Rifampicin exposure reveals within-host Mycobacterium tuberculosis diversity in patients with delayed culture conversion.

Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptation to tuberculosis (TB) infection and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in two cohorts of TB patients, either with delayed Mtb culture conversion (> 2 months), or with fast culture conversion (< 2 months). We captured the genetic diversity of Mtb isolates obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed these isolates to rifampicin (RIF) prior to whole genome sequencing (WGS) analysis. Thanks to WGS, we detected at least 1 unfixed SNP within the Mtb isolates for 9/15 patients with delayed culture conversion, and non-synonymous (ns) SNPs for 8/15 patients. Furthermore, RIF exposure revealed 9 additional unfixed nsSNP from 6/15 isolates unlinked to drug resistance. By contrast, in the fast culture conversion cohort, RIF exposure only revealed 2 unfixed nsSNP from 2/20 patients. To better understand the dynamics of Mtb micro-diversity, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments mimicking the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants not necessarily linked to canonical drug-resistance, but which are prone to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management.

Synovial Extracellular Vesicles: Structure and Role in Synovial Fluid Tribological Performances.

The quality of the lubricant between cartilaginous joint surfaces impacts the joint's mechanistic properties. In this study, we define the biochemical, ultrastructural, and tribological signatures of synovial fluids (SF) from patients with degenerative (osteoarthritis-OA) or inflammatory (rheumatoid arthritis-RA) joint pathologies in comparison with SF from healthy subjects. Phospholipid (PL) concentration in SF increased in pathological contexts, but the proportion PL relative to the overall lipids decreased. Subtle changes in PL chain composition were attributed to the inflammatory state. Transmission electron microscopy showed the occurrence of large multilamellar synovial extracellular vesicles (EV) filled with glycoprotein gel in healthy subjects. Synovial extracellular vesicle structure was altered in SF from OA and RA patients. RA samples systematically showed lower viscosity than healthy samples under a hydrodynamic lubricating regimen whereas OA samples showed higher viscosity. In turn, under a boundary regimen, cartilage surfaces in both pathological situations showed high wear and friction coefficients. Thus, we found a difference in the biochemical, tribological, and ultrastructural properties of synovial fluid in healthy people and patients with osteoarthritis and arthritis of the joints, and that large, multilamellar vesicles are essential for good boundary lubrication by ensuring a ball-bearing effect and limiting the destruction of lipid layers at the cartilage surface.

Physiological and pathological roles of FATP-mediated lipid droplets in Drosophila and mice retina.

Increasing evidence suggests that dysregulation of lipid metabolism is associated with neurodegeneration in retinal diseases such as age-related macular degeneration and in brain disorders such as Alzheimer's and Parkinson's diseases. Lipid storage organelles (lipid droplets, LDs), accumulate in many cell types in response to stress, and it is now clear that LDs function not only as lipid stores but also as dynamic regulators of the stress response. However, whether these LDs are always protective or can also be deleterious to the cell is unknown. Here, we investigated the consequences of LD accumulation on retinal cell homeostasis under physiological and stress conditions in Drosophila and in mice. In wild-type Drosophila, we show that dFatp is required and sufficient for expansion of LD size in retinal pigment cells (RPCs) and that LDs in RPCs are required for photoreceptor survival during aging. Similarly, in mice, LD accumulation induced by RPC-specific expression of human FATP1 was non-toxic and promoted mitochondrial energy metabolism in RPCs and non-autonomously in photoreceptor cells. In contrast, the inhibition of LD accumulation by dFatp knockdown suppressed neurodegeneration in Aats-metFB Drosophila mutants, which carry elevated levels of reactive oxygen species (ROS). This suggests that abnormal turnover of LD may be toxic for photoreceptors cells of the retina under oxidative stress. Collectively, these findings indicate that FATP-mediated LD formation in RPCs promotes RPC and neuronal homeostasis under physiological conditions but could be deleterious for the photoreceptors under pathological conditions.

Skeletal muscle insulin resistance is induced by 4-hydroxy-2-hexenal, a by-product of n-3 fatty acid peroxidation.

