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.

Eicosanoid Signaling in Insect Immunology: New Genes and Unresolved Issues.

This paper is focused on eicosanoid signaling in insect immunology. We begin with eicosanoid biosynthesis through the actions of phospholipase A2, responsible for hydrolyzing the C18 polyunsaturated fatty acid, linoleic acid (18:2n-6), from cellular phospholipids, which is subsequently converted into arachidonic acid (AA; 20:4n-6) via elongases and desaturases. The synthesized AA is then oxygenated into one of three groups of eicosanoids, prostaglandins (PGs), epoxyeicosatrienoic acids (EETs) and lipoxygenase products. We mark the distinction between mammalian cyclooxygenases and insect peroxynectins, both of which convert AA into PGs. One PG, PGI2 (also called prostacyclin), is newly discovered in insects, as a negative regulator of immune reactions and a positive signal in juvenile development. Two new elements of insect PG biology are a PG dehydrogenase and a PG reductase, both of which enact necessary PG catabolism. EETs, which are produced from AA via cytochrome P450s, also act in immune signaling, acting as pro-inflammatory signals. Eicosanoids signal a wide range of cellular immune reactions to infections, invasions and wounding, including nodulation, cell spreading, hemocyte migration and releasing prophenoloxidase from oenocytoids, a class of lepidopteran hemocytes. We briefly review the relatively scant knowledge on insect PG receptors and note PGs also act in gut immunity and in humoral immunity. Detailed new information on PG actions in mosquito immunity against the malarial agent, Plasmodium berghei, has recently emerged and we treat this exciting new work. The new findings on eicosanoid actions in insect immunity have emerged from a very broad range of research at the genetic, cellular and organismal levels, all taking place at the international level.

EFFICACY AND MODES OF ACTION OF RESISTANCE INDUCERS ON TWO WHEAT SPECIES AGAINST MYCOSPHAERELLA GRAMINICOLA.

Plant resistance inducers could be an alternative to conventional fungicides to control in a more durable and environmentally friendly manner fungal pathogens. Here, we tested the protection efficacy and the modes of action of four resistance inducers (R1, R2, R3 and R4) against the causal agent of Septoria tritici blotch, Mycosphaerella graminicola, the most frequently occurring pathogen on wheat crops worldwide. The four inducers were tested on two wheat cultivars, Premio (a French bread wheat cultivar) and Karim (a Tunisian durum wheat cultivar), each inoculated with a bread-wheat or a durum-wheat adapted isolate; respectively. All inducers exhibited in the greenhouse a significant protection level on both cultivars regarding disease symptoms (necrosis and chlorosis) and sporulation (pycnidium density). The most efficient inducer was R3 which showed 84% symptom reduction, while the less efficient one was R2 with only a 39% reduction. None of the studied inducers showed direct biocide effect against the fungus, except R4 which displayed a significant in planta inhibition of spore germination. Further investigations revealed that all inducers elicited the plant defence enzymes peroxidase and lipoxygenase, but the activity levels varied depending on the considered inducer. In addition, the effect of resistance inducers on the infection process and the fungal cell-wall degrading enzymes xylanases and glucanases was also investigated. Our study allowed us to find out four efficient resistance inducers on wheat against M. graminicola and to establish data about the modes of action of these inducers.

MECHANISMS INVOLVED IN MYCORRHIZAL WHEAT PROTECTION AGAINST POWDERY MILDEW.

