on the third day of the oestrous cycle or after insemination and divided into isthmus and ampullary parts. This study demonstrated lower mRNA (in the isthmus and ampulla; p<0.05, p<0.001, respectively) and protein prostaglandin endoperoxide synthase 2 expression (in the isthmus; p<0.001) in gilts treated with human chorionic gonadotrophin/equine chorionic gonadotrophin (hCG/eCG) compared with Group II that were inseminated only. In addition, hCG and eCG treatment decreased mPGES-1 mRNA levels in Groups III and IV, in both the isthmus (p<0.01 in III, p<0.001 in IV) and ampulla (p<0.001). The prostaglandin E2 content of oviductal tissues was significantly lower in Groups III (p<0.05) and IV (p<0.01 in isthmus, p<0.0001 in ampulla) compared with Group II. mRNA and protein levels of PGFS in Group IV in the oviductal isthmus were higher (p<0.01) compared with the non-treated Group II. In effect, the amount of prostaglandin F2α in oviductal tissues of gilts treated with hCG/eCG was significantly elevated (p<0.001 in isthmus of Groups III and IV; p<0.05 in ampulla of Group IV). Differential expression of the factors analysed in gilts treated with exogenous gonadotrophins suggests that hCG/eCG stimulation affects prostaglandins synthesis pathway. These changes can alter oviduct functions and in turn affect gamete maturation and fertilization as well as development of embryos and their transport to the uterus.

Effects of PGF2α on the Expression of Uterine Activation Proteins in Pregnant Human Myometrial Cells From Upper and Lower Segment

The lower and upper segments of the uterus may play different roles in the process of parturition. The switch from pregnancy to delivery involves changes in expression of uterine activation proteins (UAPs). Prostaglandin (PG) F2α has multiple and complex roles in the birth process in addition to its vital contractile role. The purpose of this study was to investigate whether PGF2α regulates the expression of UAPs in human myometrium and to compare PGF2α actions in lower and upper segments. Cultured human myometrial cells from upper and lower segments were treated with PGF2α. Western blotting was used to determine the levels of connexin 43 (CX-43), prostaglandin endoperoxide synthase-2 (PTGS-2; cyclooxygenase-2), oxytocin receptor (OTR), and PGF2α receptor (PTGFR) in the cells. The small interfering RNA approach was used to knock down PTGFR. PGF2α dose dependently increased CX-43 and PTGS-2 while decreasing PTGFR in upper and lower segments. PGF2α increased OTR in the lower segment while decreasing it in the upper segment. PGF2α lost its effects on PTGS-2 and OTR in PTGFR knockdown cells, but its effect on CX-43 remained. AL8810, a specific antagonist of PTGFR, reversed the actions of PGF2α on UAPs except for CX-43 in the lower segment. Indomethacin reversed the PGF2α-induced effects on CX-43 and PTGS-2, but it did not alter PGF2α-induced PTGFR and OTR expression. The stimulatory effects of PGF2α were enhanced in the presence of IL-1β, which reversed the inhibitory effect of PGF2α on PTGFR. PGF2α regulates UAPs in both upper and lower segment cells through either direct or indirect pathways, indicating that PGF2α uniquely participates in uterine preparation for the onset of labor.

Evaluation and Theoretical Study on the Anti-inflammatory Mechanism of 1-Nitro-2-phenylethane

In this study, 1-nitro-2-phenylethane was evaluated with respect to its effects in edema models of acute inflammation induced with carrageenan, dextran, and croton oil. 1-Nitro-2-phenylethane produced inhibition of rat paw edema induced by carrageenan and dextran at the doses of 25 and 50 mg/kg. The same doses caused an inhibition of croton oil-induced ear edema in mice. Our results suggest that 1-nitro-2-phenylethane has anti-inflammatory activity, probably of peripheral origin, acting in the synthesis and/or release of inflammatory mediators. A conformational study of 1-nitro-2-phenylethane was carried out using density functional theory calculations, showing three different groups of conformers corresponding to energy minimum geometries. The stereoelectronic repulsions are responsible for conformational preferences and the one most stable conformer. The prostaglandin endoperoxide synthase mechanism is related more to electrophilic than nucleophilic properties.

