Prostaglandin Synthase Activity Research Articles

Prostaglandin synthase activity of sigma- and mu-class glutathione transferases in a parasitic trematode, Clonorchis sinensis.

Sigma-class glutathione transferase (GST) proteins with dual GST and prostaglandin synthase (PGS) activities play a crucial role in the establishment of Clonorchis sinensis infection. Herein, we analyzed the structural and enzymatic properties of sigma-class GST (CsGST-σ) proteins to obtain insight into their antioxidant and immunomodulatory functions in comparison with mu-class GST (CsGST-μ) proteins. CsGST-σ proteins conserved characteristic structures, which had been described in mammalian hematopoietic prostaglandin D2 synthases. Recombinant forms of these CsGST-σ and CsGST-μ proteins expressed in Escherichia coli exhibited considerable degrees of GST and PGS activities with substantially different specific activities. All recombinant proteins displayed higher affinities toward prostaglandin H2 (PGS substrate; average Km of 30.7 and 3.0 μm for prostaglandin D2 [PGDS] and E2 synthase [PGES], respectively) than those toward CDNB (GST substrate; average Km of 1,205.1 μm). Furthermore, the catalytic efficiency (Kcat/Km) of the PGDS/PGES activity was higher than that of GST activity (average Kcat/Km of 3.1, 0.7, and 7.0×10-3 s-1μm-1 for PGDS, PGES, and GST, respectively). Our data strongly suggest that the C. sinensis sigma- and mu-class GST proteins are deeply involved in regulating host immune responses by generating PGD2 and PGE2 in addition to their roles in general detoxification.

Gastroprotektivne karakteristike krioprezerviranog ekstrakta placente kod kombinovanog delovanja niskih temperatura i inhibicije ciklooksigenaze

Introduction. Non-steroidal anti-inflammatory drugs (NSAIDs) are ranked first among the world's effective anti-inflammatory and analgesic drugs with anticipated side effects. That is why the prevention of development of adverse reactions associated with NSAIDs, in particular, of those related to their ulcerogenicity, remains a serious global problem. Aims. To characterize the mechanisms of gastrocytoprotective activity of cryopreserved placenta in combined action of low temperatures and diclofenac sodium. Material and methods. The study was performed on 42 male rats weighing 200-220 g. Acute diclofenac sodium-induced gastropathy was reproduced by a single injection of the latter in rats at the dose of 50 mg/kg. The content of malonic dialdehyde, catalase activity, prostaglandin synthase activity and the content of nitrogen monoxide metabolites in homogenates of gastric mucosa were determined by spectrophotometric method. Results and discussion. The study showed that prophylactic administration of placental cryoextract in rats with diclofenac sodium-induced gastropathy is associated with increased activity of antioxidant system in gastric mucosa as demonstrated by an elevated catalase activity by 40.0% as compared with control rats. Modulation of antioxidant-prooxidant homeostasis is believed to be one of the principal mechanisms of gastrocytoprotective action in combined action of low temperatures and cryoextract of the placenta. This is shown by a statistically significant (p < 0.05) 2.2-fold increase of antioxidant-prooxidant index in the study group as compared with rats with diclofenac sodium-induced gastropathy. Administration of placental cryoextract was found to increase prostaglandin synthase activity in rats with diclofenac sodium-induced gastropathy by two times as compared with control rats, which would reduce iatrogenic prostaglandin deficiency in gastric mucosa. Also, the combined action of low temperatures and of placenta cryoextract was associated with a statistically significant (p < 0.05) increase in the level of metabolites of nitrogen monoxide (by 70.1%) as compared with rats with diclofenac sodium-induced gastropathy. Conclusions. Modulation of prooxidant-antioxidant homeostasis in gastric mucosa and increase in contents of nitrogen monoxide metabolites and prostaglandin synthase activity are the leading mechanisms of gastroprotective activity of cryopreserved placental extract in diclofenac sodium-induced gastropathy.

Inhibition of phospholipase A2 and prostaglandin synthase activities as possible mechanistic insight into the anti-inflammatory activity of Brenania brieyi methanol and chloroform fractions

Inhibition of phospholipase A2 and prostaglandin synthase activities as possible mechanistic insight into the anti-inflammatory activity of Brenania brieyi methanol and chloroform fractions

Characterization of a novel glycosylated glutathione transferase of Onchocerca ochengi, closest relative of the human river blindness parasite.

