Substitution of the mononuclear, non-heme iron cofactor in lipoxygenases for structural studies.

This Chapter describes methods for the biosynthetic substitution of the mononuclear, non-heme iron in plant and animal lipoxygenases (LOXs). Substitution of this iron center for a manganese ion results in an inactive, yet faithful structural surrogate of the LOX enzymes. This metal ion substitution permits structural and dynamical studies of enzyme-substrate complexes in solution and immobilized on lipid membrane surfaces. Representative procedures for two LOXs, soybean lipoxygenase (SLO) from plants and human epithelial 15-lipoxygenase-2 (15-LOX-2) from mammals, are described as examples.

NAD+ deficiency primes defense metabolism via 1O2-escalated jasmonate biosynthesis in plants.

Nicotinamide adenine dinucleotide (NAD+) is a redox cofactor and signal central to cell metabolisms. Disrupting NAD homeostasis in plant alters growth and stress resistance, yet the underlying mechanisms remain largely unknown. Here, by combining genetics with multi-omics, we discover that NAD+ deficiency in qs-2 caused by mutation in NAD+ biosynthesis gene-Quinolinate Synthase retards growth but induces biosynthesis of defense compounds, notably aliphatic glucosinolates that confer insect resistance. The elevated defense in qs-2 is resulted from activated jasmonate biosynthesis, critically hydroperoxidation of α-linolenic acid by the 13-lipoxygenase (namely LOX2), which is escalated via the burst of chloroplastic ROS-singlet oxygen (1O2). The NAD+ deficiency-mediated JA induction and defense priming sequence in plants is recapitulated upon insect infestation, suggesting such defense mechanism operates in plant stress response. Hence, NAD homeostasis is a pivotal metabolic checkpoint that may be manipulated to navigate plant growth and defense metabolism for stress acclimation.

The structural and digestive properties of indica rice starch-fatty acid complexes.

The structural and digestive properties of indica rice starch-fatty acid complexes and the effects of lipoxygenase on the structural and digestive properties of the complexes were examined in this study. The complexes were characterized by scanning electron microscopy, X-ray diffraction, Fourier transform-infrared spectroscopy and Raman spectroscopy. The results showed that indica rice starch had the highest molecular chain order and the highest crystallinity, and the crystallization disappeared after gelatinization, and the formation of indica rice starch-fatty acid complexes promoted the transformation of starch crystal structure from A-type to V-type. Lipoxygenase reduced the regularity of starch molecular crystal structure in the complexes, while enzyme protein improved the order of starch molecular structure in the complexes. The regularity of starch crystal structure in the complexes could improve with the increase of composite temperature and the increase of fatty acid unsaturation. In vitro digestibility and in vitro digestion kinetics showed that the formation of indica rice starch-fatty acid complexes reduced the digestibility of indica rice starch to a certain extent. The RDS content of indica rice starch was 66.42 ± 0.39 %, and lipoxygenase reduced the reduction of rapidly digested starch content during complexes digestion, while enzyme protein increased the content of resistant starch.

Oxidation Kinetics of Fluorescent Membrane Lipid Peroxidation Indicators.

The oxidation of the cellular membrane through lipid peroxidation (LPO) is linked to aging and disease. Despite the physiological importance, the chemical mechanisms underlying LPO and oxidative reactions in membranes in general remain incompletely understood, and challenges exist in translating LPO inhibitor efficacies from in vitro to in vivo. The complexity of LPO, including multiple oxidation reactions in complex membrane environments and the difficulty in quantifying reaction kinetics, underlies these difficulties. In this work, we developed a robust and straightforward method for quantifying the oxidation rate kinetics of fluorescent molecules and determined the oxidation kinetics of widely fluorophores used as indicators of membrane LPO, diphenylhexatriene (DPH), BODIPY-C11, and Liperfluo. The measurement is initiated by lipoxygenase, which provides chemical specificity and enables a straightforward interpretation of oxidation kinetics. Our results reveal that the membrane composition significantly impacts the observed kinetics oxidation in DPH and BODIPY-C11 but not Liperfluo. Reaction mechanisms for their lipid peroxide-induced oxidation are proposed. This work provides a foundation for the quantitative analysis of LPO with fluorescence and extricating the complexity of oxidation reactions within membranes.