Divergent Expression of IL-1 Receptor Antagonists in CD34+ Fibrocytes and Orbital Fibroblasts in Thyroid-associated Ophthalmopathy: Contribution of Fibrocytes to Orbital Inflammation

Thyroid-associated ophthalmopathy (TAO) manifests as inflammation of orbital connective tissue. Bone marrow-derived CD34⁺ fibrocytes infiltrate the orbit in TAO where they become CD34⁺ orbital fibroblasts. They express thyroid-specific antigens and thus may contribute to inflammation. Evidence suggests that orbital susceptibility to TAO may involve IL-1, which is modulated by IL-1 receptor antagonists, designated secreted (sIL-1RA) and intracellular (icIL-1RA). We sought to characterize the expression of sIL-1RA and icIL-1RA in TAO orbital fibroblasts compared to CD34⁺ fibrocytes. Patients with TAO and healthy donors were recruited from an academic medical center clinical practice. Real-time PCR, cytokine-specific ELISA, gene promoter activities, transcriptional analysis, mRNA stability, and cytometric cell sorting were performed. Orbital fibroblasts treated with IL-1β exhibit greater inductions of IL-1α, IL-1β, and prostaglandin endoperoxide H synthase-2 transcripts than do fibrocytes. Fibrocytes express dramatically higher basal levels of both icIL-1RA and sIL-1RA. When treated with IL-1β, icIL-1RA is induced in orbital fibroblasts but not sIL-1RA, whereas in fibrocytes, sIL-1RA is dominantly up-regulated. These inductions result from increased steady-state levels of respective mRNAs, enhanced transcript stabilities, and modestly increased gene transcription. Robust responses of TAO orbital fibroblasts to IL-1β are a consequence of low-level sIL-1RA expression. This results in poorly opposed actions of IL-1β. In contrast, circulating fibrocytes express high levels of sIL-1RA, which are diminished as these cells transition to orbital fibroblasts. These findings identify an explanation for the inflammatory phenotype exhibited by TAO orbital fibroblasts and provide a potential target for altering disease susceptibility.

Association between the 8473T>C Polymorphism of PTGS2 and Prostate Cancer Risk: A Metaanalysis Including 24,716 Subjects

Background: Prostaglandin endoperoxide synthase 2 (PTGS2) is involved in prostate cancer (PCa) by stimulating cell proliferation, promoting angiogenesis, inhibiting apoptosis, and mediating immune suppression. 8473T>C, located in the 3' UTR of the PTGS2 gene, has been considered to influence PCa risk. Methods: We searched Medline, PubMed, Elsevier, and Web of Science (updated to February 5, 2012) using the following search terms: ‘8473T>C' or ‘rs5275', ‘genetic variant' or ‘polymorphism', ‘prostate cancer', ‘cancer', ‘PTGS2' or ‘COX-2'. Odds ratios with 95% confidence intervals were assessed by using fixed or random effect models. Both funnel plot and Egger's test were used to assess the publication bias. Results: Finally, 5 case control studies were included. Overall, no evidence was observed of a relationship between the 8473T>C and PCa risk in any genetic model. No significant association was found in the studies whose controls conform to the Hardy-Weinberg equilibrium. In the stratified analysis, significant association was detected in other populations (except for Caucasians), which were based on hospitals. Conclusion: The 8473T>C polymorphism may have little association with PCa risk among Caucasians, but might be involved in PCa risk in other ethnicities. Nevertheless, more well-designed studies with a larger sample size including different ethnicities should be conducted.

Regulation of protein kinase C by nitroarachidonic acid: Impact on human platelet activation

Platelet activation represents a key event in normal hemostasis as well as during platelet plug formation related to thrombosis. Nitro-fatty acids are novel endogenously produced signaling mediators exerting pluripotent anti-inflammatory actions in cells and tissues. We have recently shown that nitroarachidonic acid inhibits thromboxane synthesis during platelet activation by affecting prostaglandin endoperoxide H synthase (PGHS). Herein, we investigated the regulation of human platelet activation by NO2AA and describe a novel mechanism involving protein kinase C (PKC) inhibition. NO2AA-mediated antiplatelet effects were characterized using mass spectrometry, confocal microscopy, flow cytometry, western blot and aggregometry. Incubation of NO2AA with human platelets caused a significant reduction in platelet sensitivity to thrombin, ADP, arachidonic acid (AA), and phorbol ester (PMA). These effects were cGMP-independent and did not involve Ca2+ store-dependent mobilization. In contrast, signaling downstream of conventional PKC activation, such as α-granule secretion and extracellular signal regulated kinase 2 activation was strongly inhibited by NO2AA. Immunofluorescence confocal microscopy confirmed NO2AA-mediated inhibition of PKCα translocation to the membrane. In summary, we demonstrate that NO2AA inhibits platelet activation through modulation of PKCα activity as a potential novel mechanism for platelet regulation in vivo.

