The microaerophilic flagellate, Trichomonas vaginalis, contains unusual acyl lipids but no detectable cardiolipin.

Previous lipid analysis of trichomonads has led to controversy as to whether these hydrogenosome-containing organisms contain cardiolipin (CL), which is a characteristic component of mitochondria. Here we report a careful lipid analysis of the sexually transmitted protist Trichomonas vaginalis. Major lipids were phosphatidylethanolamine (42%) and phosphatidylcholine (20%) with lesser amounts of phosphatidylglycerol (PG) (12%) and non-polar components. Two unusual lipids, acyl-PG (8%) and ceramide phosphorylethanolamine (2%), were also significant components. The structures of these lipids were confirmed by tandem mass spectrometry following reverse-phase high-performance liquid chromatography. This is the first time ceramide phosphorylethanolamine has been reported in a trichomonad. In contrast, CL (diphosphatidylglycerol) could not be detected either by two-dimensional thin-layer chromatography or by mass spectrometry. These data are discussed in relation to the organism's phylogenetic origin as a parasite showing secondary adaptation to microaerobic conditions.

Interleukin-13-induced MUC5AC is regulated by 15-lipoxygenase 1 pathway in human bronchial epithelial cells.

RATIONALE: 15-Lipoxygenase-1 (15LO1) and MUC5AC are highly expressed in asthmatic epithelial cells. IL-13 is known to induce 15LO1 and MUC5AC in human airway epithelial cells in vitro. Whether 15LO1 and/or its product 15-HETE modulate MUC5AC expression is unknown. OBJECTIVES: To determine the expression of 15LO1 in freshly harvested epithelial cells from subjects with asthma and normal control subjects and to determine whether IL-13-induced 15LO1 expression and activation regulate MUC5AC expression in human bronchial epithelial cells in vitro. METHODS: Human airway epithelial cells from subjects with asthma and normal subjects were evaluated ex vivo for 15LO1 and MUC5AC expression. The impact of 15LO1 on MUC5AC expression in vitro was analyzed by inhibiting 15LO1 through pharmacologic (PD146176) and siRNA approaches in human bronchial epithelial cells cultured under air-liquid interface. We analyzed 15 hydroxyeicosatetraenoic acid (15-HETE) by liquid chromatography/UV/mass spectrometry. MUC5AC and 15LO1 were analyzed by real-time RT-PCR, immunofluoresence, and Western blot. MEASUREMENTS AND MAIN RESULTS: Epithelial 15LO1 expression increased with asthma severity (P < 0.0001). 15LO1 significantly correlated with MUC5AC ex vivo and in vitro. IL-13 increased 15LO1 expression and stimulated formation of two molecular species of 15-HETE esterified to phosphotidylethanolamine (15-HETE-PE). Inhibition of 15LO1 suppressed 15-HETE-PE and decreased MUC5AC expression in the presence of IL-13 stimulation. The addition of exogenous 15-HETE partially restored MUC5AC expression. CONCLUSIONS: Epithelial 15LO1 expression increases with increasing asthma severity. IL-13 induction of 15-HETE-PE enhances MUC5AC expression in human airway epithelial cells. High levels of 15LO1 activity could contribute to the increases of MUC5AC observed in asthma.

Eicosanoids: generation and detection in mammalian cells.

Eicosanoids are 20-carbon lipids generated by the oxidation of arachidonic acid that are involved in physiological signaling in virtually all organ systems. Three primary enzymatic pathways are responsible for their synthesis in mammalian cells: lipoxygenase, cyclooxygenase, and cytochrome P450. They signal through receptor-dependent pathways, and their dysregulation is central to numerous pathological states including cancer and inflammation. Recent advances in their detection and analysis using mass spectrometry have made the study of these molecules more accessible to the research community in general. This review focuses on the available methods for the detection and analysis of eicosanoids and aims to act as a guide for those wishing to approach the analysis of eicosanoids for their own research.

The identification and role of a novel eicosanoid in the reproductive behaviour of barnacles (Balanus balanus).