AIMS/HYPOTHESIS: Oxidative stress is involved in the pathophysiology of insulin resistance and its progression towards type 2 diabetes. The peroxidation of n-3 polyunsaturated fatty acids produces 4-hydroxy-2-hexenal (4-HHE), a lipid aldehyde with potent electrophilic properties able to interfere with many pathophysiological processes. The aim of the present study was to investigate the role of 4-HHE in the development of insulin resistance. METHODS: 4-HHE concentration was measured in plasma from humans and rats by GC-MS. Insulin resistance was estimated in healthy rats after administration of 4-HHE using hyperinsulinaemic-euglycaemic clamps. In muscle cells, glucose uptake was measured using 2-deoxy-D-glucose and signalling pathways were investigated by western blotting. Intracellular glutathione was measured using a fluorimetric assay kit and boosted using 1,2-dithiole-3-thione (D3T). RESULTS: Circulating levels of 4-HHE in type 2 diabetic humans and a rat model of diabetes (obese Zucker diabetic fatty rats), were twice those in their non-diabetic counterparts (33 vs 14 nmol/l, p < 0.001), and positively correlated with blood glucose levels. During hyperinsulinaemic-euglycaemic clamps in rats, acute intravenous injection of 4-HHE significantly altered whole-body insulin sensitivity and decreased glucose infusion rate (24.2 vs 9.9 mg kg-1 min-1, p < 0.001). In vitro, 4-HHE impaired insulin-stimulated glucose uptake and signalling (protein kinase B/Akt and IRS1) in L6 muscle cells. Insulin-induced glucose uptake was reduced from 186 to 141.9 pmol mg-1 min-1 (p < 0.05). 4-HHE induced carbonylation of cell proteins and reduced glutathione concentration from 6.3 to 4.5 nmol/mg protein. Increasing intracellular glutathione pools using D3T prevented 4-HHE-induced carbonyl stress and insulin resistance. CONCLUSIONS/INTERPRETATION: 4-HHE is produced in type 2 diabetic humans and Zucker diabetic fatty rats and blunts insulin action in skeletal muscle. 4-HHE therefore plays a causal role in the pathophysiology of type 2 diabetes and might constitute a potential therapeutic target to taper oxidative stress-induced insulin resistance.

Oxygenation of polyunsaturated fatty acids and oxidative stress within blood platelets.

The oxygenation metabolism of arachidonic acid (ArA) has been early described in blood platelets, in particular with its conversion into the potent labile thromboxane A2 that induces platelet aggregation and vascular smooth muscle cells contraction. In addition, the primary prostaglandins D2 and E2 have been mainly reported as inhibitors of platelet function. The platelet 12-lipoxygenase (12-LOX) product, i.e. the hydroperoxide 12-HpETE, appears to stimulate platelet ArA metabolism at the level of its release from membrane phospholipids through phospholipase A2 (cPLA2) and cyclooxygenase (COX-1) activities, the first enzymes in prostanoid production cascade. Also, 12-HpETE may regulate the oxygenation of other polyunsaturated fatty acids (PUFA) by platelets, especially that of eicosapentaenoic acid (EPA). On the other hand, the reduced product of 12-HpETE, 12-HETE, is able to antagonize TxA2 action. This is even more obvious for the 12-LOX end-products from docosahexaenoic acid (DHA), 11- and 14-HDoHE. In addition, 12-HpETE plays a key role in platelet oxidative stress as observed in pathophysiological conditions, but may be regulated by DHA with a bimodal way according to its concentration. Other oxygenated products of PUFA, especially omega-3 PUFA, produced outside platelets may affect platelet functions as well.

Docosahexaenoic acid-containing choline phospholipid modulates LPS-induced neuroinflammation in vivo and in microglia in vitro.