In France, the Ecophyto 2018 national action plan will set out to reduce the use of pesticides by 50% by 2018, if possible. To achieve this goal, the use of arbuscular mycorrhizal (AM) fungi could be a potential alternative method allowing the control of crop diseases. The inoculation by AM fungi has been demonstrated to protect plants against soil-borne pathogens, but little is known about their effectiveness against aerial pathogens, such as the biotrophic fungus Blumeria graminis f.sp. tritici (Bgt) causing wheat (Triticum aestivum) powdery mildew. In the present study, wheat plants were grown in pots, under controlled conditions. Using various phosphorus (P) concentrations, the effectiveness of three AM inocula (Rhizophagus irregularis (Ri), Funneliformis mosseae (Fm)) and Solrize, a mixture of Ri and Fm) in Orvantis wheat cultivar, were tested. After 42 days of culture, mycorrhizal (M) and non-mycorrhizal (NM) wheat plants were infected by Bgt. A satisfactory mycorrhizal rate was obtained with the phosphorus concentration P/5 (P corresponding to the dose used in wheat fields in = 62 mg/L). Our work shows, for the first time, (i) a protective effect of AM inoculation against wheat powdery mildew, reaching up to 73% with Fm inocula, and (ii) its ability to induce a systemic resistance in wheat. Thereafter, we investigated mechanisms involved in this protection. Control plants, M plants, infected plants by Bgt, and M-infected plants were compared at: (i) cytological level, our results revealed that papillae and whole-fluorescent cells presence was induced, conversely fungal haustorium formation in epidermal cells was reduced within M plants leaves (ii) enzymatic level-by assessing defense enzyme activities (lipoxygenase, peroxidase) known as defense markers were measured 24, 48, 72 and 96 hours after infection (hai). The importance of these activities in the defense pathways induced in wheat by AM fungi will be discussed.

RNA-Seq analysis reveals candidate genes for ontogenic resistance in Malus-Venturia pathosystem.

Ontogenic scab resistance in apple leaves and fruits is a horizontal resistance against the plant pathogen Venturia inaequalis and is expressed as a decrease in disease symptoms and incidence with the ageing of the leaves. Several studies at the biochemical level tried to unveil the nature of this resistance; however, no conclusive results were reported. We decided therefore to investigate the genetic origin of this phenomenon by performing a full quantitative transcriptome sequencing and comparison of young (susceptible) and old (ontogenic resistant) leaves, infected or not with the pathogen. Two time points at 72 and 96 hours post-inoculation were chosen for RNA sampling and sequencing. Comparison between the different conditions (young and old leaves, inoculated or not) should allow the identification of differentially expressed genes which may represent different induced plant defence reactions leading to ontogenic resistance or may be the cause of a constitutive (uninoculated with the pathogen) shift toward resistance in old leaves. Differentially expressed genes were then characterised for their function by homology to A. thaliana and other plant genes, particularly looking for genes involved in pathways already suspected of appertaining to ontogenic resistance in apple or other hosts, or to plant defence mechanisms in general. IN THIS WORK, FIVE CANDIDATE GENES PUTATIVELY INVOLVED IN THE ONTOGENIC RESISTANCE OF APPLE WERE IDENTIFIED: a gene encoding an "enhanced disease susceptibility 1 protein" was found to be down-regulated in both uninoculated and inoculated old leaves at 96 hpi, while the other four genes encoding proteins (metallothionein3-like protein, lipoxygenase, lipid transfer protein, and a peroxidase 3) were found to be constitutively up-regulated in inoculated and uninoculated old leaves. The modulation of the five candidate genes has been validated using the real-time quantitative PCR. Thus, ontogenic resistance may be the result of the corresponding up- and down-regulation of these genes.

Alginic acid isolated from Sargassum wightii exhibits anti-inflammatory potential on type II collagen induced arthritis in experimental animals.

The present study evaluated the anti-inflammatory potential of alginic acid isolated from the brown algae Sargassum wightii in type II collagen induced arthritic rats, a well established arthritic model that resembles more closely to human rheumatoid arthritis in its clinical, pathological, immunological and histological aspects. Type II collagen induced arthritic rats showed increased activities of inflammatory marker enzymes like cycloxygenase-2 (COX-2), lipoxygenase (5-LOX), xanthine oxidase (XO) and myeloperoxidase (MPO) along with increased concentration of rheumatoid factor (RF), ceruloplasmin and C-reactive protein (CRP). Treatment with alginic acid significantly reduced the activities of COX-2 and 5-LOX along with reduction in MPO, XO, RF and CRP. Alginic acid treatment reverted to the altered levels of hematological parameters like RBC count, WBC count and ESR in arthritic rats. Concentrations of proinflammatory cytokines like IL-1 ß, TNF α and IL-6 were significantly higher in arthritic rats which were reduced on treatment with alginic acid. Increased activities of lysosomal enzymes that manifest the systemic damage during arthritis were significantly reduced by the treatment with alginic acid which indicates the reduction in the rupture and degradation of connective tissue. Histopathology of knee joint tissues showed that extensive bone degradation and synovial hyperplasia along with infiltrating cells and treatment with alginic acid reversed the histopathological changes which indicate the protective potential of alginic acid in rheumatoid arthritis.