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.

Inhibition of Phospholipase A2 and Prostaglandin Synthase Activities as Possible Mechanisms for the Anti-Inflammatory Effect of Cucumis sativus Fruit Homogenate

Inhibition of Phospholipase A2 and Prostaglandin Synthase Activities as Possible Mechanisms for the Anti-Inflammatory Effect of Cucumis sativus Fruit Homogenate

Purification of native Sigma class glutathione transferase from Fasciola hepatica

Fascioliasis is a parasitic disease of grazing livestock and a threat to global food security by significantly reducing the production value of sheep, goats and cattle. Moreover, the zoonotic parasite is also a re-emerging food borne threat to human populations. Driven by climate change, the prevalence of fascioliasis is set to increase. Efforts to control the main causative agent, Fasciola hepatica, are hampered by short lived chemotherapy approaches that are becoming increasingly obsolete due to therapeutic failure and resistance. A protective vaccine is urgently needed. A recombinant form of Sigma class glutathione transferase (Hematopoietic Prostaglandin D synthase) from F. hepatica (FhGSTS1) with confirmed prostaglandin synthase activity shows immune-modulation activity via suppression of Th17 responses in host dendritic cells. In vaccine trials recombinant FhGSTS1 reduces liver pathology but not worm burden. Native FhGSTS1 is yet to be tested for immune-modulation activities or for vaccine potential, primarily due to the technical difficulty in purifying FhGST-S1 away from the other more abundant GST members in F. hepatica cytosol. This paper reports a pipeline for the purification of native FhGSTS1 using a two-step process consisting of glutathione-agarose affinity and cationic exchange chromatography. The methodology allows for the isolation of purified and active FhGSTS1 or Sigma GSTs from other sources for analytical biochemical and immunological studies.

Anti-Inflammatory Effects of The Chloroform Extract of Annona muricata Leaves on Phospholipase A2 and Prostaglandin Synthase Activities

This study ascertained the mechanisms of the anti-inflammatory activity of the total lipid (chloroform) extract of Annona muricata leaves. The plant material was extracted with a mixture of chloroform and methanol (2:1) and partitioned with 0.2 volume water. The chloroform extract was investigated for its effect on the in vitro activities of phospholipase A2, prostaglandin synthase and membrane stabilization. The extract significantly (p < 0.05) inhibited phospholipase A2 activity in a concentration-related manner compared to the control, with a range of 0.2 - 0.6 mg/ml inhibiting the enzyme activity by 23.91 - 43.48%. Effect of the extract on prostaglandin synthase activity showed a significant (p < 0.05) inhibition of enzyme activity at the doses 0.1, 0.5 and 1.0 mg/ml compared to the control. The highest percentage inhibition (87.46%) attained at 0.5 mg/ml was comparable to that of 1.0 mg/ml indomethacin. At various concentrations (0.1-0.8 mg/ml), the chloroform extract also significantly (p < 0.05) inhibited heat and hypotonicityinduced haemolysis of human red blood cells (HRBCs) compared to the control. The highest percentage inhibition of heat-induced haemolysis (53.03%) was obtained at 0.4 mg/ml of the extract while the highest percentage inhibition of hypotonicity-induced haemolysis (77.91%) was obtained at 0.8 mg/ml. This study thus confirmed that the mode of action of this extract of Annona muricata leaves on inflammation could be through the inhibition of phospholipase A2 and prostaglandin synthase activities and by membrane stabilization.

In-vitro stability and aggregatory effect of ethanol extract leaves of Sida acuta Burm F. on human erythrocyte.