Synthesis, characterization and reactivity of a Mn(III)-hydroxido complex as a biomimetic model for lipoxygenase.

Manganese hydroxido (Mn-OH) complexes supported by a tripodal N,N',N″-[nitrilotris(ethane-2,1-diyl)]tris(P,P-diphenylphosphinic amido) ([poat]3-) ligand have been synthesized and characterized by spectroscopic techniques including UV-vis and electron paramagnetic resonance (EPR) spectroscopies. X-ray diffraction (XRD) methods were used to confirm the solid-state molecular structures of {Na2[MnIIpoat(OH)]}2 and {Na[MnIIIpoat(OH)]}2 as clusters that are linked by the electrostatic interactions between the sodium counterions and the oxygen atom of the ligated hydroxido unit and the phosphinic (P=O) amide groups of [poat]3-. Both clusters feature two independent monoanionic fragments in which each contains a trigonal bipyramidal Mn center that is comprised of three equatorial deprotonated amide nitrogen atoms, an apical tertiary amine, and an axial hydroxido ligand. XRD analyses of {Na[MnIIIpoat(OH)]}2 also showed an intramolecular hydrogen bonding interaction between the MnIII-OH unit and P=O group of [poat]3-. Crystalline {Na[MnIIIpoat(OH)]}2 remains as clusters with Na+---O interactions in solution and is unreactive toward external substrates. However, conductivity studies indicated that [MnIIIpoat(OH)]- generated in situ is monomeric and reactivity studies found that it is capable of cleaving C-H bonds, illustrating the importance of solution-phase speciation and its direct effect on chemical reactivity. Synopsis: Manganese-hydroxido complexes were synthesized to study the influence of H-bonds in the secondary coordination sphere and their effects on the oxidative cleavage of substrates containing C-H bonds.

Acetylsalicylic acid inhibition of the lipoxygenase pathway: Implications for HIV prevention.

BACKGROUND: 1.5 million new HIV infections occurred in 2021, suggesting new prevention methods are needed. Inflammation increases the risk for HIV acquisition by attracting HIV target cells to the female genital tract (FGT). In a pilot study, acetylsalicylic acid (ASA/Aspirin) decreased the proportion of FGT HIV target cells by 35 %. However, the mechanism remains unknown. METHODS: Women from Nairobi, Kenya took low-dose ASA (81 mg) daily for 6-weeks. Free oxylipins in the plasma were quantified by high-performance liquid chromatography-tandem mass spectroscopy. RESULTS: Oxylipins from 9 fatty acid substrates were detected, with more than one analyte from 4 substrates reduced post-ASA. Summary analysis found ASA downregulated cyclooxygenase and lipoxygenase but not cytochrome P450 activity with a lower n-6/n-3 oxylipin profile, reflecting reduced inflammation post-ASA. CONCLUSIONS: Inflammation is associated with increased lipoxygenase activity and HIV risk. Our data suggests ASA reduces inflammation through downregulation of oxylipins. Understanding how ASA reduces inflammation may lead to novel HIV prevention approaches.

12-Hydroxyeicosatetraenoic acid is the only enzymatically produced HETE increased under brain ischemia.