BACKGROUND: Neuroinflammatory processes are considered a double-edged sword, having both protective and detrimental effects in the brain. Microglia, the brain's resident innate immune cells, are a key component of neuroinflammatory response. There is a growing interest in developing drugs to target microglia and control neuroinflammatory processes. In this regard, docosahexaenoic acid (DHA), the brain's n-3 polyunsaturated fatty acid, is a promising molecule to regulate pro-inflammatory microglia and cytokine production. Several works reported that the bioavailability of DHA to the brain is higher when DHA is acylated to phospholipid. In this work, we analyzed the anti-inflammatory activity of DHA-phospholipid, either acetylated at the sn-1 position (AceDoPC, a stable form thought to have superior access to the brain) or acylated with palmitic acid at the sn-1 position (PC-DHA) using a lipopolysaccharide (LPS)-induced neuroinflammation model both in vitro and in vivo. METHODS: In vivo, adult C57Bl6/J mice were injected intravenously (i.v.) with either AceDoPC or PC-DHA 24 h prior to LPS (i.p.). For in vitro studies, immortalized murine microglia cells BV-2 were co-incubated with DHA forms and LPS. AceDoPC and PC-DHA effect on brain or BV-2 PUFA content was assessed by gas chromatography. LPS-induced pro-inflammatory cytokines interleukin IL-1ß, IL-6, and tumor necrosis factor (TNF) α production were measured by quantitative PCR (qPCR) or multiplex. IL-6 receptors and associated signaling pathway STAT3 were assessed by FACS analysis and western-blot in vitro. RESULTS: In vivo, a single injection of AceDoPC or PC-DHA decreased LPS-induced IL-6 production in the hippocampus of mice. This effect could be linked to their direct effect on microglia, as revealed in vitro. In addition, AceDoPC or PC-DHA reduced IL-6 receptor while only AceDoPC decreased IL-6-induced STAT3 phosphorylation. CONCLUSIONS: These results highlight the potency of administered DHA-acetylated to phospholipids-to rapidly regulate LPS-induced neuroinflammatory processes through their effect on microglia. In particular, both IL-6 production and signaling are targeted by AceDoPC in microglia.

Omega-3 polyunsaturated fatty acids and oxygenated metabolism in atherothrombosis.

Numerous epidemiological studies and clinical trials have reported the health benefits of omega-3 polyunsaturated fatty acids (PUFA), including a lower risk of coronary heart diseases. This review mainly focuses on the effects of alpha-linolenic (ALA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on some risk factors associated with atherothrombosis, including platelet activation, plasma lipid concentrations and oxidative modification of low-density lipoproteins (LDL). Special focus is given to the effects of marine PUFA on the formation of eicosanoids and docosanoids, and to the bioactive properties of some oxygenated metabolites of omega-3 PUFA produced by cyclooxygenases and lipoxygenases. The antioxidant effects of marine omega-3 PUFA at low concentrations and the pro-oxidant effects of DHA at high concentrations on the redox status of platelets and LDL are highlighted. Non enzymatic peroxidation end-products deriving from omega-3 PUFA such as hydroxy-hexenals, neuroketals and EPA-derived isoprostanes are also considered in relation to atherosclerosis. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Protein "amyloid-like" networks at the phospholipid membrane formed by 4-hydroxy-2-nonenal-modified mitochondrial creatine kinase.

4-Hydroxy-2-nonenal (4-HNE) is a reactive aldehyde and a lipid peroxidation product formed in biological tissues under physiological and pathological conditions. Its concentration increases with oxidative stress and induces deleterious modifications of proteins and membranes. Mitochondrial and cytosolic isoforms of creatine kinase were previously shown to be affected by 4-HNE. In the present study, we analyzed the effect of 4-HNE on mitochondrial creatine kinase, an abundant protein from the mitochondrial intermembrane space with a key role in mitochondrial physiology. We show that this effect is double: 4-HNE induces a step-wise loss of creatine kinase activity together with a fast protein aggregation. Protein-membrane interaction is affected and amyloid-like networks formed on the biomimetic membrane. These fibrils may disturb mitochondrial organisation both at the membrane and in the inter membrane space.

Bis(monoacylglycero)phosphate accumulation in macrophages induces intracellular cholesterol redistribution, attenuates liver-X receptor/ATP-Binding cassette transporter A1/ATP-binding cassette transporter G1 pathway, and impairs cholesterol efflux.