Lipids as markers of induced resistance in wheat: a biochemical and molecular approach.

Our work aimed at a global investigation of the lipid metabolism during the induction of resistance in wheat (Triticum aestivum) against powdery mildew (Blumeria graminis f.sp. tritici). More specifically, the effect of salicylic acid, known as playing a key role in the activation of defence reactions against pathogens in plants, has been investigated. After salicylic acid infiltration, accumulation of phosphatidic acid was observed that could be due to the phospholipase C pathway since an up-regulation of a phospholipase C-encoding gene expression as well as an accumulation of diacylglycerol were observed. The phosphatidic acid accumulation could also result from the phospholipase D pathway since a reduction of phosphatidylethanolamine content occurred. The response to salicylic acid at the octadecanoid pathway level was also investigated: both a lipoxygenase-encoding gene expression and lipoxygenase enzymatic activity were induced by salicylic acid simultaneously with a decrease of the linolenic acid content. Finally, a lipid transfer protein-encoding gene expression was also up-regulated upon salicylic acid infiltration. These observations indicate that lipid metabolism could be considered as a marker of elicitation in wheat.

Interaction between eicosanoids and the complement system in salmonid fish.

Both eicosanoid generation and the complement system have long evolutionary histories predating the emergence of the vertebrates over 500 myr ago. This study investigated the interplay between these two systems in an example of a bony fish, the rainbow trout (Oncorhynchus mykiss). Specifically, it examined whether purified complement fragments including C3a-1 and zymosan-activated serum, stimulate the biosynthesis of any of these eicosanoids by trout macrophages. Incubation of macrophages with zymosan pre-incubated with normal trout serum resulted in the phagocytosis of such particles and the generation of both intra- and extra-cellularly located lipoxygenase and cyclooxygenase products. Both eicosanoid generation and phagocytosis levels were significantly elevated following incubation of zymosan in trout serum in comparison with heat-inactivated (60°C for 30 min) trout serum and saline alone. A combined mass spectrometry/high performance liquid chromatography approach was employed to conclusively demonstrate the presence of the cyclooxygenase product, prostaglandin E (PGE) in the culture supernatants of ionophore-challenged macrophages. Incubation of trout macrophages with zymosan-activated trout serum (i.e. no zymosan present) failed to stimulate PGE generation. Similarly, incubation of these cells for up to 60 min with C3a-1 (4 or 50 nM) failed to generate significant amounts of PGE or lipoxygenase products such as leukotriene B(4/5) or lipoxin A(4/5). Longer term (6 & 24h) incubation of macrophages with C3a-1 (4 nM) resulted in a time dependent increase in the generation of PGE but not leukotriene B in culture supernatants. No conclusive evidence that the increase in PGE generation was caused by changes in the expression of either cyclooxygenase-1 or -2 was found.

The pepper 9-lipoxygenase gene CaLOX1 functions in defense and cell death responses to microbial pathogens.

Lipoxygenases (LOXs) are crucial for lipid peroxidation processes during plant defense responses to pathogen infection. A pepper (Capsicum annuum) 9-LOX gene, CaLOX1, which encodes a 9-specific lipoxygenase, was isolated from pepper leaves. Recombinant CaLOX1 protein expressed in Escherichia coli catalyzed the hydroperoxidation of linoleic acid, with a K(m) value of 113. 9 mum. Expression of CaLOX1 was differentially induced in pepper leaves not only during Xanthomonas campestris pv vesicatoria (Xcv) infection but also after exposure to abiotic elicitors. Transient expression of CaLOX1 in pepper leaves induced the cell death phenotype and defense responses. CaLOX1-silenced pepper plants were more susceptible to Xcv and Colletotrichum coccodes infection, which was accompanied by reduced expression of defense-related genes, lowered lipid peroxidation, as well as decreased reactive oxygen species and lowered salicylic acid accumulation. Infection with Xcv, especially in an incompatible interaction, rapidly stimulated LOX activity in unsilenced, but not CaLOX1-silenced, pepper leaves. Furthermore, overexpression of CaLOX1 in Arabidopsis (Arabidopsis thaliana) conferred enhanced resistance to Pseudomonas syringae pv tomato, Hyaloperonospora arabidopsidis, and Alternaria brassicicola. In contrast, mutation of the Arabidopsis CaLOX1 ortholog AtLOX1 significantly increased susceptibility to these three pathogens. Together, these results suggest that CaLOX1 and AtLOX1 positively regulate defense and cell death responses to microbial pathogens.