Hydroxyeicosatetraenoic acids (HETE) are dramatically increased under brain ischemia and significantly affect post-ischemic recovery. However, the exact mechanism of HETE increase and their origin under ischemia are poorly understood. HETE might be produced de novo through lipoxygenase (LOX) -dependent synthesis with possible esterification into a lipid storage pool, or non-enzymatically through free radical oxidation of esterified arachidonic acid (20:4n6). Because HETE synthesized through LOX exhibit stereospecificity, chiral analysis allows separation of enzymatic from non-enzymatic pools. In the present study, we analyzed free HETE stereoisomers at 30 sec, 2 min, and 10 min of ischemia. Consistent with previous reports, we demonstrated a significant, gradual increase in all analyzed HETE over 10 min of brain ischemia, likely attributed to release of the esterified pool. The R/S ratio for 5-HETE, 8-HETE, and 15-HETE was not different from a racemic standard mix, indicating their non-enzymatic origin, which was in opposition to the inflamed tissue used as a positive control in our study. However, 12(S)-HETE was the predominant isoform under ischemia, indicating that ∼90 % of 12-HETE are produced enzymatically. These data demonstrate, for the first time, that 12-LOX is the major LOX isoform responsible for the enzymatic formation of the inducible HETE pool under ischemia. We also confirmed the requirement for enzyme inactivation with high-energy focused microwave irradiation (MW) for accurate HETE quantification and validated its application for chiral HETE analysis. Together, our data suggest that 12-LOX and HETE-releasing enzymes are promising targets for HETE level modulation upon brain ischemia.

Anti-inflammatory potential of aconitine produced by endophytic fungus Acremonium alternatum.

Argemone mexicana belonging to family Papaveraceae is a traditional medicinal plant widely utilized by tribal people in India for treating various ailments like skin infections, wounds and inflammation. This plant is very rich in alkaloidal content, which has a great potential in the treatment of anti-inflammatory disorders. Therapeutically promising bioactive molecules are often produced by endophytic fungi associated with medicinal plants. In this investigation, endophytic fungi were isolated from various parts of A. mexicana and screened for alkaloidal content. Among these, one of the fungal isolate, Acremonium alternatum AMEF-5 producing maximum alkaloids showed significant anti-inflammatory activity. Fractionation of this crude fungal extract through column chromatography yielded eight fractions, which were further screened for anti-inflammatory activities. Fraction 3 exhibited significant anti-inflammatory activity by the inhibition of lipoxygenase enzyme (IC50 15.2 ± 0.09 µg/ml), scavenging of the nitric oxide radicals (IC50 11.38 ± 0.35 µg/ml), protein denaturation (IC50 14.93 ± 0.4 µg/ml), trypsin inhibition (IC50 12.06 ± 0.64 µg/ml) and HRBC stabilization (IC50 11.9 ± 0.22 µg/ml). The bioactive alkaloid in fraction 3 was identified as aconitine which was confirmed by UV, FTIR, HPLC, HRMS, 1H NMR, and 13C NMR analysis. This study demonstrates that endophytic fungi serve a potential source for sustainable production of therapeutically important alkaloids.

In-situ co-immobilization of lipase, lipoxygenase and L-cysteine within a metal-amino acid framework for conversion of soybean oil into higher-value products.

We propose a co-immobilized chemo-enzyme cascade system to mitigate random intermediate diffusion from the mixture of individual immobilized catalysts and achieve a one-pot reaction of multi-enzyme and reductant. Catalyzed by lipase and lipoxygenase, unsaturated lipid hydroperoxides (HPOs) were synthesized. 13(S)-hydroperoxy-9Z, 11E-octadecadienoic acid (13-HPODE), one compound of HPOs, was subsequently reduced to 13(S)-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) by cysteine. Upon the optimized conditions, 75.28 mg of 13-HPODE and 4.01 mg of 13-HODE were produced from per milliliter of oil. The co-immobilized catalysts exhibited improved yield compared to the mixture of individually immobilized catalysts. Moreover, it demonstrated satisfactory durability and recyclability, maintaining a relative HPOs yield of 78.5% after 5 cycles. This work has achieved the co-immobilization of lipase, lipoxygenase and the reductant cysteine for the first time, successfully applying it to the conversion of soybean oil into 13-HODE. It offers a technological platform for transforming various oils into high-value products.

Multifactorial optimization enables the identification of a greener method to produce (+)-nootkatone.

