8S-lipoxygenase products activate peroxisome proliferator-activated receptor alpha and induce differentiation in murine keratinocytes.

To determine the function and mechanism of action of the 8S-lipoxygenase (8-LOX) product of arachidonic acid, 8S-hydroxyeicosatetraenoic acid (8S-HETE), which is normally synthesized only after irritation of the epidermis, transgenic mice with 8-LOX targeted to keratinocytes through the use of a loricrin promoter were generated. Histological analyses showed that the skin, tongue, and stomach of transgenic mice are highly differentiated, and immunoblotting and immunohistochemistries of skin showed higher levels of keratin-1 expression compared with wild-type mice. The labeling index, however, of the transgenic epidermis was twice that of the wild-type epidermis. Furthermore, 8S-HETE treatment of wild-type primary keratinocytes induced keratin-1 expression. Peroxisome proliferator activated receptor alpha (PPARalpha) was identified as a crucial component of keratin-1 induction through transient transfection with expression vectors for PPARalpha, PPARgamma, and a dominant-negative PPAR, as well as through the use of known PPAR agonists. From these studies, it is concluded that 8S-HETE plays an important role in keratinocyte differentiation and that at least some of its effects are mediated by PPARalpha.     

Stereocontrol of arachidonic acid oxygenation by vertebrate lipoxygenases: newly cloned zebrafish lipoxygenase 1 does not follow the Ala-versus-Gly concept

Animal lipoxygenases (LOXs) are classified according to their specificity of arachidonic acid oxygenation, and previous sequence alignments suggested that S-LOXs contain a conserved Ala at a critical position at the active site but R-LOXs carry a Gly instead. Here we cloned, expressed, and characterized a novel LOX isoform from the model vertebrate Danio rerio (zebrafish) that carries a Gly at this critical position, classifying this enzyme as putative arachidonic acid R-LOX. Surprisingly, the almost exclusive arachidonic acid oxygenation product was 12S-H(p)ETE (hydro(pero)xyeicosatetraenoic acid), and extensive mutation around Gly-410 failed to induce R-lipoxygenation. This finding prompted us to explore the importance of the corresponding amino acids in other vertebrate S-LOXs. We found that Ala-to-Gly exchange in human 15-LOX2 and human platelet 12-LOX induced major alterations in the reaction specificity with an increase of specific R-oxygenation products. For mouse 5-LOX and 12/15-LOX from rabbits, men, rhesus monkeys, orangutans, and mice, only minor alterations in the reaction specificity were observed. For these enzymes, S-HETE (hydroxyeicosatetraenoic acid) isomers remained the major oxygenation products, whereas chiral R-HETEs contributed only 10-30% to the total product mixture. Taken together these data indicate that the Ala-versus-Gly concept may not always predict the reaction specificity of vertebrate LOX isoforms.     

Biological role of hepoxilins: upregulation of phospholipid hydroperoxide glutathione peroxidase as a cellular response to oxidative stress?

The 12S-lipoxygenase (12S-LOX) pathway of arachidonic acid (AA) metabolism is bifurcated at 12(S)-hydroperoxy-5Z,8Z,10E (12S-HpETE) in the reduction route to form 12S-hydroxy-eicosatetraenoic acid (12S-HETE) and in 8(S/R)-hydroxy-11(S),12S-trans-epoxyeicosa-5Z,9E,14Z-trienoic acid (HXA3) synthase pathway, previously known as isomerization route, to form hepoxilins. Earlier we showed that the HXA3 formation is restricted to cellular systems devoid of hydroperoxide reducing enzymes, e.g. GPxs, thus causing a persistent oxidative stress situation. Here, we show that HXA3 at as low as 100 nM concentration upregulates phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA and protein expressions, whereas other metabolites of AA metabolism 12S-HpETE and 12S-HETE failed to stimulate the PHGPx. Moreover, the decrease in 12S-HpETE below a threshold value of the hydroperoxide tone causes both suppression of the overall 12S-LOX activity and a shift from HXA3 formation towards 12S-HETE formation. We therefore propose that under persistent oxidative stress the formation of HXA3 and the HXA3-induced upregulation of PHGPx constitute a compensatory defense response to protect the vitality and functionality of the cell.     