The role of specific Toxoplasma gondii molecules in manipulation of innate immunity.

Infection with the parasite Toxoplasma gondii stimulates an innate immune response in the host. T. gondii also induces alterations in infected monocytes and dendritic cells that probably contribute to its ability to disseminate and ultimately to establish persistent infection. Recent progress has linked specific parasite molecules to immune stimulation or the ability of the parasite to subvert intracellular signaling pathways in infected cells to evade immunity.

Dual-function protein in plant defence: seed lectin from Dolichos biflorus (horse gram) exhibits lipoxygenase activity.

Plant-pathogen interactions play a vital role in developing resistance to pests. Dolichos biflorus (horse gram), a leguminous pulse crop of the subtropics, exhibits amazing defence against attack by pests/pathogens. Investigations to locate the possible source of the indomitable pest resistance of D. biflorus, which is the richest source of LOX (lipoxygenase) activity, have led to a molecule that exhibits LOX-like functions. The LOX-like activity associated with the molecule, identified by its structure and stability to be a tetrameric lectin, was found to be unusual. The evidence for the lectin protein with LOX activity has come from (i) MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, (ii) N-terminal sequencing, (iii) partial sequencing of the tryptic fragments of the protein, (iv) amino acid composition, and (v) the presence of an Mn2+ ion. A hydrophobic binding site of the tetrameric lectin, along with the presence of an Mn2+ ion, accounts for the observed LOX like activity. This is the first ever report of a protein exhibiting both haemagglutination and LOX-like activity. The two activities are associated with separate loci on the same protein. LOX activity associated with this molecule adds a new dimension to our understanding of lectin functions. This observation has wide implications for the understanding of plant defence mechanisms against pests and the cellular complexity in plant-pathogen interactions that may lead to the design of transgenics with potential to impart pest resistance to other crops.

Mechanism of chronic urticaria exacerbation by aspirin.

In some patients with chronic idiopathic urticaria (CIU), aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit cyclooxygenase 1 (COX-1) precipitate wheals and swelling. There is no in vitro diagnostic, and diagnosis can be established only by provocation challenges with aspirin or other NSAIDs. Skin reactions triggered by aspirin are associated with the inhibition of cyclooxygenase, specifically COX-1, but not COX-2, and are characterized by overproduction of cysteinyl leukotrienes (cys-LTs). Aspirin and other NSAIDs should be avoided, but highly specific COX-2 inhibitors, known as coxibs, are well tolerated and can probably be safely used. Evidence has been accumulated that these reactions are due to the interference of aspirin-like drugs with arachidonic-acid metabolism. In this article, we discuss the mechanism of these reactions, and the characteristic course of aspirin-induced urticaria and its management.

Cysteinyl leukotrienes are secretagogues in atrophic coeliac and in normal duodenal mucosa of children.

OBJECTIVE: The pathophysiology of intestinal inflammation and diarrhoea is complex and involves the arachidonic acid cascade. Prostaglandins induce chloride secretion in healthy subjects and in patients with coeliac disease. Leukotrienes (LTs) are also known inflammatory mediators which have been shown to stimulate ion secretion in ileum and colon of rats and rabbits. The aim of this study was to determine the effects of leukotrienes C(4) (LTC(4)) and D(4) (LTD(4)) in normal and atrophic intestinal mucosa in children. MATERIAL AND METHODS: Routine paediatric intestinal biopsies were obtained from 109 children. Sixty-seven control biopsies and 42 biopsies from children with different stages of coeliac disease were mounted in a modified Ussing chamber. Potential difference (Pd) was measured continuously and tissue resistance (R(t)) and the generated current (I(m)) were calculated. RESULTS: In morphologically normal mucosa of duodenum, LTC(4) and LTD(4) increased Pd and I(m) in a dose-dependent manner. The increase was more pronounced in the distal than in the proximal part, similar to the response to prostaglandin E(2). The induced current was chloride-mediated, since replacement of Cl(-) with SO(4)(2-) in the bathing solution eliminated the response to the LTs. The LTC(4)-induced secretion was significantly decreased in atrophic mucosa, but the response was partially restored after preincubation with the cyclooxygenase inhibitor indomethacin. CONCLUSIONS: The results showed that LTC(4) and LTD(4) are secretagogues in the duodenal mucosa from healthy children by inducing a net chloride secretion. Restoration of the response in coeliac disease by cyclooxygenase inhibition suggests interactions between the different pathways of the arachidonic cascade in the intestinal mucosa.

