Prostaglandin E 2 (PGE 2 ) is one of the most important biologically active prostanoids found throughout the gastrointestinal tract. Despite the fact that PGE 2 regulates many physiological functions of the gut including mucosal protection, gastrointestinal secretion and motility, it is implicated in the pathophysiology of inflammatory bowel diseases (IBD) and colorectal neoplasia. The varied biological functions exerted by PGE 2 are through the pharmacologically distinct, G-protein coupled plasma membrane receptors termed EP receptors. Disruptions of various prostanoid receptor genes have helped in unravelling the physiological functions of these receptors. To date, all four subtypes of EP receptors have been individually knocked out in mice and various phenotypes have been reported for each subtype. Similarly, in vitro and in vivo studies using EP receptor agonists and antagonists have helped in uncoupling the diverse functions of PGE 2 signalling involving distinct EP receptors in the gut. In this review, we will summarize and conceptualize the salient features of EP receptor subtypes, their regional functions in the gut and how expressions of EP receptors are altered during disease states.

The compositions and physical states of the Iver phospholipids of marine and freshwater fish adapted to relatively constant but radically different temperatures were investigated. Fish adapted to low temperature (5-10°C) accumulated more unsaturated fatty acids than those in a warm (25-27°C) environment. There were no measurable differences in the gross fatty acid compositions of the total liver phospholipids fom Ident thermal environments. Docosahexaenoic acid (22:6) Poikilotherms are often subjected to temperature stress or seasonal variation of temperature. These organisms exploit diversity in lipid structures to fashion membranes to prevailing ambient temperatures in such a manner that they become more fluid in a cold-acclimatized state and less fluid in a warmth-acclimatized state (1, 2). Sinensky (3) has termed this response to temperature "homeoviscous adaptation". The extent ofhomeoviscous efficacy with which cells compensate membrane fluidity in response to changes in ambient temperature is only partial (4,5). However, this response is rather rapid in the carp liver endoplasmic reticulum (6) and erythrocytes (7). Restructuring of the polar headgroup composition of phospholipid classes, together with modification of the unsaturation of their fatty acyl chains, may ensure that the components present are best suited to function within the constraints imposed by a prevailing thermal environment. One rational explanation of these responses is that the melting points of the fatty acids decrease with an increasing number of double bonds in the molecule, resulting in a more fluid structure in membranes rich in polyunsaturated fatty acids. Accepting this hypothesis, one would expect a marked difference between fish species inhabiting regions of ex-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.tremely different but relatively constant temperatures. Phospholipids from specific membranes of an Arctic marine fish proved more unsaturated than those of subtropical freshwater fish, and these differences were reflected in the microviscosity ofthese structures (8). The present article compares the liver phospholipid compositions of a number of marine and freshwater fish, and a feasible mechanism is proposed to explain the adaptational phenomena at a membrane structural level.MATERIALS AND METHODS Fish. The following fish were involved in this study. Warm-adapted marine fish (WAMF) included Carnax calla, Epinephelus bleekeri, Lutaganus sebeae, Mugil passia, Nemipterus hexodon, Nemipterusjavonicus, Pomadysys hasta, Sardinella longiceps, and Tachisurus yella from the South China Sea and the southwest coast of India (20-27C). Cold-adapted marine fish (CAMF) included Clupea harengus, Scorpaenichthys marmoratus, Hexaganus stelleri, Hipoglossus hipoglossus, Onchorhyncus kisutch, Onchorhyncus tshawsytscha, Ophioidon melanus, and Sebastosomus melanus from the...