An Adaptation of Prostaglandin Endoperoxide H Synthases in Binding to Endogenous Ligands and Nonsteroidal Anti-Inflammatory Drugs

Prostaglandins play critical roles in a number of physiological and pathophysiological processes. These molecules are involved in regulating blood flow to organs, initiating platelet aggregation in blood clotting, and mainly mediating the classical symptoms of inflammation. Biosynthesis of prostaglandins is initiated by prostaglandin endoperoxide H synthases (PGHS) −1 and −2, which catalyze the conversion of arachidonic acid to PGH2. PGHSs are homodimers comprised of ∼72 kDa monomers of identical primary sequence and recently have been investigated in the respect of allosteric communications between two subunits during ligand binding. The nonsteroidal anti-inflammatory drugs show multiple inhibition modalities against PGHSs. Their time-dependent and subunit-preferential bindings to PGHSs complicate the interactions of PGHSs with the cognate substrates and their regulations by natural and synthetic ligands. Accordingly, a paradigm shift in the analysis of PGHS-ligand complex during PGHS catalysis is required and their theoretical revaluation and biological functioning in vivo are described in this paper.

Nitro-Fatty Acids: Formation, Redox Signaling, and Therapeutic Potential

Nitrated derivatives of unsaturated fatty acids (nitro-fatty acids) are being formed and detected in human plasma, cell membranes, and tissue, triggering signaling cascades via covalent and reversible post-translational modifications of susceptible nucleophilic amino acids in transcriptional regulatory proteins and enzymes. Nitro-fatty acids modulate metabolic as well as inflammatory signaling pathways, including the p65 subunit of nuclear factor κB and the transcription factor peroxisome proliferator-activated receptor-γ. Moreover, nitro-fatty acids can activate heat shock as well as phase II antioxidant responses. As electrophiles, they also activate the Nuclear factor erythroid 2-related factor 2 pathway. We first discuss the mechanisms of nitro-fatty acid formation as well as their key chemical and biochemical properties, including their capacity to release nitric oxide and exert antioxidant actions. The electrophilic properties of nitro-fatty acids to activate anti-inflammatory signaling pathways are discussed in detail. A critical issue is the influence of nitroarachidonic acid on prostaglandin endoperoxide H synthases, modulating inflammatory processes through redirection of arachidonic acid metabolism and signaling. Based on this information, we analyze in vivo data supporting nitro-fatty acids as promising pharmacological tools to prevent inflammatory diseases associated with oxidative and nitrative stress conditions. A key future issue is to evaluate whether nitro-fatty acid supplementation would be useful for human diseases linked to inflammation as well as their potential toxicity when administered by long periods of time.

Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.

257 EFFECTS OF FIBROBLAST GROWTH FACTOR 8 ON CUMULUS EXPANSION AND NUCLEAR MATURATION IN BOVINE CUMULUS - OOCYTE COMPLEXES