Abstract Aim: This study aims to evaluate the membrane stability, Phospholipase A2 activity, prostaglandin synthase activity and aggregatory effect of ethanol leaves extract of Sida acuta Burm F. using an in-vitro heamolytic assay. Materials and methods: Two milliliters of blood collected from a healthy volunteer who has not taken drugs for last two weeks and ethanol extracts leaves of Sida acuta were used in the assay. Results: Apparently there was platelet aggregation at all but there was no minimal aggregation to denote in samples 1 and 2. There was a decrease in the supernatants of sample 1 of hypotonicity-induced haemolysis of human red blood cells incubated in normal saline but there was a high increase in the supernatants of sample 2 incubated with hypotonic solution. Inclusion of the extract concentrations 2, 4 and 8 mg/ml in the incubation medium (hypotonic solution) in samples 3, 4 and 5 decreased in a concentration dependent fashion. The absorption of haemolysed human red blood cells in isotonic solution showed the OD of 540nm:630nm to 2.15 for methaemoglobin while the OD of 540nm:570nm was 0.97 for deoxyhaemoglobin. In hypotonic solution where haemoglobin was released, the ratio of 540nm:630nm was 4.41 and 540nm:570nm was 1.20. The inclusion of the extract concentrations 2, 4 and 8 mg/ml in samples 3, 4 and 5 caused the ratio to decreased from1.69, 1.58 and 0.41 for methaemoglobin and 1.08, 0.86 and 0.41 for deoxyhaemoglobin. There was a decrease in activity of phospholipase A2 in samples 3, 4, 5 and 9 with the extract concentrations 2, 6, 12 and 16 mg/ml respectively but no decrease was observed in samples 6,7, 8 and 10, though no human red blood cells was included in samples 6, 7, 8, 9 and 10. There was a significant reduction (p  0.05) in prostaglandin synthase activity in sample 4 with extract concentration 25 mg/ml but a non-significant decrease (p  0.05) was observed in samples 3 and 5. Conclusion: The extract showed a significant inhibition of in-vitro haemolysis and could have a potential therapeutic effect on disease processes causing destabilization of biological membranes.

Insights into the Prostanoid Pathway in the Ovary Development of the Penaeid Shrimp Penaeus monodon

The prostanoid pathway converts polyunsaturated fatty acids (PUFAs) into bioactive lipid mediators, including prostaglandins, thromboxanes and prostacyclins, all of which play vital roles in the immune and reproductive systems in most animal phyla. In crustaceans, PUFAs and prostaglandins have been detected and often associated with female reproductive maturation. However, the presence of prostanoid biosynthesis genes remained in question in these species. In this study, we outlined the prostanoid pathway in the black tiger shrimp Penaeus monodon based on the amplification of nine prostanoid biosynthesis genes: cytosolic phospholipase A2, hematopoietic prostaglandin D synthase, glutathione-dependent prostaglandin D synthase, prostaglandin E synthase 1, prostaglandin E synthase 2, prostaglandin E synthase 3, prostaglandin F synthase, thromboxane A synthase and cyclooxygenase. TBLASTX analysis confirmed the identities of these genes with 51-99% sequence identities to their closest homologs. In addition, prostaglandin F2α (PGF2α), which is a product of the prostaglandin F synthase enzyme, was detected for the first time in P. monodon ovaries along with the previously identified PUFAs and prostaglandin E2 (PGE2) using RP-HPLC and mass-spectrometry. The prostaglandin synthase activity was also observed in shrimp ovary homogenates using in vitro activity assay. When prostaglandin biosynthesis was examined in different stages of shrimp ovaries, we found that the amounts of prostaglandin F synthase gene transcripts and PGF2α decreased as the ovaries matured. These findings not only indicate the presence of a functional prostanoid pathway in penaeid shrimp, but also suggest a possible role of the PGF2α biosynthesis in shrimp ovarian development.

Crystal structure of a Bombyx mori sigma-class glutathione transferase exhibiting prostaglandin E synthase activity

BackgroundGlutathione transferases (GSTs) are members of a major family of detoxification enzymes. Here, we report the crystal structure of a sigma-class GST of Bombyx mori, bmGSTS1, to gain insight into the mechanism catalysis. MethodsThe structure of bmGSTS1 and its complex with glutathione were determined at resolutions of 1.9Å and 1.7Å by synchrotron radiation and the molecular replacement method. ResultsThe three-dimensional structure of bmGSTS1 shows that it exists as a dimer and is similar in structure to other GSTs with respect to its secondary and tertiary structures. Although striking similarities to the structure of prostaglandin D synthase were also detected, we were surprised to find that bmGSTS1 can convert prostaglandin H2 into its E2 form. Comparison of bmGSTS1 with its glutathione complex showed that bound glutathione was localized to the glutathione-binding site (G-site). Site-directed mutagenesis of bmGSTS1 mutants indicated that amino acid residues Tyr8, Leu14, Trp39, Lys43, Gln50, Met51, Gln63, and Ser64 in the G-site contribute to catalytic activity. ConclusionWe determined the tertiary structure of bmGSTS1 exhibiting prostaglandin E synthase activity. General significanceThese results are, to our knowledge, the first report of a prostaglandin synthase activity in insects.