Eicosanoids mediate Manduca sexta cellular response to the fungal pathogen Beauveria bassiana: a role for the lipoxygenase pathway.

Many studies have documented the involvement of eicosanoids in insect cellular immune responses to bacteria. The use of the fungal pathogen Beauveria bassiana as a nodulation elicitor, with inhibition of phospholipase A(2) by dexamethasone, extends the principle to fungi. This study also provides the first evidence of involvement of the lipoxygenase (LOX) pathway rather than the cyclooxygenase (COX) pathway in synthesis of the nodulation mediating eicosanoid(s). The LOX product, 5(S)-hydroperoxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid (5-HPETE), substantially reversed nodulation inhibition caused by dexamethasone and the LOX inhibitors, caffeic acid and esculetin. The COX product, prostaglandin H(2) (PGH(2)), did not reverse the nodulation inhibition by dexamethasone or the COX inhibitor, ibuprofen. None of the inhibitors tested had a significant effect on the phagocytosis of B. bassiana blastospores in vitro. Hemocyte phenoloxidase activity was reduced by dexamethasone, esculetin, and the COX inhibitor, indomethacin. The rescue candidates 5-HPETE and PGH(2) did not reverse the inhibition.

Accumulation of chloroplast-targeted lipoxygenase in passion fruit leaves in response to methyl jasmonate.

Wounding caused local and systemic induction of lipoxygenase (LOX) activity in passion fruit (Passiflora edulis f. flavicarpa) leaves, while exposing intact plants to methyl jasmonate (MJ) vapor provoked a much stronger response. Western blot analysis of these leaf protein extracts using polyclonal antibodies against cucumber LOX, revealed an accumulation of a 90 kDa protein, consistent with LOX enzymatic assays. The inducible LOX was purified to apparent homogeneity, and in vitro analysis of LOXactivity using linoleic acid as substrate showed that it possesses C-13 specificity. Immunocytochemical localization studies using leaf tissue from MJ-treated plants demonstrated that the inducible LOX was compartmented in large quantities in the chloroplasts of mesophyll cells, associated with the stroma. The results suggest that the wound response in passion fruit plants may be mediated by a chloroplast 13-LOX, a key enzyme of the octadecanoid defense-signaling pathway.

Isolation of lipoxygenase isoforms from Glycine max embryo axes based on apparent cross-reactivity with anti-myosin antibodies.

Three lipoxygenase isoforms were isolated from Glycine max embryo axes. A number of proteins around 97 kDa cross-reacted with several anti-actin and anti-myosin antibodies and these were used to follow their purification through gel filtration, hydroxyapatite and anion exchange columns. The 97-kDa cross-reactive material eluted in the unbound fractions of the last anion exchange column, and displayed two components of pI's 6.2 and 6.3. Further phase partition of this fraction in TX-114 yielded a hydrophobic 97 kDa protein. Additionally, a 95-kDa protein was retained and eluted from this last column. Partial peptide sequences indicated that the 95 kDa protein was soybean lipoxygenase-1, the first 97 kDa protein was lypoxygenase-3, and the hydrophobic 97 kDa protein was lipoxygenase-2. Several possible reasons for the cross-reactivity with the antibodies are discussed. To our knowledge, this is the first example of individual lipoxygenase isoforms isolated from soybean embryo axes.

Modulation of the host response in periodontal therapy.

This paper was prepared by the Research, Science, and Therapy Committee of the American Academy of Periodontology to provide the dental profession an overview of current and potential methods to modulate the host response in the treatment of periodontal diseases. Specifically, it discusses components of periodontal disease pathogenesis (i.e., immune and inflammatory responses, excessive production of matrix metalloproteinases and arachidonic acid metabolites, and regulation of bone metabolism) and their modulation.

Changes of lipoxygenase and fatty acid hydroperoxide lyase activities in bell pepper fruits during maturation.