The natural aroma compound (+)-nootkatone was obtained in selective conversions of up to 74 mol% from inexpensive (+)-valencene substrate by using a comparatively greener biocatalytic process developed based on modifications of the previously published Firmenich method. Buffer identity and concentration, pH, temperature and downstream work-up procedures were optimized to produce a crude product in which >90 % of (+)-valencene had been converted, with high chemoselectivity observed for (+)-nootkatone production. Interestingly, the biotransformation was carried out efficiently at temperatures as low as 21 ºC. Surprisingly, the best results were obtained when an acidic pH in the range of 3-6 was applied, as compared to the previously published procedure in which it appeared to be necessary to buffer the pH optimally and fixed throughout at 8.5. Furthermore, there was no need to maintain a pure oxygen atmosphere to achieve good (+)-nootkatone yields. Instead, air bubbled continuously at a low rate through the reaction mixture via a submerged glass capillary was sufficient to enable the desired lipoxygenase-catalyzed oxidation reactions to occur efficiently. No valencene epoxide side-products were detected in the organic product extract by a standard GCMS protocol. Only traces of the anticipated corresponding α- and ß-nootkatol intermediates were routinely observed.

Localization and activity of lipoxygenase in the ovule of Larix kaempferi (Lamb.) Carr. during female gametophyte maturation.

KEY MESSAGE: Lipoxygenase activity and localization vary throughout the development of Larix kaempferi ovules, with the highest enzyme activity observed in ovules at the cellular stage and the most intense immunogold reaction noted at the mature archegonium stage of gametophyte development. Lipoxygenases are a family of oxidoreductases with a significant role in biological systems, widespread in living organisms e.g. mammals, fish, corals, plants, mosses, algae, fungi, yeasts, and bacteria. Lipoxygenase activity in plants leads to the formation of phytooxylipins, i.e. signaling molecules, which play a crucial role in many significant physiological processes such as male and female gametophyte maturation, germination and seedling growth, pathogen resistance, abiotic stress response, fruit ripening, and senescence. The activity and localization of lipoxygenase change during plant growth and development. The localization of lipoxygenase in a developing ovule of Larix kaempferi was analyzed using the immunogold labeling method, and the activity was determined spectrophotometrically with linolenic acid as a substrate. Among the investigated stages, the immunogold reaction was the most intense at the mature archegonium stage in the ovule. Lipoxygenase was found in all parts of the L. kaempferi ovule. The largest number of immunogold particles was detected in the integument cells of all the analyzed stages of ovule development. Only one isoform of lipoxygenase with an optimum at pH 8 was active in the ovules during female gametophyte maturation. The highest enzyme activity was determined at the cellular stage, whereas the mature archegonium stage was characterized by its lowest level, which means that LOX activity in developing ovules of the Japanese larch is not correlated with the number of antibody-labeled molecules of the enzyme.

Effects of ultraviolet C on the quality and aroma volatile in peach fruit during postharvest storage.

The study investigated the impact of UV-C irradiation on peach fruit quality during postharvest storage, with a focus on aroma changes and the mechanisms involving lipoxygenase metabolism. Results showed that UV-C irradiation at a dosage of 1.5 kJ/m2 was found to preserve the quality attributes of peach fruit during ambient storage, as evidenced by high flesh firmness, inhibition of weight loss and respiration rate, as well as high values of L* and ascorbic acid. Meanwhile, UV-C irradiation led to an increase in the contents of aroma-related volatiles, particularly esters and lactones, compared to non-irradiated fruit. Our results suggested that the enhanced emission of aroma-related volatiles in UV-C irradiated peach fruit was linked to elevated levels of unsaturated fatty acids. Besides, UV-C induced the expressions and activities of enzymes in the lipoxygenase pathway, thus promoting the synthesis of esters and lactones, which contribute to the enhanced aroma in peach fruit.

A multifunctional nanoplatform for chemotherapy and nanocatalytic synergistic cancer therapy achieved by amplified lipid peroxidation.