OBJECTIVE: Endosomal signature phospholipid bis(monoacylglycero)phosphate (BMP) has been involved in the regulation of cellular cholesterol homeostasis. Accumulation of BMP is a hallmark of lipid storage disorders and was recently reported as a noticeable feature of oxidized low-density lipoprotein-laden macrophages. This study was designed to delineate the consequences of macrophage BMP accumulation on intracellular cholesterol distribution, metabolism, and efflux and to unravel the underlying molecular mechanisms. APPROACH AND RESULTS: We have developed an experimental design to specifically increase BMP content in RAW 264.7 macrophages. After BMP accumulation, cell cholesterol distribution was markedly altered, despite no change in low-density lipoprotein uptake and hydrolysis, cholesterol esterification, or total cell cholesterol content. The expression of cholesterol-regulated genes sterol regulatory element-binding protein 2 and hydroxymethylglutaryl-coenzyme A reductase was decreased by 40%, indicative of an increase of endoplasmic reticulum-associated cholesterol. Cholesterol delivery to plasma membrane was reduced as evidenced by the 20% decrease of efflux by cyclodextrin. Functionally, BMP accumulation reduced cholesterol efflux to both apolipoprotein A1 and high-density lipoprotein by 40% and correlated with a 40% decrease in mRNA contents of ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, and liver-X receptor α and ß. Foam cell formation induced by oxidized low-density lipoprotein exposure was exacerbated in BMP-enriched cells. CONCLUSIONS: The present work shows for the first time a strong functional link between BMP and cholesterol-regulating genes involved in both intracellular metabolism and efflux. We propose that accumulation of cellular BMP might contribute to the deregulation of cholesterol homeostasis in atheromatous macrophages.

Characterization and biological effects of di-hydroxylated compounds deriving from the lipoxygenation of ALA.

We have recently described a di-hydroxylated compound called protectin DX (PDX) which derives from docosahexaenoic acid (DHA) by double lipoxygenation. PDX exhibits anti-aggregatory and anti-inflammatory properties, that are also exhibited by similar molecules, called poxytrins, which possess the same E,Z,E conjugated triene geometry, and are synthesized from other polyunsaturated fatty acids with 22 or 20 carbons. Here we present new biological activities of di-hydroxylated metabolites deriving from α-linolenic acid (18:3n-3) treated by soybean 15-lipoxygenase (sLOX). We show that 18:3n-3 is converted by sLOX into mainly 13(S)-OH-18:3 after reduction of the hydroperoxide product. But surprisingly, and in contrast to DHA which is metabolized into only one di-hydroxylated compound, 18:3n-3 leads to four di-hydroxylated fatty acid isomers. We report here the complete characterization of these compounds using high field NMR and GC-MS techniques, and some of their biological activities. These compounds are: 9(R),16(S)-dihydroxy-10E,12E,14E-octadecatrienoic acid, 9(S),16(S)-dihydroxy-10E,12E,14E-octadecatrienoic acid, 9(S),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic acid, and 9(R),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic acid. They can also be synthesized by the human recombinant 15-lipoxygenase (type 2). Their inhibitory effect on blood platelet and anti-inflammatory properties were compared with those already reported for PDX.

Expanding the horizons of lipidomics. Towards fluxolipidomics.

This short review takes into consideration the status of lipidomics as issued from almost a decade of development. Because of the huge number of molecular species analyzed, there is a trend in subdividing lipidomics according to subdomains, in particular relating to the function of molecules. It is also pointed out that lipid imaging without the use of exogenous probes will help making relationships between molecular structures and the topography of lipid assemblies, especially in cellular compartments. Finally, a fluxomics approach is proposed for lipid molecular species, both in terms of compartments and biochemical metabolism. The example of fluxolipidomics of essential fatty acids toward their enzyme-dependent oxygenated metabolites and further toward their degradation products is developed.

Dietary oxidized n-3 PUFA induce oxidative stress and inflammation: role of intestinal absorption of 4-HHE and reactivity in intestinal cells.