The Sigma Class Glutathione Transferase from the Liver Fluke Fasciola hepatica

BackgroundLiver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised.Methodology/Principal FindingsIn this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection.Conclusions/SignificanceWe have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity.

A contemporary viewpoint on ‘aspirin resistance’

Aspirin is an irreversible inhibitor of platelet prostaglandin synthase activity, and is the most widely prescribed drug for the secondary prevention of cardiovascular disease. In recent years, clinical and laboratory evidence has shown significant individual variability in the response to aspirin and its link to clinical outcome. The term ‘aspirin resistance’ has been introduced to describe situations when clinical or ex-vivo effects of aspirin are less than expected. The accumulating evidence of increased risk of major adverse clinical events (MACE) associated with ‘aspirin resistance’ in the settings of acute coronary syndrome (ACS), stroke, and peripheral arterial disease has stimulated the search for ways of overcoming aspirin resistance. Existence of the link between high on-treatment platelet reactivity and atherothrombotic events suggests the common mechanisms for atherosclerosis progression and thrombotic complications with the platelets, being a key cellular interface between coagulation and inflammation.This review article provides a contemporary view on ‘aspirin resistance’ and discusses its definition, clinical importance, and possible mechanisms in light of recent data on the role of platelets in atherothrombosis.

Low-dose acetylsalicylic acid inhibits the secretion of interleukin-6 from white adipose tissue

Chronically elevated interleukin-6 (IL-6) is implicated in obesity-associated pathologies, where a proportion of this cytokine is derived from adipose tissue. Proinflammatory prostaglandins, which regulate this cytokine elsewhere, are also produced by this tissue. To investigate whether constitutively active cyclooxygenase (COX)/prostaglandin (PG) pathway in white adipose tissue (WAT) is responsible for basal IL-6 production. The effect of acetylsalicylic acid (ASA), an inhibitor of COX, on IL-6 was assessed in human subjects and mice. COX, downstream PG synthase (PGS) activity and PG receptor signalling were determined in subcutaneous (SC), gonadal (GN) WAT and adipocytes. In obese humans, low-dose ASA (150 mg day(-1) for 10 days) inhibited systemic IL-6 and reduced IL-6 release from SC WAT ex vivo (0.2 mM). Similarly, in mice, ASA (0.2 and 2.0 mg kg(-1)) suppressed SC WAT 6-keto-PGF(1alpha) (a stable metabolite of prostacyclin) and IL-6 release. Although both COX isoforms are comparably expressed, prostacyclin synthase expression is higher in GN WAT, with levels of activity correlating directly with IL-6. Both ASA (5 mM) and NS-398 (COX-2 selective inhibitor <or=1 microM), but not SC-560 (COX-1 selective inhibitor <or=1 microM), attenuated IL-6 release from murine WAT in vitro and abolished its depot differences. Prostacyclin receptor (IP) and, to a lesser extent, PGE(2) (EP2 and EP4) receptor agonists elevated the release of IL-6 from adipocytes. In adipose tissue, constitutive COX-2-coupled prostacyclin triggers the release of basal IL-6, which in obese subjects is significantly dampened by ASA ingestion, thus offering a novel, modifiable pathway to regulate the potentially pathological component of this cytokine.