Traditional cancer chemotherapy suffers from low efficacy and severe side effects, limiting its use as a first-line treatment. To address this issue, we investigated a novel way to induce lipid peroxidation (LPO), which plays an essential role in ferroptosis and may be useful against cancer cells and tumors. In this study, a pH-responsive synergistic cancer therapy nanoplatform was prepared using CaCO3 co-loaded with oleanolic acid (OA) and lipoxygenase (LOX), resulting in the formation OLCaP NP. This nanoplatform exhibited good drug release properties in an acidic tumor environment owing to the presence of CaCO3. As a result of acidic stimulation at tumor sites, the OLCaP NP released OA and LOX. OA, a chemotherapeutic drug with anticancer activity, is already known to promote the apoptosis of cancer cells, and LOX is a natural enzyme that catalyzes the oxidation of polyunsaturated fatty acids, leading to the accumulation of lipid peroxides and promoting the apoptosis of cancer cells. More importantly, OA upregulated the expression of acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), which promoted enzyme-mediated LPO. Based on our combined chemotherapy and nanocatalytic therapy, the OLCaP NP not only had remarkable antitumor ability but also upregulated ACSL4 expression, allowing further amplification of LPO to inhibit tumor growth. These findings demonstrate the potential of this nanoplatform to enhance the therapeutic efficacy against tumors by inducing oxidative stress and disrupting lipid metabolism, highlighting its clinical potential for improved cancer treatment. STATEMENT OF SIGNIFICANCE: This study presents a novel nanoplatform that combines oleanolic acid (OA), a chemotherapeutic drug, and lipoxygenase (LOX), which oxidizes polyunsaturated fatty acids to trigger apoptosis, for targeted cancer therapy. Unlike traditional treatments, our nanoplatform exhibits pH-responsive drug release, specifically in acidic tumor environments. This innovation enhances the therapeutic effects of OA and LOX, upregulating acyl-CoA synthetase long-chain family member 4 expression and amplifying lipid peroxidation to promote tumor cell apoptosis. Our findings significantly advance the existing literature by demonstrating a synergistic approach that combines chemotherapy and nanocatalytic therapy. The scientific impact of this work lies in its potential to improve cancer treatment efficacy and specificity, offering a promising strategy for clinical applications and future research in cancer therapy.

Arabidopsis Transcriptomics Reveals the Role of Lipoxygenase2 (AtLOX2) in Wound-Induced Responses.

In wounded Arabidopsis thaliana leaves, four 13S-lipoxygenases (AtLOX2, AtLOX3, AtLOX4, AtLOX6) act in a hierarchical manner to contribute to the jasmonate burst. This leads to defense responses with LOX2 playing an important role in plant resistance against caterpillar herb-ivory. In this study, we sought to characterize the impact of AtLOX2 on wound-induced phytohormonal and transcriptional responses to foliar mechanical damage using wildtype (WT) and lox2 mutant plants. Compared with WT, the lox2 mutant had higher constitutive levels of the phytohormone salicylic acid (SA) and enhanced expression of SA-responsive genes. This suggests that AtLOX2 may be involved in the biosynthesis of jasmonates that are involved in the antagonism of SA biosynthesis. As expected, the jasmonate burst in response to wounding was dampened in lox2 plants. Generally, 1 h after wounding, genes linked to jasmonate biosynthesis, jasmonate signaling attenuation and abscisic acid-responsive genes, which are primarily involved in wound sealing and healing, were differentially regulated between WT and lox2 mutants. Twelve h after wounding, WT plants showed stronger expression of genes associated with plant protection against insect herbivory. This study highlights the dynamic nature of jasmonate-responsive gene expression and the contribution of AtLOX2 to this pathway and plant resistance against insects.

Tert-Butylhydroquinone retards longan fruit deterioration by regulating membrane lipid and energy metabolisms.

Longan fruit deteriorates rapidly after harvest, which limits its storability. This study aimed to investigate the effect of tert-butylhydroquinone (TBHQ) on quality maintenance, membrane lipid metabolism, and energy status of longan fruit during 25 °C storage. Compared with control fruit, TBHQ treatment maintained better marketable fruit rate and suppressed activities of phospholipase D (PLD), lipase, and lipoxygenase (LOX), and downregulated expressions of DlPLD, DlLOX, and Dllipase. TBHQ also increased the ratio of unsaturated fatty acids to saturated fatty acids (U/S) and the index of unsaturated fatty acids (IUFA). In addition, higher levels of ATP, ADP, energy charge, NADP+/ NADPH as well as higher activities of H+-ATPase, Ca2+-ATPase and NADK were also observed in TBHQ-treated fruit. These results suggested that TBHQ may maintain postharvest quality of longan fruit by regulating membrane lipid and energy metabolisms.