A comparison of the structural orders of membranes of a mixed brain-cell population isolated from Cyprinus carpio L. acclimated to either summer (23-250C) or winter (50C) revealed a high degree of compensation (80%) for temperature, as assayed by electron spin resonance spectroscopy. The cells rapidly forget their thermal history and adjust the physical properties of the membranes when shifted to the other extreme of temperature either in vivo or in vitro. Phospholipids separated from both types of animals exhibit only around 10% compensation. Arachidonic and docosahexaenoic acids are the major polyunsaturated fatty acids in the brains, but the fatty acid composition of the brain total phospholipids does not vary with adaptation to temperature. Separation of phosphatidylcholines and phosphatidylethanolamines into molecular species revealed a 2-to 3-fold accumulation of 18:1/ 22:6, 18:1/20:4, and 18:1/18:1 species in the latter; 18:0/22:6 showed an opposite tendency. Molecular species composition of phosphatidylcholines did not vary with the temperature. The same trends of changes were seen with brains offreshwater fish from subtropical (Coda cada L.) or boreal (Acerina cernua) regions. It is concluded that the gross amount of docosahexaenoic acid (22:6) plays only a minor role in adjusting the membrane physical properties to temperature. Factors other than lipids might be involved in the adaptation processes. Due to their specific molecular architecture, molecules such as 18:1/22:6, 18:1/20:4, or 18:1/18:1 phosphatidylethanolamine might prevent the contraction of membranes in the cold and may provide an environment for some other components involved in the temperature regulation ofphysical properties of nerve cell membranes.Most poikilotherms respond to thermal changes by adapting the physical properties of their membranes to the new situation to preserve the functional and structural integrity of these structures, a phenomenon that Sinensky (1) termed "homeoviscous adaptation." The homeoviscous efficacy, the extent to which the cells compensate for temperature changes, varies among the tissues and membranes (2, 3). Adjustment of the physicochemical properties of the membranes to the temperature is expected to be rapid and reversible to ensure proper functioning under fluctuating thermal conditions in fish. Wodtke and Cossins (4) have shown that the fluidity of the mitochondria in fish liver follows changes in the environmental temperature. It has also been demonstrated that the plasma membrane of carp erythrocyte rapidly adjusts to temperature under both in vivo and in vitro conditions (5, 6). The functions of neural tissue are highly dependent on membrane processes. Adaptation of the physical state of the synaptic vesicles in fish brain (2, 7), of the synaptic vesicles, mitochondria, and myelin fractions of an air-breathing, subtropical fish, Channa punctatus (8), and of the synaptosomal and myelin fraction of carp brain (9) has been described. Changes in environmental temperature have been shown to ca...

Phospholipids from livers of carps (Cyprinus carpio L.) adapted to winter (5 degrees C) and summer (25 degrees C) temperatures were isolated, and the fatty acid composition of total phospholipids, as well as molecular species composition of diacyl phosphatidylcholines and ethanolamines, were determined. Order parameter of 5-doxyl stearic acid and steady-state fluorescence anisotropy of different anthroyloxy fatty acids--[2-, 12(N-9-anthroyloxy)stearic acid and 16(N-9-anthroyloxy)palmitic acid--embedded in native and synthetic (16:0/16:0, 16:0/22:6, 18:0/22:6, 18:1/22:6, 20:4/20:4, 22:6/22:6 phosphatidylcholines and 16:0/18:1, 18:1/22:6 phosphatidylethanolamines) phospholipid vesicles was also determined between -30 and 30 degrees C and 5 and 30 degrees C, respectively. There is an accumulation of 1-monoenoic, 2-polyenoic diacyl phosphatidylcholine and ethanolamine with a concomitant reduction of 1-stearoyl,2-docosahexaenoyl species in the cold-adapted state. Despite a 30% accumulation of long-chain polyunsaturated fatty acids in phospholipids in cold, there is only a 5 degrees C downshift in the solid-gel to liquid-crystalline phase transition temperature (-8 vs. -13 degrees C). Vesicles from total phospholipids of cold-adapted fish proved to be more disordered in all segments than from the warm-adapted ones when assayed using 2,12-(N-9-anthroyloxy)stearic and 16-(N-9-anthroyloxy)palmitic acid. Vesicles made from purified phosphatidylcholines showed the same pattern, but they were more disordered than the corresponding total phospholipids. This could be modelled using mixed phospholipid vesicles made of synthetic 16:0/22:6 phosphatidylcholine (75%) and either 18:1/22:6 phosphatidylethanolamine (25%) vs. 16:0/18:1 phosphatidylethanolamine (25%) and comparison of the anisotropy parameters of 100% 16:0/22:6 and 100% 18:1/22:6 phosphatidylcholine vesicles. Mixing either 16:0/18:1 (25%) or 18:1/22:6 (25%) phosphatidylethanolamines to 18:0/22:6 (75%) phosphatidylcholine shifted down or up, respectively, the transition temperature of vesicles compared to 100% 18:0/22:6 vesicles assayed by electron spin resonance spectroscopy using 5-doxylstearic acid. It is concluded that it is not the gross amount of long-chain polyunsaturated fatty acids in phospholipids, but rather their specific combination with cis delta 9 monounsaturated fatty acids in the position sn-1, especially in phosphatidylethanolamines, that is important in determining the physical properties of biomembranes in relation to adaptational temperature.