Dietary intake of long-chain n-3 PUFA is now widely advised for public health and in medical practice. However, PUFA are highly prone to oxidation, producing potentially deleterious 4-hydroxy-2-alkenals. Even so, the impact of consuming oxidized n-3 PUFA on metabolic oxidative stress and inflammation is poorly described. We therefore studied such effects and hypothesized the involvement of the intestinal absorption of 4-hydroxy-2-hexenal (4-HHE), an oxidized n-3 PUFA end-product. In vivo, four groups of mice were fed for 8 weeks high-fat diets containing moderately oxidized or unoxidized n-3 PUFA. Other mice were orally administered 4-HHE and euthanized postprandially versus baseline mice. In vitro, human intestinal Caco-2/TC7 cells were incubated with 4-hydroxy-2-alkenals. Oxidized diets increased 4-HHE plasma levels in mice (up to 5-fold, P < 0.01) compared with unoxidized diets. Oxidized diets enhanced plasma inflammatory markers and activation of nuclear factor kappaB (NF-κB) in the small intestine along with decreasing Paneth cell number (up to -19% in the duodenum). Both in vivo and in vitro, intestinal absorption of 4-HHE was associated with formation of 4-HHE-protein adducts and increased expression of glutathione peroxidase 2 (GPx2) and glucose-regulated protein 78 (GRP78). Consumption of oxidized n-3 PUFA results in 4-HHE accumulation in blood after its intestinal absorption and triggers oxidative stress and inflammation in the upper intestine.

Poxytrins, a class of oxygenated products from polyunsaturated fatty acids, potently inhibit blood platelet aggregation.

Docosahexaenoic acid (DHA), an important component of marine lipids, exhibits anti-inflammatory activity related to some of its oxygenated metabolites, such as neuroprotectin/protectin D1 [NPD1/PD1; 10(R),17(S)-dihydroxy-docosa-4Z,7Z, 11E,13E,15Z,19Z-hexaenoic acid] produced through the 15-lipoxygenase pathway. However, other metabolites from DHA can be produced through this pathway, and other polyunsaturated fatty acids (PUFAs) of nutritional value may be oxygenated as well. Their biological activities remain unknown. Isomers of protectin D1 were synthesized using soybean lipoxygenase and tested for their ability to inhibit human blood platelet aggregation. A geometric isomer called PDX, previously described with the 11E,13Z,15E geometry, instead of 11E,13E,15Z in PD1, inhibited platelet aggregation at submicromolar concentrations when induced by either collagen, arachidonic acid, or thromboxane. The inhibition occurred at the level of both the cyclooxygenase activity and thromboxane receptor site. Interestingly, all the metabolites tested exhibiting the E,Z,E-conjugated triene were active, whereas E,E,Z trienes (as in PD1) or all-trans (E,E,E) trienes were inactive. We conclude that PDX and other oxygenated products from PUFAs of nutritional interest, having the E,Z,E-conjugated triene motif and collectively named poxytrins (PUFA oxygenated trienes), might have antithrombotic potential.

Low concentrations of reactive gamma-ketoaldehydes prime thromboxane-dependent human platelet aggregation via p38-MAPK activation.

Oxidative stress has been strongly implicated in pathological processes. Isoketals are highly reactive gamma-ketoaldehydes of the isoprostanes pathway of free radical-induced peroxidation of arachidonic acid that are analogous to cyclooxygenase-derived levuglandins. Because aldehydes, that are much less reactive than isoketals, have been shown to trigger platelet activation, we investigated the effect of one isoketal (E(2)-IsoK) on platelet aggregation. Isoketal potentiated aggregation and the formation of thromboxane B(2) in platelets challenged with collagen at a concentration as low as 1 nM. Moreover, the potentiating effect of 1 nM isoketal on collagen-induced platelet aggregation was prevented by pyridoxamine, an effective scavenger of gamma-ketoaldehydes. Furthermore, we provide evidence for the involvement of p38 mitogen-activated protein kinase in isoketal-mediated platelet priming, suggesting that isoketals may act upstream the activation of collagen-induced cytosolic phospholipase A(2). Additionally, the incubation of platelets with 1 nM isoketal led to the phosphorylation of cytosolic phospholipase A(2). The cytosolic phopholipase A(2) inhibitors AACOCF3 and MAFP both fully prevented the increase in isoketal-mediated platelet aggregation challenged with collagen. These results indicate that isoketals could play an important role in platelet hyperfunction observed in pathological states such as atherosclerosis and thrombosis through the activation of the endogenous arachidonic acid cascade.

Plasma membrane microdomains from hybrid aspen cells are involved in cell wall polysaccharide biosynthesis.