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-γ-linolenic acid (DGLA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbβ3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane.     

Formation of lipoxin B by the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate

The pure reticulocyte lipoxygenase converts 15LS-hydroxy-5,8,11,13(Z,Z,Z,E)-icosatetraenoic acid (15LS-HETE) methyl ester to a complex mixture of products containing 5DS,14LR,15LS-trihydro(pero)xy-6E,++ +8Z,10E,12E-icosatetraenoate methyl ester (lipoxin B methyl ester), 5DS,15LS-DiH(P)ETE methyl ester and four 8,15LS-DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy-icosatetraenoic acid]. After a short incubation period (15 min) 5DS,15LS-DiH(P)ETE methyl ester was found to be the main product, whereas after a 3-h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400-MHz 1H-NMR. Atmospheric oxygen was incorporated at carbon-5 and carbon-14 into the major product. 5DS,15LS-DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15LS-HETE and 5DS,15LS-DiHETE. The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.     

Expression of 5-oxoETE receptor in prostate cancer cells: critical role in survival

Previously, we reported that metabolism of arachidonic acid through the 5-lipoxygenase (5-LOX) pathway plays an important role in the survival and growth of human prostate cancer cells. Inhibition of 5-LOX by pharmacological inhibitors triggers apoptosis in prostate cancer cells within hours of treatment, which is prevented by the metabolites of arachidonate 5-lipoxygenase, 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE), and its dehydrogenated derivative, 5-oxoeicosatetraenoic acid (5-oxoETE). These findings suggested that 5-lipoxygenase metabolites are critical survival factors of prostate cancer cells. However, molecular mechanisms by which 5(S)-HETE and its derivative 5-oxoETE exert their effects on prostate cancer cell survival are yet to be understood. Here, we report that human prostate cancer cells differentially express a G-protein-coupled 5-oxoETE receptor (5-oxoER) in them. Blocking expression of 5-oxoER by short-interfering RNA (siRNA) significantly reduced the viability of prostate cancer cells, suggesting that 5-oxoER is critical for prostate cancer cell survival, and that the 5-LOX metabolite, 5-oxoETE, controls survival of prostate cancer cells through its own G-protein-coupled receptor, 5-oxoER.     

Structure, biochemistry and biology of hepoxilins: an update

Hepoxilins are biologically relevant epoxy-hydroxy eicosanoids synthesized through the 12S-lipoxygenase (12S-LOX) pathway of the arachidonic acid (AA) metabolism. The pathway is bifurcated at the level of 12S-hydroperoxy-eicosatetraenoic acid (12S-HpETE), which can either be reduced to 12S-hydro-eicosatetraenoic acid (12S-HETE) or converted to hepoxilins. The present review gives an update on the biochemistry, biology and clinical aspects of hepoxilin-based drug development. The isolation, cloning and characterization of a rat leukocyte-type 12S-LOX from rat insulinoma RINm5F cells revealed a 12S-LOX possessing an intrinsic 8S/R-hydroxy-11,12-epoxyeicosa-5Z,9E,14Z-trienoic acid (HXA(3)) synthase activity. Site-directed mutagenesis studies on rat 12S-LOX showed that the HXA(3) synthase activity was impaired when the positional specificity of AA was altered. Interestingly, amino acid Leu353, and not conventional sequence determinants Met419 and Ile418, was found to be a crucial sequence determinant for AA oxygenation. The regulation of HXA(3) formation is dependent on the cellular overall peroxide tone. Cellular glutathione peroxidases (cGPxs) compete with HXA(3) synthase for 12S-HpETE as substrate either to reduce to 12S-HETE or to convert to HXA(3), respectively. Therefore, RINm5F cells, which are devoid of GPxs, are capable of converting AA or 12S-HpETE to HXA(3) under basal conditions, whereas cells overexpressing cGPx are unable to do so. HXA(3) exhibits a myriad of biological effects, most of which are associated with the stimulation of intracellular calcium or the transport of calcium across the membrane. The activation of HXA(3)-G-protein-coupled receptors explains many of the extracellular effects of HXA(3), including AA- and diacylglycerol (DAG) release in human neutrophils, insulin secretion in rat pancreatic beta-cells or islets, and synaptic actions in the brain. The availability of stable analogs of HXA(3), termed 10-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid derivatives (PBTs), recently made several animal studies possible and explored the role of HXA(3) as a therapeutic in treatment of diseases. Thus, PBT-3 induced apoptosis in K562 tumour cells and inhibited growth of K562 CML solid tumours in nude mice. HXA(3) inhibited bleomycin-evoked lung fibrosis and inflammation in mice and the raised insulin level in the circulation of rats. At low glucose concentrations (0-3 mm), HXA(3) also stimulated insulin secretion in RINm5F cells through the activation of IRE1alpha, an endoplasmic reticulum-resident kinase. The latter regulates the protein folding for insulin biosynthesis. In conclusion, HXA(3)-mediated signaling may be involved in normal physiological functions, and hepoxilin-based drugs may serve as therapeutics in diseases such as type II diabetes and idiopathic lung fibrosis.     