The intestinal protozoan parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide. However, almost nothing is known about the molecules secreted by the parasite that modulate host immune responses or epithelial barrier function in the colon. Herein, we describe the isolation and characterization of a cyclooxygenase (COX)-like enzyme in E. histolytica that is responsible for the biosynthesis of prostaglandin (PG)E 2 . PGE 2 produced by ameba was constitutive but highly dependent on exogenous arachidonic acid substrate. COX-like activity and the immunoreactive protein were localized to the nuclear fraction of E. histolytica. The COX-like protein (72 kDa) was microsequenced and cloned by reverse transcriptase PCR. Ameba COX showed little homology with COX-1͞2 enzymes from different species at the nucleotide and amino acid levels. Surprisingly, the arachidonatebinding domain and heme-coordinating and catalytic sites, which are conserved in other species, were absent in ameba. Ameba COX expressed in Escherichia coli demonstrated COX-like enzyme activity in vitro by converting arachidonic acid into PGE 2 but not into PGD 2 or PGF2␣. COX activity was inhibited with 1 mM aspirin but not with indomethacin or COX-1͞2-specific inhibitors. Taken together, these studies reveal that E. histolytica produces PGE 2, by means of a previously undescribed ancestral COX-like enzyme, which could play a major role in pathogenesis and immune evasion.

Background and purpose: Prostaglandin (PG) E2 and interleukin (IL)-8 are simultaneously increased during the inflammation that characterizes numerous pathologies such as inflammatory bowel disease. IL-8 is a potent neutrophil chemo-attractant and activator, and can initiate and/or exacerbate tissue injury. PGE2 signals principally through prostanoid receptors of the EP2 and/or EP4 subtypes to promote cAMP-dependent cellular functions. The aim of this study was to identify the role of the EP2 and EP4 receptor subtype(s) on two human colonic epithelial cell lines (Caco-2 and T84), in regulating PGE2-induced IL-8 production.Experimental approach: To identify the causative receptor, we knocked-down and over-expressed EP2 and EP4 receptor subtypes in colonic epithelial cells and studied the effect of several selective EP2/EP4 receptor agonists and antagonists. The inductions of IL-8 and EP receptor mRNA and protein expression were determined by real-time PCR and western blot analysis. The affinity of PGE2 and Bmax values for the EP2 and EP4 receptor on colonic epithelial cells were determined by radioligandbinding assays with [ 3 H]PGE2. Key results: PGE2 had the highest affinity for the EP4 receptor subtype and promoted a robust stimulation of cAMP-dependent IL-8 synthesis. This effect was mimicked by a selective EP4 receptor agonist, ONO-AE1-329, and abolished by silencing the EP4 receptor gene by using siRNA techniques, a selective EP4 receptor antagonist (ONO-AE3-208) and a selective inhibitor (Rp-cAMP) of cAMP-dependent protein kinase. Conclusions and implications: These findings suggest that initiation and progression of colonic inflammation induced by IL-8 could be mediated, at least in part, by PGE2 acting via the EP4 receptor subtype.