Recent data indicate that fibroblast growth factor (FGF) signalling regulates oocyte developmental competence. Fibroblast growth factor 10 enhanced nuclear maturation, cumulus expansion, and embryo development in cattle (Zhang et al. 2010 Reproduction 140, 815–826). Like FGF10, FGF8 is expressed in the bovine oocyte, but whereas FGF10 activates FGF receptors (FGFR) 1B and 2B with higher affinity, FGF8 preferentially activates FGF receptor (FGFR) 2C, FGFR3C, and FGFR4. The involvement of FGF8 in the regulation of bovine cumulus–oocyte complex (COC) maturation has remained unknown. This study aimed to assess the effects of FGF8 supplementation in the in vitro maturation medium on nuclear maturation, degree of cumulus expansion, and expression of the genes necessary for expansion in bovine COC. Groups of 20 immature COC (grades 1 and 2) aspirated from 3- to 8-mm follicles were cultured in 200-µL drops of TCM-199 supplemented with FSH (1 µg mL–1), LH (10 IU mL–1), pyruvate (22 µg mL–1), amikacin (75 µg mL–1), and graded doses of recombinant human FGF8 (Peprotech, Rocky Hill, NJ, USA; 0, 1, 10, and 100 ng mL–1) for 22 h at 38.5°C and 5% CO2. After culture, COC were visually classified according to the degree of cumulus expansion (grades 1 to 3, indicating absent, moderate, and full expansion, respectively). Oocytes were mechanically separated from cumulus cells and stained with Hoechst 33342 to assess meiosis progression. Total RNA was extracted from cumulus cells using RNeasy (Qiagen, Venlo, the Netherlands), and 100 ng of RNA was reverse-transcribed using Omniscript (Qiagen). Expression levels of messenger RNA encoding genes necessary for cumulus expansion [prostaglandin endoperoxide synthase 2 (PTGS2), hyaluronan synthase 2 (HAS2), pentraxin 3 (PTX3) and tumour necrosis factor-stimulated gene-6 protein (TSG6)] were assessed by real-time PCR, with cyclophilin (CYCA) as the housekeeping gene. Data were derived from 5 replicates. Maturation and expansion data were transformed to arcsine, and gene expression data were log transformed. Effects of treatments were tested by ANOVA, and means were compared with the Tukey-Kramer honestly significant difference test. The FGF8 at 10 and 100 ng mL–1 reduced the proportion of oocytes reaching metaphase II (70, 64.8, 52.8, and 36% for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively; P = 0.005) and increased the proportion of oocytes in metaphase I at 22 h of culture (30, 35.2, 47.2, and 64% for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively; P = 0.004). Fibroblast growth factor 8 did not affect the degree of cumulus expansion as visually assessed. However, FGF8 at 10 and 100 ng mL–1 increased the messenger RNA abundance of PTGS2 [P = 0.0002; relative values (±SEM) of 0.69 ± 0.10, 0.63 ± 0.11, 1.56 ± 0.44, and 1.67 ± 0.20 for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively] and HAS2 [P = 0.0002; relative values (±SEM) of 1.38 ± 0.15, 1.37 ± 0.24, 3.58 ± 0.61, and 4.14 ± 0.27 for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively] in cumulus cells. In conclusion, the present data suggest the involvement of FGF8 in the mechanisms regulating transcription of expansion-inducing genes in cattle. In contrast with previous findings with FGF10, FGF8 inhibited nuclear maturation, suggesting different actions for different FGF in the regulation of COC maturation. Further research is needed to clarify the roles of FGF8 in the bovine COC. Supported by FAPESP.

Inhibitory Effects of Phenylpropanoids Isolated from the Bark of Ailanthus altissima on COX-2 Activity

Prostaglandin (PG) and thromboxane biosynthesis involves the catalyzed conversion of arachidonic acid, by the sequential actions of cyclooxygenases (COXs) and prostaglandin endoperoxide synthase (PGHS), to prostaglandin H2 (PGH2). 1 Three isozymes of COX (COX-1, COX-2, and COX-3) are known to date, and COX-1 and COX-2 have been well defined. COX-1 is constitutively expressed in many organs or tissues, while COX-2 is induced by various stimuli. 2 However, recent molecular-biological studies show that this simple paradigm has many exceptions. For example, COX-1 can be regulated during development, 3 whereas COX-2 is constitutively expressed in the brain 4 and in reproductive tissues. 5 Often, both isozymes are involved under physiological and pathophysiological conditions, whereas under other conditions they play distinctly different roles. These COX-1 and COX-2 are the key enzymes during prostaglandin (PG) biosynthesis, and the inhibition of PG synthesis is the strategy underlying current anti-inflammatory therapies. Furthermore, accumulating evidence indicates that COX-2 is involved in many inflammatory processes and in the pathogeneses of various cancers, which suggests that COX-2 plays a key role in inflammation and tumorigenesis. 6,7 Thus, the direct inhibition of COX-2 has been actively pursued as

Requirement for the histone deacetylase Hdac3 for the inflammatory gene expression program in macrophages

Histone deacetylases (HDACs) regulate inflammatory gene expression, as indicated by the potent antiinflammatory activity of pan-HDAC inhibitors. However, the specific contribution of each of the 11 HDAC proteins to the inflammatory gene expression program is unknown. Using an integrated genomic approach, we found that Hdac3-deficient macrophages were unable to activate almost half of the inflammatory gene expression program when stimulated with LPS. A large part of the activation defect was attributable to loss of basal and LPS-inducible expression of IFN-β, which maintains Stat1 protein levels in unstimulated cells and acts in an autocrine/paracrine manner after stimulation to promote a secondary wave of Stat1-dependent gene expression. Loss of Hdac3-mediated repression of nuclear receptors led to hyperacetylation of thousands of genomic sites and associated gene derepression. The up-regulation of the constitutively expressed prostaglandin endoperoxide synthase, Ptgs1 (Cox-1), a nuclear receptor target, had a causative role in the phenotype because its chemical inhibition reverted, albeit partially, the Ifn-β activation defect. These data indicate a central role for Hdac3 in inflammation and may have relevance for the use of selective Hdac inhibitors as antiinflammatory agents.