Expression Of 5-lipoxygenase Research Articles

Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (FoxLOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-(2)H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the (2)H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser(348) in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser(348), binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.

Enhanced Neuroprotection of Acetyl-11-Keto-β-Boswellic Acid (AKBA)-Loaded O-Carboxymethyl Chitosan Nanoparticles Through Antioxidant and Anti-Inflammatory Pathways.

Acetyl-11-keto-β-boswellic acid (AKBA), a main active constituent from Boswellia serrata resin, is a novel candidate for therapy of cerebral ischemia-reperfusion (I/R) injury. Nevertheless, its poor solubility in aqueous solvent, bioavailability, and rapid clearance limit its curative efficacy. To enhance its potency, in our study, AKBA-loaded o-carboxymethyl chitosan nanoparticle (AKBA-NP) delivery system was synthesized. The transmission electron microscopy and transmission electron microscope images of AKBA-NPs suggested that particle size was 132 ± 18nm, and particles were spherical in shape with smooth morphology. In pharmacokinetics study, AKBA-NPs apparently increases the area under the curve of plasma concentration-time and prolonged half-life compared with AKBA. The tissue distribution study confirmed that AKBA-NPs had a better brain delivery efficacy in comparison with AKBA. The results from our pharmacodynamic studies showed that AKBA-NPs possess better neuroprotection compared with AKBA in primary neurons with oxygen-glucose deprivation (OGD) model and in animals with middle cerebral artery occlusion (MCAO) model. Additionally, AKBA-NPs modulate antioxidant and anti-inflammatory pathways more effectively than AKBA by increasing nuclear erythroid 2-related factor 2 and heme oxygenase-1 expression, and by decreasing nuclear factor-kappa B and 5-lipoxygenase expression. Collectively, our results suggest that AKBA-NPs serve as a potent delivery vehicle for AKBA in cerebral ischemic therapy.

Leukotriene B4—leukotriene B4 receptor axis promotes oxazolone‐induced contact dermatitis by directing skin homing of neutrophils and CD8+ T cells

Leukotriene B4 (LTB4 ) is a lipid mediator that is rapidly generated in inflammatory sites, and its functional receptor, BLT1, is mostly expressed on immune cells. Contact dermatitis is a common inflammatory skin disease characterized by skin oedema and abundant inflammatory infiltrates, primarily including neutrophils and CD8(+) T cells. The role of the LTB4 -BLT1 axis in contact dermatitis remains largely unknown. In this study, we found up-regulated gene expression of 5-lipoxygenase and leukotriene A4 hydrolase, two critical enzymes for LTB4 synthesis, BLT1 and elevated LTB4 levels in skin lesions of oxazolone (OXA)-induced contact dermatitis. BLT1 deficiency or blockade of LTB4 and BLT1 by the antagonists, bestatin and U-75302, respectively, in the elicitation phase caused significant decreases in ear swelling and skin-infiltrating neutrophils and CD8(+) T cells, which was accompanied by significantly reduced skin expression of CXCL1, CXCL2, interferon-γ and interleukin-1β. Furthermore, neutrophil depletion during the elicitation phase of OXA-induced contact dermatitis also caused significant decreases in ear swelling and CD8(+) T-cell infiltration accompanied by significantly decreased LTB4 synthesis and gene expression of CXCL2, interferon-γ and interleukin-1β. Importantly, subcutaneous injection of exogenous LTB4 restored the skin infiltration of CD8(+) T cells in neutrophil-depleted mice following OXA challenge. Collectively, our results demonstrate that the LTB4 -BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils, which are a major source of LTB4 that sequentially direct CD8(+) T-cell homing to OXA-challenged skin. Hence, LTB4 and BLT1 could be potential therapeutic targets for the treatment of contact dermatitis.

Burkholderia phytofirmans PsJN induces long-term metabolic and transcriptional changes involved in Arabidopsis thaliana salt tolerance.