Background2-Hydroxyoleic acid is a synthetic fatty acid with potent anti-cancer activity which does not induce undesired side effects. However, the molecular and cellular mechanisms by which this compound selectively kills human glioma cancer cells without killing normal cells is not fully understood. The present study was designed to determine the molecular bases underlying the potency against 1321N1, SF-767 and U118 human glioma cell lines growth without affecting non cancer MRC-5 cells.Methodology/Principal FindingsThe cellular levels of endoplasmic reticulum (ER) stress, unfolded protein response (UPR) and autophagy markers were determined by quantitative RT-PCR and immunoblotting on 1321N1, SF-767 and U118 human glioma cells and non-tumor MRC-5 cells incubated in the presence or absence of 2OHOA or the ER stress/autophagy inducer, palmitate. The cellular response to these agents was evaluated by fluorescence microscopy, electron microscopy and flow cytometry. We have observed that 2OHOA treatments induced augments in the expression of important ER stress/UPR markers, such as phosphorylated eIF2α, IRE1α, CHOP, ATF4 and the spliced form of XBP1 in human glioma cells. Concomitantly, 2OHOA led to the arrest of 1321N1 cells in the G2/M phase of the cell cycle, with down-regulation of cyclin B1 and Cdk1/Cdc2 proteins in the three glioma cell lines studied. Finally, 2OHOA induced autophagy in 1321N1, SF-767 and U118 cells, with the appearance of autophagic vesicles and the up-regulation of LC3BI, LC3BII and ATG7 in 1321N1 cells, increases of LC3BI, LC3BII and ATG5 in SF-767 cells and up-regulation of LC3BI and LC3BII in U118 cells. Importantly, 2OHOA failed to induce such changes in non-tumor MRC-5 cells.Conclusion/SignificanceThe present results demonstrate that 2OHOA induces ER stress/UPR and autophagy in human glioma (1321N1, SF-767 and U118 cell lines) but not normal (MRC-5) cells, unraveling the molecular bases underlying the efficacy and lack of toxicity of this compound.

Entamoeba histolytica pathogenesis in the colon occurs in a stepwise fashion. It begins with colonization of the mucin layer, which is followed by stimulation of a proinflammatory response that causes nonspecific tissue damage that may facilitate parasite invasion of the underlying colonic mucosa. Unfortunately, the parasite and/or host factors that stimulate a proinflammatory response in the gut are poorly understood. In this study, we found that live E. histolytica or secretory or proteins (SP) and soluble ameba components (SAP) can markedly increase interleukin-8 (IL-8) mRNA expression and protein production in colonic epithelial cells. The IL-8-stimulating molecule produced by live amebae was identified as prostaglandin E 2 (PGE 2 ) as trophozoites treated with cyclooxygenase inhibitors inhibited the biosynthesis of PGE 2 and eliminated IL-8 production induced by live parasites or ameba components. Moreover, using specific prostaglandin EP2 and EP4 receptor agonists and antagonists, we found that PGE 2 binds exclusively through EP4 receptors in colonic epithelial cells to stimulate IL-8 production. Silencing of EP4 receptors with EP4 small interfering RNA completely eliminated SP-and SAP-induced IL-8 production. These studies identified bioactive PGE 2 as a one of the major virulence factors produced by E. histolytica that can stimulate the potent neutrophil chemokine and activator IL-8, which can trigger an acute host inflammatory response. Thus, the induction of IL-8 production in response to E. histolytica-derived PGE 2 may be a mechanism that explains the initiation and amplification of acute inflammation associated with intestinal amebiasis.Entamoeba histolytica is an enteric protozoan parasite and the fourth leading cause of death due to a parasite (26). Humans are the only known host for E. histolytica, and about 50 million people are affected worldwide each year. The pathogenesis of amebiasis is believed to be a multistep and multifactorial process. Although a large number of studies have attempted to unravel the factors or molecules responsible for the pathogenesis of amebiasis, the processes involved in pathogenesis are not well understood. In most infected individuals, E. histolytica trophozoites exist as commensals. However, in a small percentage of infections, amebae can elude luminal and epithelial barrier host defense mechanisms and invade the intestinal mucosa, causing ulcers and amebic colitis. Even though host inflammatory responses play an important role in the onset and progression of invasive amebiasis, little is known about the parasite factors that initiate this event. Even less is known about the parasite components that are secreted or released in the gut and can modulate colonic epithelial cell functions.