Regional biosynthesis of prostaglandins and hydroxyeicosatetraenoic acids from arachidonic acid in the rat stomach tissue

The present study was conducted to determine regional differences in the biosynthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs) in the rat stomach tissue (fundus, corpus and pyloric antrum) from radioactive arachidonic acid (AA). The radioactive metabolites were validated by RP-HPLC using non-radioactive AA as substrate. PGE(2) was the major prostanoid in the tissue(.) The relative ratio of PGE(2):PGF(2)alpha:PGD(2) in the whole stomach was 1:0.5:0.1. Regionally, the fundus biosynthesized the largest amount of all three cyclo-oxygenase products. Among the lipoxygenase metabolites, 15S-HETE was the predominant product, while 12S-HETE was found to be the lowest. The relative ratio of 15S-HETE:5S-HETE:12S-HETE in the whole stomach was 1:0.6:0.4. Interestingly, the generation of lipoxygenase products was the highest in the pyloric antrum when compared to fundus or corpus. Thus, the regional differences in the biosyntheses of gastric PGs and monohydroxy fatty acids may be relevant to our understanding of corresponding differences in mucosal resistance or susceptibility to gastric disease.     

ent-Labdane glycosides from the aquatic plant Potamogeton lucens and analytical evaluation of the lipophilic extract constituents of various Potamogeton species

Two new ent-labdane glycosides, one known furano-ent-labdane and a new hydroxylated fatty acid were isolated from the dichloromethane extract of the freshwater aquatic plant Potamogeton lucens. The new compounds were assigned the structures of beta-d-glucopyranosyl-8(17),13-ent-labdadien-16,15-olid-18-oate, 18-beta-d-glucopyranosyloxy-8(17),13-ent-labdadien-16,15-olide and 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trien-oic acid by spectroscopic means. The algicidal activity of these compounds was tested against Raphidocelis subcapitata. Based on our previous study of Potamogeton pectinatus, other constituents were identified in P. lucens by LC-UV-MS, LC-NMR and GC-MS. The lipophilic extract profiles of both species are presented. Two other species, Potamogeton perfoliatus and P. crispus, were also investigated by analytical comparison of their non-polar extracts. The distribution of ent-labdanes characterized in Potamogeton is summarized.     

Comparative activity of natural mono-, di- and tri-hydroxy derivatives of arachidonic acid on guinea-pig lungs: myotropic effect and stimulation of cyclooxygenase activity