Salinity is one of the major limitations for food production worldwide. Improvement of plant salt-stress tolerance using plant-growth promoting rhizobacteria (PGPR) has arisen as a promising strategy to help overcome this limitation. However, the molecular and biochemical mechanisms controlling PGPR/plant interactions under salt-stress remain unclear. The main objective of this study was to obtain new insights into the mechanisms underlying salt-stress tolerance enhancement in the salt-sensitive Arabidopsis thaliana Col-0 plants, when inoculated with the well-known PGPR strain Burkholderia phytofirmans PsJN. To tackle this, different life history traits, together with the spatiotemporal accumulation patterns for key metabolites and salt-stress related transcripts, were analyzed in inoculated plants under short and long-term salt-stress. Inoculated plants displayed faster recovery and increased tolerance after sustained salt-stress. PsJN treatment accelerated the accumulation of proline and transcription of genes related to abscisic acid signaling (Relative to Dessication, RD29A and RD29B), ROS scavenging (Ascorbate Peroxidase 2), and detoxification (Glyoxalase I 7), and down-regulated the expression of Lipoxygenase 2 (related to jasmonic acid biosynthesis). Among the general transcriptional effects of this bacterium, the expression pattern of important ion-homeostasis related genes was altered after short and long-term stress (Arabidopsis K+ Transporter 1, High-Affinity K+ Transporter 1, Sodium Hydrogen Exchanger 2, and Arabidopsis Salt Overly Sensitive 1). In all, the faster and stronger molecular changes induced by the inoculation suggest a PsJN-priming effect, which may explain the observed tolerance after short-term and sustained salt-stress in plants. This study provides novel information about possible mechanisms involved in salt-stress tolerance induced by PGPR in plants, showing that certain changes are maintained over time. This opens up new venues to study these relevant biological associations, as well as new approaches to a better understanding of the spatiotemporal mechanisms involved in stress tolerance in plants.

Abstract 210: Biosynthesis of D-series Resolvin by Isolated Vascular Cells and Tissues

Introduction: Resolvin-D1 (RvD1) and other specialized pro-resolving lipid mediators (SPM) are synthesized in-vivo from docosahexaenoic acid (DHA) through transcellular pathways involving leukocytes. We investigated if vascular tissues, in the absence of inflammatory cells, can contribute to the local production of SPM. Methods: Primary cultures of human saphenous vein endothelial (EC) and smooth muscle (SMC) cells were supplemented with DHA in cell culture media (10% serum) for 4h-24h. Freshly harvested rabbit aorta was incubated intact or following gentle EC denudation in medium with or without DHA for 48h. RvD1 levels were quantified by ELISA, and lipoxygenase (LO) expression by western blotting. Results: In the absence of DHA supplementation, EC and SMC produced undetectable levels of RvD1. DHA treatment produced a dose and time-dependent increase in RvD1 production by EC and SMC (10.1 ±1.0 pg, 7.4 ±0.2 pg respectively; 1000nM DHA; 24h; Fig A, B). 5-LO expression was demonstrated in both cell types, however DHA induced increased 5-LO expression in EC (Fig C) but not in SMC. DHA-treated intact rabbit aorta segments produced 0.24±0.05 pg RvD1/mg tissue versus 0.13±0.01 pg RvD1/mg tissue in media alone. Moreover, EC-denuded aortas produced significantly less RvD1 (Fig D). Conclusions: Human vascular cells and rabbit vascular tissue can biosynthesize RvD1 de novo from its precursor DHA, signifying a potentially important local source of SPM in the vasculature.

Expression of 5-lipoxygenase in human tissues and its association with disease

5-Lipoxygenase, one of lipoxygenase isozymes, is a well-studied oxidative metabolism enzyme. It widely exists in various human tissues and cells, participates in the oxidative metabolism of endogenous and exogenous chemicals, and produces a variety of metabolites, all of which contribute to the occurrence of human diseases, such as inflammation, asthma, atherosclerosis, and tumor and so on. The expression of 5-lipoxygenase is at low level in normal human tissues while at high level in abnormal tissues. 5-Lipoxygenase is closely related to many kinds of diseases in human ovary, brain, cardiovascular system, lung, liver, pancreas and other tissues. The abnormal expression of 5-lipoxygenase tends to promote the development of the disease.