Enzymatic formation of prostaglandin D 2, E 2, and F 2α in the parasitic protozoan Trypanosoma brucei

African trypanosomiasis (sleeping sickness in humans or nagana in domestic animals) is caused by infection with African trypanosomes and is characterized by a marked increase in prostaglandin (PG) production. Although PGs are known to cause pathogenesis of African trypanosomiasis, little is known about the mechanism of up-regulation of PG production during this disease. In the present study, we investigated the metabolism of arachidonic acid in African trypanosomes and found that by using EIA and gas chromatography mass spectrometry (GC-MS), Trypanosoma brucei produces and secretes PGD 2, E 2, and F 2α. Interestingly, PGs were differentially produced by the different life stages of the parasite. PGF 2α was the most abundant PG produced by the cell lysates. Moreover, PG synthase activities were recovered in the cytosolic fraction and were abolished by heating (100 °C, 5 min). Prostaglandin (PG) formation in African trypanosomes is catalyzed by enzymatic systems, which are markedly different from their mammalian counterparts that are localized in the microsomal fraction. The release of PGs from live trypanosomes into the culture media indicates that T. brucei may well produce PGs that are, in part, responsible for the symptoms observed in patients with African trypanosomiasis.

Inflammation, reproduction, cancer and all that…. The regulation and role of the inducible prostaglandin synthase

Discovery of a second, inducible prostaglandin synthase provides explanations for many previously puzzling observations, but also raises new questions about prostanoid synthesis. A cis-acting sequence closely related to the cyclic AMP response element has been shown to play a role in both basal and induced prostaglandin synthase 2 gene expression. Aspirin and other currently available non-steroidal anti-inflammatory drugs that inhibit prostaglandin synthase activity do not effectively discriminate between the inducible prostaglandin synthase 2 and constitutive prostaglandin synthase 1 enzymes. Identification of a second prostaglandin synthase, induced by inflammatory stimuli, initiated a search for isoform-specific inhibitors. Use of prostaglandin synthase 2 specific inhibitors and antisense oligonucleotides has led to the suggestion that specific ligands activate alternative pathways of prostanoid production, using one of the prostaglandin synthase isoforms preferentially. The coupling mechanisms by which these pathways are activated in response to alternative stimuli should provide additional routes of intervention in prostanoid production.

Prostaglandin synthase activity of fetal sheep cotyledons at 122 days of gestation and term: expression of prostaglandin synthetic capacity in fetal cotyledonary tissue near labor is location-dependent.

An activity assay is described quantification of prostaglandin synthase (PGHS) in sheep placental cotyledon under initial velocity conditions through measurement of the stable product prostaglandin E2 (PGE2). The effects of temperature, time, and substrate concentration on initial reaction velocity, and lipoxygenase and PGHS product formation in cotyledonary tissue, were examined in detail. We used this activity assay to determine whether or not an increase of active PGHS by placental location within the uterus might contribute selected prostaglandins (PG) for the directed initiation of parturition. Sheep cotyledon tissue was collected (n = 6 animals) from the ventral aspect of the uterine body, mid-horn, and horn tip at 122 days of gestation (dga), and from the same locations in the ventral body and horn tip at 142-145 dga (in animals at term but not in labor; n = 4). At 122 dga, there was no increase in active PGHS in cotyledonary tissue from the horn tip, mid-horn, or uterine body. By 142-145 dga, the horn showed significantly (p < 0.01) more enzyme activity than the body. At the same time, production of PGE2, expressed as a percentage of total eicosanoids, had not changed significantly. The development of an increase in PGHS toward the uterine tip implies that variations in regional PG production may contribute to the progression of labor.

Mechanical stimulation of skeletal muscle cells mitigates glucocorticoid‐induced decreases in prostaglandin production and prostaglandin synthase activity

The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content in tissue cultured, avian skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the effects of Dex and mechanical stretch on prostaglandin production and prostaglandin H synthase (PGHS) activity were examined. In static cultures, 10(-8) M Dex reduced PGF2 alpha production 55-65% and PGE2 production 84-90% after 24-72 h of incubation. Repetitive 10% stretch-relaxations of non-Dex-treated cultures increased PGF2 alpha efflux 41% at 24 h and 276% at 72 h, and increased PGE2 production 51% at 24 h and 236% at 72 h. Mechanical stimulation of Dex-treated cultures increased PGF2 alpha production 162% after 24 h, returning PGF2 alpha efflux to the level of non-Dex-treated cultures. At 72 h, stretch increased PGF2 alpha efflux 65% in Dex-treated cultures. Mechanical stimulation of Dex-treated cultures also increased PGE2 production at 24 h, but not at 72 h. Dex reduced PGHS activity in the muscle cultures by 70% after 8-24 h of incubation, and mechanical stimulation of the Dex-treated cultures increased PGHS activity by 98% after 24 h. Repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by mitigating the Dex-induced declines in PGHS activity and prostaglandin production.