The activity of natural 5,6-Dihydroxy-eicosatetraenoic acid (5,6-DiHETE; 2 isomers), 5S,15S-DiHETE, 8S,15S-DiHETE, 5S,12S-DiHETE, delta 6-trans-leukotriene B4, 12-epi-delta 6-trans-leukotriene B4, omega-hydroxy-leukotriene B4, omega-carboxy-leukotriene B4, 15S-hydroxyeicosatetraenoic acid (15S-HETE), 12S-HETE, 5S-HETE and 12S-hydroxy-heptadecatrienoic acid was compared to LTB4 on the guinea-pig lung parenchymal strip and on the release of prostaglandins and thromboxanes by the perfused guinea-pig lungs. The omega-hydroxy-LTB4 appeared more potent than LTB4 both for inducing a contraction and for releasing prostanoids whereas the omega-carboxy-LTB4 was much less active on the parenchyma and did not release prostanoids at the dose used. All other hydroxy acids tested were either very weakly active or inactive in the two systems used with the exception of the 5,6-DiHETEs which showed significant activity. These di-hydroxy acids induced contractions of the lung parenchymal strip which could be blocked by FPL-55712 but were inactive on the guinea-pig ileum. The 5S-HETE, 12S-HETE and 15S-HETE were also tested for possible myotropic activity on selected smooth muscle preparations. Our results provide further informations on the structural requirements for LTB4 (and other hydroxy acids) actions on the guinea-pig lungs.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Interactions of 12-lipoxygenase with phospholipase A2 isoforms following platelet activation through the glycoprotein VI collagen receptor

Recent studies implicate the collagen receptor, glycoprotein VI (GPVI) in activation of platelet 12-lipoxygenase (p12-LOX). Herein, we show that GPVI-stimulated 12-hydro(peroxy)eicosatetraenoic acid (H(P)ETE) synthesis is inhibited by palmityl trifluromethyl ketone or oleyloxyethylphosphocholine , but not bromoenol lactone, implicating secretory and cytosolic, but not calcium-independent phospholipase A2 (PLA2) isoforms. Also, following GPVI activation, 12-LOX co-immunoprecipitates with both cytosolic and secretory PLA2 (sPLA2). Finally, venoms containing sPLA2 acutely activate p12-LOX in a dose-dependent manner. This study shows that platelet 12-H(P)ETE generation utilizes arachidonate substrate from both c- and sPLA2 and that 12-LOX functionally associates with both PLA2 isoforms.     

Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia

Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.     

Diterpenoids from Calceolaria inamoena

Four 9-epi-ent-labdanes were isolated from the aerial parts of Calceolaria inamoena. Their structures, 2beta-hydroxy-9-epi-ent-labda8(17)-13(E)dien-15-oic acid, 2beta-hydroxy-9-epi-ent-labda8(17)-13(Z)dien-15-oic acid, 2beta-hydroxy-9-epi-ent-labda8(17)-13(E)dien-15-al and 2beta-hydroxy-9-epi-ent-labda-8(17)-13(Z)dien-15-al, were established by spectroscopic methods including by analysis of 2 dimensional heteronuclear correlation experiments 1H/13C (normal and long range), and NOE and gs-sel-1D-NOESY and TOCSY of their methyl ester or acetyl derivatives.     

Thiol antioxidant and thiol-reducing agents attenuate 15-deoxy-delta 12,14-prostaglandin J2-induced heme oxygenase-1 expression

Heme oxygenase-1 (HO-1) is induced as a beneficial and adaptive response in cells and tissues exposed to oxidative stress. Herein we examined how various eicosanoids affect the induction of HO-1, and the possible mechanism underlying 15-deoxy-Delta(12,14)- prostaglandin J(2) (15d-PGJ(2))-induced HO-1 expression. PGH(2), PGD(2) and its metabolites of the PGJ(2) series, and PGA(1) markedly induced the protein expression of HO-1. Arachidonic acid (AA), docosahexaenoic acid (DHA), PGE(2), PGF(2 alpha), and thromboxane B(2) (TXB(2)) were shown to have no effect on the induction of HO-1. 15d-PGJ(2) was the most potent activator achieving significance at 5 microM. Although 15d-PGJ(2) significantly activated the MAPKs of JNK and ERK, the activation of JNK and ERK did not contribute to the induction of HO-1 as determined using transfection of dominant-negative plasmids and MAPKs inhibitors. Additional experiment indicated that 15d-PGJ(2) induced HO-1 expression through peroxisome proliferator-activated receptor (PPAR)-independent pathway. 15d-PGJ(2) significantly decreased the intracellular level of reduced glutathione; and the thiol antioxidant, N-acetyl-L-cysteine (NAC), and the thiol-reducing agent, dithiothreitol (DTT), inhibited the induction of HO-1 by 15d-PGJ(2). Finally, NAC and DTT exhibited significant inhibition of HO-1 mRNA and HO-1 promoter reporter activity induced by 15d-PGJ(2). These results suggest that thiol antioxidant and reducing agents attenuate the expression of HO-1 induced by 15d-PGJ(2), and that the cellular thiol-disulfide redox status may be linked to HO-1 activation.     