Detergent-resistant plasma membrane microdomains [DRMs (detergent-resistant membranes)] were isolated recently from several plant species. As for animal cells, a large range of cellular functions, such as signal transduction, endocytosis and protein trafficking, have been attributed to plant lipid rafts and DRMs. The data available are essentially based on proteomics and more approaches need to be undertaken to elucidate the precise function of individual populations of DRMs in plants. We report here the first isolation of DRMs from purified plasma membranes of a tree species, the hybrid aspen Populus tremula x tremuloides, and their biochemical characterization. Plasma membranes were solubilized with Triton X-100 and the resulting DRMs were isolated by flotation in sucrose density gradients. The DRMs were enriched in sterols, sphingolipids and glycosylphosphatidylinositol-anchored proteins and thus exhibited similar properties to DRMs from other species. However, they contained key carbohydrate synthases involved in cell wall polysaccharide biosynthesis, namely callose [(1-->3)-beta-D-glucan] and cellulose synthases. The association of these enzymes with DRMs was demonstrated using specific glucan synthase assays and antibodies, as well as biochemical and chemical approaches for the characterization of the polysaccharides synthesized in vitro by the isolated DRMs. More than 70% of the total glucan synthase activities present in the original plasma membranes was associated with the DRM fraction. In addition to shedding light on the lipid environment of callose and cellulose synthases, our results demonstrate the involvement of DRMs in the biosynthesis of important cell wall polysaccharides. This novel concept suggests a function of plant membrane microdomains in cell growth and morphogenesis.

Anti-inflammatory and anti-virus potential of poxytrins, especially protectin DX.

Poxytrins (Pufa Oxygenated Trienes) are dihydroxy derivatives from polyunsaturated fatty acids (PUFA) with adjacent hydroxyl groups to a conjugated triene having the specific E,Z,E geometry. They are made by the double action of one lipoxygenase or the combined actions of two lipoxygenases, followed by reduction of the resulting hydroperoxides with glutathione peroxidase. Because of their E,Z,E conjugated triene, poxytrins may inhibit inflammation associated with cyclooxygenase (COX) activities, and reactive oxygen species (ROS) formation. In addition of inhibiting COX activities, at least one poxytrin, namely protectin DX (PDX) from docosahexaenoic acid (DHA), has also been reported as able to inhibit influenza virus replication by targeting its RNA metabolism.

Cell wall polysaccharide synthases are located in detergent-resistant membrane microdomains in oomycetes.

The pathways responsible for cell wall polysaccharide biosynthesis are vital in eukaryotic microorganisms. The corresponding synthases are potential targets of inhibitors such as fungicides. Despite their fundamental and economical importance, most polysaccharide synthases are not well characterized, and their molecular mechanisms are poorly understood. With the example of Saprolegnia monoica as a model organism, we show that chitin and (1-->3)-beta-d-glucan synthases are located in detergent-resistant membrane microdomains (DRMs) in oomycetes, a phylum that comprises some of the most devastating microorganisms in the agriculture and aquaculture industries. Interestingly, no cellulose synthase activity was detected in the DRMs. The purified DRMs exhibited similar biochemical features as lipid rafts from animal, plant, and yeast cells, although they contained some species-specific lipids. This report sheds light on the lipid environment of the (1-->3)-beta-d-glucan and chitin synthases, as well as on the sterol biosynthetic pathways in oomycetes. The results presented here are consistent with a function of lipid rafts in cell polarization and as platforms for sorting specific sets of proteins targeted to the plasma membrane, such as carbohydrate synthases. The involvement of DRMs in the biosynthesis of major cell wall polysaccharides in eukaryotic microorganisms suggests a function of lipid rafts in hyphal morphogenesis and tip growth.

Effects of high and low inspired fractions of oxygen on horse erythrocyte membrane properties, blood viscosity and muscle oxygenation during anaesthesia.