In-situ enzyme-initiated production of hexanal from sunflower oil and its release from double emulsion electrospun bio-active membranes.

The aim of the presented study was to evaluate the release of the enzymatically initiated production of hexanal from double emulsion electrospun bio-active membranes at a temperature of fruit storage. Among different formulations of water-in-oil (W1/O) primary emulsions, the emulsion composed of 12% w/v Tween20 and 0.1 M NaCl in water (W1) and 6% of poly(glycerol) poly(ricinoleate) dissolved in sunflower oil (O) using W1/O ratio of 80/20 (w/w) (Tween20-NaCl/6% PGPR) was selected, for further incorporation of enzymes, based on the lowest average droplet size (391.0 ± 15.6 nm), low polydispersity index (0.255 ± 0.07), and good gravitational stability also after 14 days. Both enzymes, lipase and lipoxygenase are needed to produce hexanal (up to 58 mg/L). Additionally, double emulsions were prepared with sufficient conductivity and viscosity using different W1/O to W2 ratios for electrospinning. From the selected electrospun membrane, up to 4.5 mg/L of hexanal was released even after 92 days.

The CsDof1.8-CsLIPOXYGENASE09 module regulates C9 aroma production in cucumber.

Nine-carbon aldehydes and their relative alcohols (C9 aromas) are the main aroma compounds of cucumber (Cucumis sativus L.) fruits and provide a unique cucumber-like note. However, the key regulators of C9 aroma accumulation in cucumber fruit are poorly characterized. Based on C9 aroma dynamic analysis and transcriptome analysis during fruit development of two different cucumber inbred lines, Q16 and Q24, Lipoxygenase09 (CsLOX09) was identified as a candidate gene for C9 aroma accumulation. Additionally, Q24 with higher CsLOX09 expression accumulated more C9 aromas than Q16. To verify the function of CsLOX09, Cslox09 homozygote knockout lines were created. C9 aroma content decreased by 80.79% to 99.16% in these mutants compared to the wild type. To further explore the reasons for the difference in CsLOX09 expression between Q16 and Q24 fruits, a co-expression network was constructed by integrating the C9 aroma-associated metabolism and transcriptomic data. Eighteen candidate transcription factors were highly correlated with the expression of CsLOX09. DNA binding with One Finger 1.8 (CsDof1.8) was confirmed to bind directly to the A/TAAAG motif of the CsLOX09 promoter through dual-luciferase, yeast one-hybrid, chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assays. Furthermore, C9 aroma content and CsLOX09 expression were significantly increased in the CsDof1.8 overexpression lines. Overall, these data elucidate the metabolic regulation of C9 aromas in cucumber and provide a foundation for facilitating the regulation of flavor in cucumber breeding.

A non-invasive method for phenotyping scab-tolerant apple plants using volatile organic compounds.

One of the most devastating diseases of apples is scab, caused by the fungus Venturia inaequalis. Most commercial apple varieties are susceptible to this disease; only a few are resistant. Breeding approaches are being used to develop better apple varieties that are resistant to scab. Volatile organic compounds (VOCs) contribute greatly to a plant's phenotype, and their emission profile largely depends on the genotype. In the non-destructive phenotyping of plants, VOCs can be used as biomarkers. In this study, we assessed non-destructively the scab tolerance potential of resistant (cv. 'Prima') and susceptible (cv. 'Oregon Spur') apple cultivars by comparing their major leaf VOC compositions and relative proportions. A comparison of the leaf VOC profiles of the two cultivars revealed 16 different VOCs, with cis-3-hexenyl acetate (3HA) emerging as a biomarker of cultivar differences. V. inaequalis growth was significantly inhibited in vitro by 3HA treatment. 3HA was significantly effective in reducing scab symptoms on V. inaequalis-inoculated leaves of 'Oregon Spur.' The resistant cultivar 'Prima' also exhibited higher lipoxygenase (LOX) activity and α-linolenic acid (ALA) levels, suggesting that V. inaequalis resistance is linked to LOX activity and 3HA biosynthesis. This study proposes 3HA as a potential biomarker for rapid non-destructive screening of scab-resistant apple germplasm of 'Prima' based on leaf VOCs.