Increased entropy production in diaphragm muscle of PPAR alpha knockout mice

Peroxisome proliferator activated receptor alpha (PPAR alpha) regulates fatty acid beta-oxidation (FAO) and plays a central role in the metabolic and energetic homeostasis of striated muscles. The thermodynamic consequences of the absence of PPAR alpha were investigated in diaphragm muscle of PPAR alpha knockout mice (KO). Statistical mechanics provides a powerful tool for determining entropy production, which quantifies irreversible chemical processes generated by myosin molecular motors and which is the product of thermodynamic force A/T (chemical affinity A and temperature T) and thermodynamic flow (myosin crossbridge (CB) cycle velocity upsilon). The behavior of both wild type (WT) and KO diaphragm was shown to be near-equilibrium and in a stationary state, but KO was farther from equilibrium than WT. In KO diaphragm, a substantial decrease in contractile function was associated with an increase in both A/T and upsilon and with profound histological injuries such as contraction band necrosis. There were no changes in PPAR delta and gamma expression levels or myosin heavy chain (MHC) patterns. In KO diaphragm, a marked increase in entropy production (A/T x upsilon) accounted for major thermodynamic dysfunction and a dramatic increase in irreversible chemical processes during the myosin CB cycle.     

Maresin Biosynthesis and Identification of Maresin 2, a New Anti-Inflammatory and Pro-Resolving Mediator from Human Macrophages

Maresins are a new family of anti-inflammatory and pro-resolving lipid mediators biosynthesized from docosahexaenoic acid (DHA) by macrophages. Here we identified a novel pro-resolving product, 13R,14S-dihydroxy-docosahexaenoic acid (13R,14S-diHDHA), produced by human macrophages. PCR mapping of 12-lipoxygenase (12-LOX) mRNA sequence in human macrophages and platelet showed that they are identical. This human 12-LOX mRNA and enzyme are expressed in monocyte-derived cell lineage, and enzyme expression levels increase with maturation to macrophages or dendritic cells. Recombinant human 12-LOX gave essentially equivalent catalytic efficiency (k_cat/K_M) with arachidonic acid (AA) and DHA as substrates. Lipid mediator metabololipidomics demonstrated that human macrophages produce a novel bioactive product 13,14-dihydroxy-docosahexaenoic acid in addition to maresin-1, 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (MaR1). Co-incubations with human recombinant 12-LOX and soluble epoxide hydrolase (sEH) demonstrated that biosynthesis of 13,14-dihydroxy-docosahexaenoic acid (13,14-diHDHA) involves the 13S,14S-epoxy-maresin intermediate produced from DHA by 12-LOX, followed by conversion via soluble epoxide hydrolase (sEH). This new 13,14-diHDHA displayed potent anti-inflammatory and pro-resolving actions, and at 1 ng reduced neutrophil infiltration in mouse peritonitis by ∼40% and at 10 pM enhanced human macrophage phagocytosis of zymosan by ∼90%. However, MaR1 proved more potent than the 13R,14S-diHDHA at enhancing efferocytosis with human macrophages. Taken together, the present findings demonstrate that macrophages produced a novel bioactive product identified in the maresin metabolome as 13R,14S-dihydroxy-docosahexaenoic acid, from DHA via conversion by human 12-LOX followed by sEH. Given its potent bioactions, we coined 13R,14S-diHDHA maresin 2 (MaR2).     