OBJECTIVES: To evaluate whether a period of hyperoxia or after a period of hypoxia produced changes attributable to reactive oxygen species in anaesthetized horses. STUDY DESIGN: Prospective randomized experimental study. ANIMALS: Six healthy (ASA I) geldings, aged 4.5-9.5 years and weighing 510-640 kg(-1). METHODS: After 30 minutes breathing air as carrier gas for isoflurane, horses were assigned randomly to breathe air as carrier gas (CG0.21) or oxygen as carrier gas (CG1.00) for a further 90 minutes. After an interval of 1 month each horse was re-anaesthetized with the other carrier gas for the 90 minute test period. Ventilation was controlled throughout anaesthesia. Arterial blood was sampled to measure gas tensions, lactate, cholesterol, vitamin E, 4-hydroxy-alkenals, 8-epi-PGF(2 alpha), half haemolysis time, half erythrolysis time, and erythrocyte membrane fluidity. Muscle blood flow and oxygenation were evaluated by near infrared spectroscopy and coloured Doppler. RESULTS: After the first 30 minutes horses were hypoxemic. Subsequently the CG1.00 group became hyperoxaemic (PaO(2) approximately 240 mmHg) whereas the CG0.21 group remained hypoxaemic (PaO(2) approximately 60 mmHg) and had increased lactate concentration. No significant changes in vitamin E, 4-hydroxy-alkenals, or 8-epi-PGF(2 alpha) concentrations were detected. During the 90 minute test period the CG0.21 group had increased resistance to free-radical-mediated lysis in erythrocytes, whereas the CG1.00 group had slightly decreased resistance of whole blood to haemolysis. CG0.21 induced a progressive muscle deoxygenation whereas CG1.00 induced an increase in muscle oxygen saturation followed by progressive deoxygenation towards baseline. CONCLUSIONS: and clinical relevance During isoflurane anaesthesia in horses, the hyperoxia induced by changing from air to oxygen induced minimal damage from reactive oxygen species. Using air as the carrier gas decreased skeletal muscle oxygenation compared with using oxygen.

Biological relevance of double lipoxygenase products of polyunsaturated fatty acids, especially within blood vessels and brain.

The double lipoxygenation of polyunsaturated fatty acids (PUFA) is possible with PUFA having at least three methylene-interrupted double bonds. Several PUFA of the omega-3/n-3 and -6 families may be converted through this route, and the products show interesting inhibitory effects on blood platelet function and cyclooxygenase activities. This review focuses on two main omega-3 PUFA of nutritional interest, namely docosahexaenoic acid (DHA/22:6n-3) and alpha linolenic acid (ALA/18:3n-3). The chemical configuration of the double lipoxygenase end-product from DHA (protectin DX) is compared with that of protectin D1 which is produced through a mono-lipoxygenation step followed by an epoxidation and epoxide hydrolysis process. The different metabolic pathways are discussed as well as the different biological activities of both protectins.

Bis-allylic Deuterated DHA Alleviates Oxidative Stress in Retinal Epithelial Cells.

Oxidative stress plays a crucial role in developing and accelerating retinal diseases including age-related macular degeneration (AMD). Docosahexaenoic acid (DHA, C22:6, n-3), the main lipid constituent of retinal epithelial cell membranes, is highly prone to radical and enzymatic oxidation leading to deleterious or beneficial metabolites for retinal tissue. To inhibit radical oxidation while preserving enzymatic metabolism, deuterium was incorporated at specific positions of DHA, resulting in D2-DHA when incorporated at position 6 and D4-DHA when incorporated at the 6,9 bis-allylic positions. Both derivatives were able to decrease DHAs' toxicity and free radical processes involved in lipid peroxidation, in ARPE-19 cells (Adult Retinal Pigment Epithelial cell line), under pro-oxidant conditions. Our positive results encouraged us to prepare lipophenolic-deuterated-DHA conjugates as possible drug candidates for AMD treatment. These novel derivatives proved efficient in limiting lipid peroxidation in ARPE-19 cells. Finally, we evaluated the underlying mechanisms and the enzymatic conversion of both deuterated DHA. While radical abstraction was affected at the deuterium incorporation sites, enzymatic conversion by the lipoxygenase 15s-LOX was not impacted. Our results suggest that site-specifically deuterated DHA could be used in the development of DHA conjugates for treatment of oxidative stress driven diseases, or as biological tools to study the roles, activities and mechanisms of DHA metabolites.