Effects of intermittent warming on aroma-related esters of 1-methylcyclopropene-treated ‘Nanguo’ pears during ripening at room temperature

Refrigeration combined with 1-methylcyclopropene (1-MCP) treatment is an effective method for the long-term storage of climacteric fruit, but this benefit is offset by a loss of aroma when the fruit is then ripened at room temperature. The pear cv. ‘Nanguo’ (Pyrus ussuriensis Maxim.) was treated with 1-MCP and continuously stored at 0±0.5°C or exposed to the same storage temperature alternated with 20±1°C for 1 d every 20 d during storage (namely, intermittent warming [IW]). Changes in aroma-related esters, levels of fatty acids, and the activity and gene expression of alcohol acyltransferase (AAT) and lipoxygenase (LOX) were investigated. Changes in the gene expression of the enzymes (1-aminocyclopropane-carboxylase (ACC) synthase (ACS) and ACC oxidase (ACO)) involved in ethylene biosynthesis and the expression of genes involved in ethylene signal transduction were also studied. Significantly higher ester content was found in IW-treated fruit. Increased activity and gene expression of AAT, LOX, and accumulated polyunsaturated fatty acids were associated with enhanced aroma quality in IW-treated fruit. The increased translation levels of PuACS1 and PuACO1 contributed to the elevated ethylene levels in IW-treated fruit. IW treatment seemed to have no significant effect on the induction of the expression of PuETR1 and merely stimulated the expression of PuCTR1 during the prior period of the shelf life at 20°C. Treatment with IW effectively prevented the loss of aroma-related esters from 1-MCP-treated ‘Nanguo’ pears, and possible mechanisms for this were discussed.

Selective inhibition of 12-lipoxygenase protects islets and beta cells from inflammatory cytokine-mediated beta cell dysfunction.

Islet inflammation leads to loss of functional pancreatic beta cell mass. Increasing evidence suggests that activation of 12-lipoxygenase leads to inflammatory beta cell loss. This study evaluates new specific small-molecule inhibitors of 12-lipoxygenase for protecting rodent and human beta cells from inflammatory damage. Mouse beta cell lines and mouse and human islets were treated with inflammatory cytokines IL-1β, TNFα and IFNγ in the absence or presence of novel selective 12-lipoxygenase inhibitors. Glucose-stimulated insulin secretion (GSIS), gene expression, cell survival and 12-S-hydroxyeicosatetraenoic acid (12-S-HETE) levels were evaluated using established methods. Pharmacokinetic analysis was performed with the lead inhibitor in CD1 mice. Inflammatory cytokines led to the loss of human beta cell function, elevated cell death, increased inflammatory gene expression and upregulation of 12-lipoxygenase expression and activity (measured by 12-S-HETE generation). Two 12-lipoxygenase inhibitors, Compounds 5 and 9, produced a concentration-dependent reduction of stimulated 12-S-HETE levels. GSIS was preserved in the presence of the 12-lipoxygenase inhibitors. 12-Lipoxygenase inhibition preserved survival of primary mouse and human islets. When administered orally, Compound 5 reduced plasma 12-S-HETE in CD1 mice. Compounds 5 and 9 preserved the function and survival of human donor islets exposed to inflammatory cytokines. Selective inhibition of 12-lipoxygenase activity confers protection to beta cells during exposure to inflammatory cytokines. These concept validation studies identify 12-lipoxygenase as a promising target in the prevention of loss of functional beta cells in diabetes.

Cotton cytochrome P450 CYP82D regulates systemic cell death by modulating the octadecanoid pathway.

Plant oxylipins are derived from unsaturated fatty acids and play roles in plant growth and development as well as defence. Although recent studies have revealed that fatty acid metabolism is involved in systemic acquired resistance, the precise function of oxylipins in plant defence remains unknown. Here we report a cotton P450 gene SILENCE-INDUCED STEM NECROSIS (SSN), RNAi suppression of which causes a lesion mimic phenotype. SSN is also involved in jasmonate metabolism and the response to wounding. Fatty acid and oxylipin metabolite analysis showed that SSN overexpression causes hyperaccumulation of hydroxide and ketodiene fatty acids and reduced levels of 18:2 fatty acids, whereas silencing causes an imbalance in LOX (lipoxygenase) expression and excessive hydroperoxide fatty acid accumulation. We also show that an unknown oxylipin-derived factor is a putative mobile signal required for systemic cell death and hypothesize that SSN acts as a valve to regulate HR on pathogen infection.