Post-copulatory behaviour in barnacles involves a violent rocking movement of the opercular valves, which is thought to contribute to the expulsion of oocytes through the oviduct into the mantle cavity where they are fertilised. We demonstrate in this study that the seminal vesicles/testis of the subtidal barnacle Balanus balanus produce a biologically active factor, barnacle muscle stimulatory factor (BMSF), which causes a significant increase in cirral and body muscular activity. BMSF was identified using a combination of high performance liquid chromatography and mass spectrometry as a novel eicosanoid/oxylipin, 8,13-dihydroxyeicosapentaenoic acid. This is rapidly inactivated under mild acid conditions to form a complex range of triene and pentaene chromophore-containing products that have only been partially identified. Injection of purified BMSF into the mantle cavity of barnacles caused the rocking movements of the opercular valves as reported following fertilisation. In excised barnacles, it also caused muscular contractions of the whole body mass. The breakdown products of BMSF, however, were without such activities. The function of BMSF in facilitating fertilisation in barnacles is comparable to the role of other eicosanoids in human reproduction, reinforcing the view that these compounds have conserved activities in both invertebrates and vertebrates.

Eicosanoid generation in the intertidal barnacle, Balanus perforatus--effect of season and reproductive status.

The nature and quantity of the principal lipoxygenase (LO) products generated by the intertidal barnacle Balanus perforatus were examined at monthly intervals and their potential involvement in reproductive events was investigated. The main mono-hydroxy products generated were found to be formed through the action of an 8-lipoxygenase (8-LO) activity and were the mono-hydroxy fatty acids, 8-hydroxyeicosapentaenoic acid (HEPE) and 8-hydroxyeicosatetraenoic acid. Generation of these products was found to be correlated with the environmental seawater temperature, although no change in either 8-LO activity or the precursor fatty acid levels in total phospholipids was found with the time of the year. Changes in fatty acid composition measured in animals collected from summer and winter conditions were found to follow the pattern expected by homeoviscous adaptation of a cold-acclimated animal. Oogenesis was found to occur in August and was linked to a significant reduction in 8-HEPE generation. Spermatozoa were found to be present year-round in the seminal vesicles although the testes became atrophic during the winter months.

At what stage in metazoan evolution did leukotriene generation first appear?--key insights from cartilaginous fish.

Leukotriene (LT) B4 is a key player in inflammatory responses in mammals. During the generation of this derivative of arachidonic acid, the unstable product of 5-lipoxygenase, termed LTA4, is converted to LTB4 by LTA4 hydrolase. Invertebrates do not generate LTs yet all vertebrates from bony fish onwards synthesize this compound. As cartilaginous fish are the most primitive living jawed vertebrates, we investigated if the leukocytes from such a fish, the Thornback ray (Raja clavata) could generate LTB4. Supernatants from ionophore-challenged leukocytes generated the 5-lipoxygenase products, 6-trans-LTB4 and 6-trans-12-epi-LTB4 but were unable to synthesize LTB4. To determine if these cells contained an active LTA4 hydrolase, LTA4 was incubated with lysates from ray leukocytes. Such preparations did not contain any demonstrable LTA4 hydrolase activity. Our findings imply at the stage of cartilaginous fish evolution over 350 million years ago that the evolution of an active LTA4 hydrolase had yet to occur.

Hepoxilins and trioxilins in barnacles: an analysis of their potential roles in egg hatching and larval settlement.

The barnacle life cycle has two key stages at which eicosanoids are believed to be involved in cellular communication pathways, namely the hatching of nauplii and the settlement of cypris larvae. Barnacle egg-hatching activity has previously been reported to reside in a variety of eicosanoids, including 8-hydroxyeicosapentaenoic acid and a number of tri-hydroxylated polyunsaturated fatty acid derivatives, the trioxilins. The production of the eicosapentaenoic acid metabolite trioxilin A4 (8,11,12-trihydroxy-5,9,14,17-eicosatetraenoic acid) by the barnacles Balanus amphitrite and Elminius modestus was confirmed using a combination of high-performance liquid chromatography and gas chromatography, both linked to mass spectrometry. In addition, both species also generated trioxilin A3 (8,11,12-trihydroxy-5,9,14-eicosatrienoic acid; an arachidonic acid-derived product), 8,11,12-trihydroxy-9,14,17-eicosatrienoic acid (a omega3 analogue of trioxilin A3; derived from omega3 arachidonic acid) and 10,13,14-trihydroxy-4,7,11,16,19-docosapentaenoic acid (a docosahexaenoic acid-derived product). In contrast to earlier reports, trioxilin A3 had no E. modestus egg-hatching activity at any of the concentrations tested (10(-9)-10(-6) mol l(-1)). The unstable epoxide precursor hepoxilin A3, however, caused significant levels of hatching at 10(-6) mol l(-1). Furthermore, the stable hepoxilin B3 analogue PBT-3 stimulated hatching at 10(-7) mol l(-1). Neither trioxilin A3, hepoxilin A3 or PBT-3 at 0.25-30 micromol l(-1) served as settlement cues for B. amphitrite cypris larvae.