Synthesis and Antioxidant Activity of N-Benzyl-2-[4-(aryl)-1H-1,2,3-triazol-1-yl]ethan-1-imine Oxides.

The synthesis, antioxidant capacity, and anti-inflammatory activity of four novel N-benzyl-2-[4-(aryl)-1H-1,2,3-triazol-1-yl]ethan-1-imine oxides 10a-d are reported herein. The nitrones 10a-d were tested for their antioxidant properties and their ability to inhibit soybean lipoxygenase (LOX). Four diverse antioxidant tests were used for in vitro antioxidant assays, namely, interaction with the stable free radical DPPH (1,1-diphenyl-2-picrylhydrazyl radical) as well as with the water-soluble azo compound AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride), competition with DMSO for hydroxyl radicals, and the scavenging of cationic radical ABTS•+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation). Nitrones 10b, 10c, and 10d, having the 4-fluorophenyl, 2,4-difluorophenyl, and 4-fluoro-3-methylphenyl motif, respectively, exhibited high interaction with DPPH (64.5-81% after 20 min; 79-96% after 60 min), whereas nitrone 10a with unfunctionalized phenyl group showed the lowest inhibitory potency (57% after 20 min, 78% after 60 min). Nitrones 10a and 10d, decorated with phenyl and 4-fluoro-3-methylphenyl motif, respectively, appeared the most potent inhibitors of lipid peroxidation. The results obtained from radical cation ABTS•+ were not significant, since all tested compounds 10a-d showed negligible activity (8-46%), much lower than Trolox (91%). Nitrone 10c, bearing the 2,4-difluorophenyl motif, was found to be the most potent LOX inhibitor (IC50 = 10 µM).

Apolipoprotein D3 and LOX product play a role in immune-priming of a lepidopteran insect, Spodoptera exigua.

Immune-priming occurs in insects after a prior pathogen exposure. However, its underlying mechanism in insects remains elusive. In the present work, immune-priming was detected in a lepidopteran insect, Spodoptera exigua. Specifically, a prior infection with a heat-killed pathogenic bacterium, Escherichia coli, led to increased survival upon the second infection of different pathogens. Plasma collected from larvae with the prior infection possessed the immune-priming factor(s) that significantly up-regulated cellular and humoral immune responses of naïve larvae. Our study also finds that variations in the timing of plasma collection for priming larvae resulted in distinct impacts on both cellular and humoral responses. However, when the active plasma exhibiting the immune-priming was heat-treated, it lost this priming activity, therefore suggesting that protein factor(s) play a role in this immune-priming. An immunofluorescence assay showed that the hemocytes collected from the immune-primed larvae highly reacted to a polyclonal antibody specific to a vertebrate lipocalin, apolipoprotein D (ApoD). Among 27 ApoD genes (Se-ApoD1 âˆ¼ Se-ApoD27) of S. exigua, Se-ApoD3 was found to be highly induced during the immune-priming, in which it was shown to be expressed in hemocytes and fat body from a fluorescence in situ hybridization analysis. RNA interference of Se-ApoD3 expression significantly impaired the immune-priming of S. exigua larvae. Moreover, the inhibition of eicosanoid biosynthesis suppressed the immune-priming, in which treatment with a lipoxygenase (LOX) inhibitor-and not treatment with a cyclooxygenase inhibitor-suppressed immune-priming. Further, an addition of LOX product such as lipoxin A4 or lipoxin B4 significantly rescued the lost immune-priming activity. Taken together, these results suggest that a complex of ApoD3 and LOX product mediates the immune-priming activity of S. exigua.