Prostaglandin biosynthesis by fat body from the tobacco hornworm, Manduca sexta

We describe prostaglandin (PG) biosynthesis by microsomal-enriched preparations of fat body from larvae of the tobacco hornworm Manduca sexta. Four major PGs were synthesized under most experimental conditions, PGA 2, PGE 2, PGD 2 and PGF 2α. PGA 2, was the predominant product under most conditions. Unlike mammals, in which PGA 2, is generally thought to arise from non-enzymatic rearrangemets of PGE 2, the fat body preparations did not convert exogenous PGE 2 into PGA 2. These findings suggest that PGA 2 is an important fat body product that is synthesized by a route that does not involve PGE 2. The PG synthase activity and the overall profile of PG synthesis were sensitive to experimental conditions, including incubation time, temperature, and protein concentration. Optimal PG biosynthesis was observed with 1 mg of microsomal-rich protein, incubated at 30°C for 1–2 min. The fat body preparation is sensitive to two non-steroidal anti-inflammatory drugs, indomethacin and naproxen, both of which inhibited PG synthesis at low dosages.

Prostaglandin G/H Synthase Isoenzyme 2 Expression in Fibroblasts - Regulation by Dexamethasone, Mitogens, and Oncogenes

Mitogens increase prostaglandin synthesis in fibroblasts. In the present studies, transcriptional activation of the prostaglandin G/H synthase isoenzyme 2 (PGHS-2) gene is shown to be responsible for this induction. Transcription of the PGHS-2 gene maximally increased within 15 mm of stimulation, and elevated cellular PGHS-2 mUNA, protein, and prostaglandin synthase activity were detected shortly thereafter. Pulse-chase experiments showed induced isoenzyme 2 is short lived, with a half-life of 22 mm in chicken embryo fibroblasts. Neoplastic transformation of fibroblasts by a variety of oncogenes induced either or both PGHS-1 and PGHS-2 mRNAs in immortalized murine NIH3T3 cells. Some preference of oncogenes such as v- src to induce PGHS2 mRNA has been previously observed. However, in this study exceptions in which v- src did not induce PGHS-2 mRNA were noted. Analysis of independent cell lines transformed by v- fes showed this oncogene induced both PGHS-1 and PGHS-2. Dexamethasone, a synthetic glucocorticoid, selectively decreased both basal and induced levels of PGHS-2 mRNA. Simultaneous addition of the steroid with mitogen reduced mitogen-induced PGHS-2 mRNA levels by 80%. However, nuclear run-on experiments failed to detect any decrease in PGHS-2 mRNA transcription. This suggested dexamethasone rapidly caused PGHS-2 mRNA destabilization. Cycloheximide blocked PGHS-2 mRNA decrease. A fibroblast cell line, RS2, was identified that contained only isoenzyme 2 and possessed PGHS activity that was more highly mitogen-inducible than in any other cell line yet described. Mitogen-inducible activity in RS2 cells was inhibited by aspirin, indicating that PGHS-2, like PGHS-1, can be inactivated by this drug.

Inhibitory activities and inhibition specificities of caffeic acid derivatives and related compounds toward 5-lipoxygenase.

Various caffeic acid derivatives were synthesized, and their effects on 5-lipoxygenase (5-LO), 12-lipoxygenase (12-LO) and prostaglandin (PG) synthase activities were investigated. Among them, caffeic acid octyl amide (5) and 1-(3,4-dihydroxyphenyl)-1-octen-3-one (11) showed very potent inhibitory activities toward 5-LO with IC50 values of 4.2 x 10(-8) and 3.5 x 10(-8) M, respectively. They were very selective inhibitors for 5-LO. Compound 11 showed non-competitive inhibition, and the two adjacent hydroxy groups attached to the benzene ring, as well as the hydrophobic alkyl side chain, were required for its strong binding to 5-LO.