Dihydroxydocosahexaenoic acids of the neuroprotectin D family: synthesis, structure, and inhibition of human 5-lipoxygenase

During aerobic oxidation of docosahexaenoic acid (DHA), soybean lipoxygenase (sLOX) has been shown to form 7,17(S)-dihydro(pero)xydocosahexaenoic acid [7,17(S)-diH(P)DHA] along with its previously described positional isomer, 10,17(S)-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid. 7,17(S)-diH(P)DHA was also obtained via sLOX-catalyzed oxidation of either 17(S)-hydroperoxydocosahexaenoic acid [17(S)-HPDHA] or 17(S)-hydroxydocosahexaenoic acid [17(S)-HDHA]. The structures of the products were elucidated by normal-phase, reverse-phase, and chiral-phase HPLC analyses and by ultraviolet, NMR, and tandem mass spectroscopy and GC-MS. 7,17(S)-diH(P)DHA was shown to have 4Z,8E,10Z,13Z,15E,19Z geometry of the double bonds. In addition, a compound apparently identical to the sLOX-derived 7,17(S)-diH(P)DHA was produced by another enzyme, potato tuber LOX, in the reactions of oxygenation of either 17(S)-HPDHA or 17(S)-HDHA. All of the dihydroxydocosahexaenoic acids (diHDHAs) formed by either of the enzymes were clearly produced through double lipoxygenation of the corresponding substrate. 7,17(S)-diHDHA inhibited human recombinant 5-lipoxygenase in the reaction of arachidonic acid (AA) oxidation. In standard conditions with 100 microM AA as substrate, the IC(50) value for 7,17(S)-diHDHA was found to be 7 microM, whereas IC(50) for 10,17(S)-DiHDHA was 15 microM. Similar inhibition by the diHDHAs was observed with sLOX, a quintessential 15LOX, although the strongest inhibition was produced by 10,17(S)-diHDHA (IC(50) = 4 microM). Inhibition of sLOX by 7,17(S)-diHDHA was slightly less potent, with an IC(50) value of 9 microM. These findings suggest that 7,17(S)-diHDHA along with its 10,17(S) counterpart might have anti-inflammatory and anticancer activities, which could be exerted, at least in part, through direct inhibition of 5LOX and 15LOX.     

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.     

Pharmacological characterization of the biosynthesis of prostanoids and hydroxyeicosatetraenoic acids in human whole blood and platelets by targeted chiral lipidomics analysis

Platelet 12-lipoxygenase(p-12-LOX) is highly expressed in human platelets, and the development of p-12-LOX inhibitors has the potential to be a novel antithrombotic tool by inhibiting thrombosis without prolonging hemostasis. A chiral liquid chromatography-mass spectrometry(LC-MS/MS) method was used to assess the impact of three commercially available LOX inhibitors[esculetin(6,7-dihydroxycoumarin), ML-355(N-2-benzothiazolyl-4-[[(2-hydroxy-3-methoxyphenyl)methyl]amino]-benzenesulfonamide), CDC(cinnamyl-3,4-dihydroxy-α-cyanocinnamate) and acetylsalicylic acid(ASA; a cyclooxygenase-1 inhibitor) on the generation of prostanoids and HETEs(hydroxyeicosatetraenoic acids) in human whole blood allowed to clot for 1 h at 37 °C(serum), platelet-rich plasma(PRP) stimulated with collagen or TRAP-6(a peptide activating thrombin receptor) and washed platelets. In serum, ML-355 did not affect eicosanoid generation, while CDC caused an incomplete reduction of 12S-HETE levels; esculetin inhibited both 12S-HETE and thromboxane(TX)B₂ production; ASA selectively affected TXB₂ production. In washed platelets stimulated with thrombin, esculetin, and CDC inhibited both 12S-HETE and TXB₂ while ML-355 was almost ineffective. In PRP, ML-355, CDC, and esculetin did not affect platelet aggregation associated with incomplete effects on eicosanoid biosynthesis. ASA alone or in combination with ticagrelor(a P2Y₁₂ blocker) affected platelet aggregation associated with profound inhibition of TXB₂ generation. P2Y₁₂ receptor signaling contributed to platelet 12S-HETE biosynthesis in response to primary agonists. In conclusion, ML-355, esculetin, and CDC were not selective inhibitors of p-12-LOX in different cellular systems. They did not affect platelet aggregation induced in PRP by collagen or TRAP-6. The characterization of 12-LOX inhibitors on eicosanoids generated in human whole blood is useful for information on their enzyme selectivity, off-target effects, and the possible influence of plasma components on their pharmacological effects.