Scorpion peptide LqhIT2 activates phenylpropanoid pathways via jasmonate to increase rice resistance to rice leafrollers

LqhIT2 is an insect-specific toxin peptide identified in Leiurus quinquestriatus hebraeus that can be toxic to lepidoptera pests. However, whether LqhIT2 induces insect resistance in rice, and how the LqhIT2 influences the biochemical metabolism of rice plants remains unknown. Here, purified LqhIT2-GST fusion protein had toxicity to rice leafrollers. Meanwhile, in vitro and field trials showed that LqhIT2 transgenic rice plants were less damaged by rice leafrollers compared to the wild type plants. Introducing LqhIT2 primed the elevated expression of lipoxygenase, a key component of the jasmonic acid biosynthetic pathway, together with enhanced linolenic acid, cis-(+)-12-oxophytodienoic acid, jasmonic acid, and jasmonic acid-isoleucine levels. In addition, phenylalanine ammonia-lyase, a key gene of the phenylpropanoid pathway, was up-regulated. Correspondingly, the contents of downstream products of the phenylpropanoid pathway such as flavonoids and lignins, were also increased in LqhIT2 transgenic plants. These changes were paralleled by decreased starch, glucose, and glucose-6-phosphate accumulation, the key metabolites of glycolysis pathway that supplies the raw material and intermediate carbon products for phenylpropanoids biosyntheses. These findings suggest that, in addition to its own toxicity against pests, LqhIT2 activate the phenylpropanoid pathway via jasmonate-mediated priming, which subsequently increases flavonoid and lignin content and improves insect resistance in rice.

Low temperature conditioning prevents loss of aroma-related esters from ‘Nanguo’ pears during ripening at room temperature

Refrigeration delays ripening and reduces decay of pear fruit, but this benefit is offset by a loss of aroma when the fruit is then ripened at room temperature. Low temperature conditioning (LTC) has been used to alleviate chilling injury (CI) of fruit, however, its effect on aroma remains unknown. Pear fruit cv. ‘Nanguo’ (Pyrus ussuriensis Maxim.) were subjected to LTC (namely, exposed to 10°C, decreased by 1°C every 3 days to 5°C, then decreased by 1°C per day to 0±0.5°C) and changes in aroma-related esters, levels of fatty acids, the activity and gene expression of alcohol acyltransferase (AAT), lipoxygenase (LOX), and alcohol dehydrogenase (ADH) were investigated. Changes in the activity and gene expression of enzymes involved in ethylene biosynthesis and the expression of genes involved in ethylene signal transduction were also studied. Significantly higher ester contents and levels of linoleic and linolenic acids were found in LTC-treated fruit. Levels of PuAAT1, PuLOX1, and PuADH3 transcripts were promoted by LTC treatment and the activity of AAT, LOX and ADH was higher in LTC-treated fruit during the shelf-life at room temperature. The increased levels of the expression and activity of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO) contributed to the elevated ethylene levels in LTC-treated fruit. The expression of genes involved in ethylene signal transduction (PuETR1 and PuCTR1) was also promoted by LTC treatment. Treatment with LTC effectively prevented the loss of aroma-related esters from refrigerated ‘Nanguo’ pears and possible mechanisms were discussed.

G.P.100 : Pilot study of flavocoxid in ambulant DMD patients

Muscle degeneration in Duchenne muscular dystrophy (DMD) is exacerbated by the endogenous inflammatory response and increased oxidative stress. A key role is played by nuclear factor (NF)-κB. We previously showed that flavocoxid, a flavonoid with antioxidant and anti-inflammatory properties, ameliorates muscle pathology and function in mdx mice. This effect seemed to be mediated by the inhibition of NF-κB, tumor necrosis factor-α, cyclooxygenase-2/5-lipoxygenase and MAPKs expression in muscle. Moreover, flavocoxid has been shown to decrement serum levels of IL-1β and TNF-α in in vivo studies. Primary end-point of this pilot study was to evaluate safety and tolerability of flavocoxid administered daily per os for one year in ambulant DMD patients. We also evaluated function, muscle strength and quality of life. The effects of flavocoxid on selected biomarkers was also assessed. We enrolled 20 patients. We did not report any treatment-related adverse event and clinically meaningful change in laboratory findings. Serum expression analysis of inflammatory cytokines showed a significant reduction of TNF-α and IL-1β and oxidative stress markers ( p p