Developmental changes in fatty acid hydroperoxide lyase (HPO lyase) and lipoxygenase (LOX) during the maturation of bell pepper fruits (Capsicum annuum L. cv. Kyonami) were examined by means of activity measurements, immunological detection of both the enzymes, and analysis of the volatile compounds formed upon homogenization of the fruits. Both the enzyme activities decreased with maturation, and immunological studies showed that the amounts of the enzymes concomitantly decreased. The amounts of six-carbon aldehydes and alcohols formed from bell pepper fruits upon homogenization also decreased during maturation, and with the fully ripened red fruits, these volatile compounds were hardly detectable. These results suggest that the major factor contributing to the changes in the composition of volatile compounds during the maturation of bell pepper fruits was changes in the amounts of HPO lyase and LOX.

Long-term elevation of cyclooxygenase-2, but not lipoxygenase, in regions synaptically distant from spreading depression.

Eicosanoids, produced from arachidonic acid by cyclooxygenases (COXs) and lipoxygenases (LIPOXs), are involved in numerous brain processes. To explore if brief and noninjurious stimuli chronically alter expression of these enzymes, we examined the induction of COX-2 and LIPOX expression following unilateral neocortical spreading depression (SD). Expression was examined over time and in regions not experiencing SD (hippocampus) but synaptically connected to the site of stimulation (cortex). One hundred six male Wistar rats had SD induced via microinjection of 0.5 M KCl (0.5 M NaCl for sham) into left parietal cortex every 9 minutes for 1 or 3 hours. One hour before SD some animals received dexamethasone (Dex), mepacrine (Mep), indomethacin (Indo), nordihydroguaiaretic acid (Ndga), phenylephrine (Pe), sodium nitroprusside (Snp) with Pe, or N omega-nitro-L-arginine methyl ester (Lnam). Animals survived for 0, 3, or 6 hours, or 1, 2, 3, 7, 14, 21, or 28 days. Brains were processed immunohistochemically for COX-2 and LIPOX, and the optical density (OD) of the left and right cortex, dentate gyrus (DG), CA3, and CA1 immunoreactivity (IR) were measured. Induction was expressed as the log of left divided by right side OD for each region. COX-2 IR in the left cortex was elevated rapidly and was sustained for 21 days following SD. COX-2 IR was also elevated in the ipsilateral hippocampus not experiencing SD, with the rank order of induction as follows: DG > CA3 > CA1. Dex, Snp, and/or Pe significantly reduced the induction of COX-2. No changes in LIPOX IR were observed. These results show that long-term changes in COX-2 expression are induced by SD and these changes decrease with synaptic distance. Benign stimuli increase COX-2 expression and thus may influence brain function for extended periods and at distant locations.

Catalysis sensitive conformational changes in soybean lipoxygenase revealed by limited proteolysis and monoclonal antibody experiments.

Soybean lipoxygenases catalyze lipid hydroperoxidation of polyunsaturated fatty acids. Putative ligand mediated conformational changes in soybean lipoxygenase 3 (L3) were studied by a combination of limited proteolysis and a series of monoclonal antibodies that recognize discontinuous epitopes and alter catalysis (inhibition and activation). Trypsin cleaved L3 (97 kDa) into C-terminal 60 kDa and N-terminal 37 kDa fragments. The 37 kDa fragment was obtained from a 38 kDa fragment formed initially. Using protein footprinting, the epitopes of the antibodies were mapped to the 37 kDa fragment. Proteolysis in the presence of a substrate analog inhibitor, oleic acid, generated the 60 and the 38 kDa fragments only. No further proteolysis of the 38 kDa fragment was seen even after prolonged incubation. This was not a detergent effect since the altered proteolysis was not obtained in the presence of SDS or Tween 20. Binding of a monoclonal antibody to L3 in the presence of oleic acid was substantially reduced providing additional evidence for a conformational change induced by the oleic acid-lipoxygenase interaction. These observations are interpreted using the recently solved three-dimensional structure of L3. It is apparent that while the protein is composed of a small N-terminal beta-barrel domain and a large principally alpha-helical C-terminal domain, proteolysis does not take place at a linking region between the two domains. The proteolysis results makes it clear that the smaller domain is connected across the entire length of the larger domain to a narrow, tongue-like projection that extends into the vicinity of the entrance to the proposed substrate binding channel.(ABSTRACT TRUNCATED AT 250 WORDS)