Characterization of Fusarium wilt resistant somaclonal variants of banana cv. Rasthali by cDNA-RAPD.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is counted among the most destructive diseases of crop plants in India. In the absence of any credible control measure to manage this disease, development of resistant cultivars is the best option. Somaclonal variations arising out of long term in vitro culture of plant tissues is an important source of genetic variability and the selection of somaclones having desired characteristics is a promising strategy to develop plants with improved characters. In the present study, we isolated a group of somaclonal variants of banana cv. Rasthali which showed efficient resistance towards Foc race 1 infection in repeated bioassays. cDNA-RAPD methodology using 96 decamer primers was used to characterize these somaclonal variants. Among the four differentially amplified bands obtained, one mapping to the coding region of a lipoxygenase gene was confirmed to be down regulated in the somaclones as compared to controls by real-time quantitative RT-PCR. Our results correlated well with earlier studies with lipoxygenase mutants in maize wherein reduced expression of lipoxygenase led to enhanced resistance towards Fusarium infection.

Markers of inflammation and oxidative stress studied in adjuvant-induced arthritis in the rat on systemic and local level affected by pinosylvin and methotrexate and their combination

Oxidative stress (OS) is important in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) and its experimental model – adjuvant arthritis (AA). Antioxidants are scarcely studied in autoimmunity, and future analyses are needed to assess its effects in ameliorating these diseases. Although there are studies about antioxidants effects on the course of RA, their role in combination therapy has not yet been studied in detail, especially on extra-articular manifestations of AA. During the 28-d administration of pinosylvin (PIN) in monotherapy and in combination with methotrexate (MTX) to AA rats, we evaluated the impact of the treatment on selected parameters. The experiment included: healthy controls, untreated AA, AA administered 50 mg/kg b.w. of PIN daily p.o., AA administered 0.4 mg/kg b.w. of MTX twice weekly p.o. and AA treated with a combination of PIN+MTX. AA was monitored using: hind paw volume, C-reactive protein, monocyte chemotactic protein-1 (MCP-1), thiobarbituric acid reactive substances (TBARS) and F2-isoprostanes in plasma, γ-glutamyltransferase activity in spleen, activity of lipoxygenase (LOX) in lung, heme oxygenase-1 (HO-1) and nuclear factor kappa B (NF-κB) in liver and lung. PIN monotherapy significantly improved the activation of NF-κB in liver and lung, HO-1 expression and activity of LOX in the lung, MCP-1 levels in plasma (on 14th d) and plasmatic levels of F2-isoprostanes. An important contribution of PIN to MTX effect was the reduction of OS (an increase of HO-1 expression in lung and reduction of plasmatic TBARS) and decrease of LOX activity in the lung.

Role of cysteinyl leukotriene signaling in a mouse model of noise-induced cochlear injury.

Noise-induced hearing loss is one of the most common types of sensorineural hearing loss. In this study, we examined the expression and localization of leukotriene receptors and their respective changes in the cochlea after hazardous noise exposure. We found that the expression of cysteinyl leukotriene type 1 receptor (CysLTR1) was increased until 3 d after noise exposure and enhanced CysLTR1 expression was mainly observed in the spiral ligament and the organ of Corti. Expression of 5-lipoxygenase was increased similar to that of CysLTR1, and there was an accompanying elevation of CysLT concentration. Posttreatment with leukotriene receptor antagonist (LTRA), montelukast, for 4 consecutive days after noise exposure significantly decreased the permanent threshold shift and also reduced the hair cell death in the cochlea. Using RNA-sequencing, we found that the expression of matrix metalloproteinase-3 (MMP-3) was up-regulated after noise exposure, and it was significantly inhibited by montelukast. Posttreatment with a MMP-3 inhibitor also protected the hair cells and reduced the permanent threshold shift. These findings suggest that acoustic injury up-regulated CysLT signaling in the cochlea and cochlear injury could be attenuated by LTRA through regulation of MMP-3 expression. This study provides mechanistic insights into the role of CysLTs signaling in noise-induced hearing loss and the therapeutic benefit of LTRA.

오디 추출물(Morus alba L.)의 혈행개선 효과

The objective of this study was to investigate the beneficial effects of mulberry extract (MBE) on blood flow improvement. The SC50 value for the DPPH radical scavenging activity of MBE was 89.36±5.46 μg/mL. Analysis of the cellular toxicity of MBE on RAW 264.7 and HepG2 cells showed no toxicity under a concentration of 2,500 μg/mL. We found that MBE inhibited the enzyme activity of cyclooxygenase (COX)-2 as well as oxidation of human LDL. Western blotting analysis showed that MBE inhibited protein expression of COX-2 and 5-lipoxygenase in RAW 264.7 cells. In addition, MBE inhibited protein expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in human umbilical vein endothelial cells. Furthermore, MBE reduced the serum levels of total cholesterol and C-reactive protein in a concentration-dependent manner. These results both in vitro and in vivo suggest that MBE can be employed for the improvement of blood flow.

Human cytomegalovirus infection induces leukotriene B4 and 5-lipoxygenase expression in human placentae and umbilical vein endothelial cells

IntroductionHuman cytomegalovirus (HCMV) can cause congenital infection with risk of neurological disability. Maternal-fetal transmission is associated with placental inflammation. 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of Leukotrienes (LTs), which are proinflammatory mediators. This study investigated the effect of HCMV infection on 5-LO expression and Leukotriene-B4 (LTB4) induction in human placentae and umbilical vein endothelial cells (HUVEC). MethodsSeven placentae from fetuses with congenital HCMV infection and brain damage and six controls were stained with HCMV-immediate-early-antigen (HCMV-IEA) and 5-LO by immunohistochemistry. 5-hydroxyeicosatetraenoic acid (5-HETE) and LTB4 were measured in culture supernatant from ex vivo HCMV-infected placental histocultures by liquid chromatography. In vitro, HCMV infected HUVEC cells were analyzed for 5-LO mRNA and protein expression by real time PCR and immunofluorescence staining. ResultsHCMV-IEA was abundant in all HCMV infected placentae but absent in control placentae. 5-LO expression was higher in endothelial and smooth muscle cells of HCMV-infected placentae, compared to control placentae.HCMV infection induced an up-regulation of LTB4 in ex vivo placental explants with higher levels of LTB4 at 72 h compared to controls (p = 0.002). In vitro, 5-LO transcript and protein expression were significantly induced in HCMV-infected HUVEC, compared to the control cultures (p = 0.036). ConclusionThe presence of HCMV coincided with high 5-LO expression in cells of in vivo HCMV infected placentae. HCMV induced up-regulation of 5-LO in both ex vivo HCMV-infected placental explants and HUVEC. HCMV induced LT-biosynthesis in congenitally infected placentae may have a role in pathogenesis of congenital HCMV disease.

Insulin‐like growth factor I (IGF‐1) suppressed foam cells via downregulation of 12/15‐lipoxygenase: potential mechanism responsible for IGF‐1‐induced atheroprotective effect (832.14)

We have shown that IGF‐1 reduced foam cells and suppressed atherosclerosis, however mechanism mediating these effects was not identified. Macrophage (MΦ) 12/15‐lipoxygenase (LOX) known to mediate oxidation of low density lipoprotein (LDL) and promote formation of foam cells contributing to atherogenesis. We hypothesized that IGF‐1 downregulates LOX and via this mechanism reduces foam cells and plaque size.IGF‐1 infusion into Apoe‐null mice (1.5 mg/kg/d, 12 weeks on a Western diet) reduced LOX plaque immunopositivity (74±9% decrease), suppressed plaque oxidative stress (64±7% decrease, immunohistochemistry with 8‐oxodG a/b), reduced plaque lipids (35%±6 decrease, Oil Red staining) and decreased atherosclerotic lesion area (IGF‐1: 0.259 ± 0.018 mm2, saline: 0.356 ± 0.031 mm2).To further define mechanisms THP‐1‐derived MΦ were exposed to IGF‐1. IGF‐1 (50 ng/ml, 16h) reduced LOX mRNA (49±6% decrease), decreased LOX protein (42±6% decrease, immunoblotting) and also IGF‐1 suppressed STAT6 transcription factor binding to LOX gene promoter region (CHIP assay with STAT6 a/b) suggesting that IGF‐1 downregulates LOX via STAT6‐dependent transcriptional mechanism. IGF‐1 reduced oxidation of LDL mediated by THP‐1 or mouse peritoneal MΦ (85±6% and 67±5% decrease, respectively; MDA assay) and also IGF‐1 decreased cellular LDL uptake (i.e. formation of foam cells) by THP‐1 or WT peritoneal MΦ (45±8% and 51±3% decrease, respectively), however both lipid uptake and IGF‐1 effect were blocked in LOX‐deficient peritoneal MΦ. These data indicate that LOX downregulation mediates IGF‐1‐induced suppression of LDL oxidation and foam cell formation.In summary, IGF‐1 reduced LOX expression in atherosclerotic plaque and in vitro in cultured MΦ. We demonstrated that LOX downregulation mediates IGF‐1‐induced reduction in foam cells representing potential mechanism mediating IGF‐1‐induced atheroprotective effect.Grant Funding Source: Supported by NIH/NHLBI

The expression and activity of 5-LOX in the large intestine of horses harbouring encysted cyathostomin larvae

Leukotrienes are products of the arachidonic acid metabolism and act as potent inflammatory mediators modulating the immune response and various physiological processes. This study evaluated the expression and activity of 5-lipoxygenase (5-LOX), the enzyme that catalyzes the first two steps in the biosynthesis of leukotrienes, in horses infected by larval cyathostomins. Tissue samples from dorsal and ventral colon, and from the cecum were collected from 16 horses slaughtered for human consumption. Samples were analyzed to estimate the burdens of encysted cyathostomin larvae and adult luminal stages, and then processed for the evaluation of biochemical parameters. No significant differences were found in the protein expression and enzymatic activity of 5-LOX between animals harbouring only adult parasites and negative horses. The protein expression and enzyme activity of 5-LOX were significantly higher in horses harbouring encysted larvae in comparison with horses free of encysted larvae. Although preliminary, these results indicate that 5-LOX is an important mediator in the course of horse cyathostominosis and further studies are warranted to unveil the possible role this enzyme plays in the pathogenesis of horse cyathostominosis, and its potential as a diagnostic marker.

Reduced macrophage selenoprotein expression alters oxidized lipid metabolite biosynthesis from arachidonic and linoleic acid

Uncontrolled inflammation is an underlying etiology for multiple diseases and macrophages orchestrate inflammation largely through the production of oxidized fatty acids known as oxylipids. Previous studies showed that selenium (Se) status altered the expression of oxylipids and magnitude of inflammatory responses. Although selenoproteins are thought to mediate many of the biological effects of Se, the direct effect of selenoproteins on the production of oxylipids is unknown. Therefore, the role of decreased selenoprotein activity in modulating the production of biologically active oxylipids from macrophages was investigated. Thioglycollate-elicited peritoneal macrophages were collected from wild-type and myeloid-cell-specific selenoprotein knockout mice to analyze oxylipid production by liquid chromatography/mass spectrometry as well as oxylipid biosynthetic enzyme and inflammatory marker gene expression by quantitative real-time polymerase chain reaction. Decreased selenoprotein activity resulted in the accumulation of reactive oxygen species, enhanced cyclooxygenase and lipoxygenase expression and decreased oxylipids with known anti-inflammatory properties such as arachidonic acid-derived lipoxin A4 (LXA4) and linoleic acid-derived 9-​oxo-octadecadienoic acid (9-oxoODE). Treating RAW 264.7 macrophages with LXA4 or 9-oxoODE diminished oxidant-induced macrophage inflammatory response as indicated by decreased production of TNFα. The results show for the first time that selenoproteins are important for the balanced biosynthesis of pro- and anti-inflammatory oxylipids during inflammation. A better understanding of the Se-dependent control mechanisms governing oxylipid biosynthesis may uncover nutritional intervention strategies to counteract the harmful effects of uncontrolled inflammation due to oxylipids.