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Leukotriene sind, neben anderen Arachidonsäuremetaboliten, wichtige Mediatoren in Entzündungsprozessen. Um ihre Rolle bei den pathophysiologischen Prozessen nach Ischämie und Reperfusion des Herzmuskels aufzuklären, wurden Wildtyp-Mäuse und 5-LOX-KO-Mäuse, die keine Leukotriene produzieren können, untersucht. Die linke vordere Herzkranzarterie wurde für 30min ligiert und 24h in vivo reperfundiert. Bei den KO-Mäusen fand sich zwar eine signifikant höhere Anzahl eingewanderter Neutrophiler im reperfundierten Gewebe, dagegen ergab sich kein signifikanter Unterschied zwischen Wildtyp- und KO-Mäusen bezüglich der resultierenden Infarktgröße und bezüglich der im Gewebe gemessenen Menge an TNF-a. Demnach lässt sich kein Einfluss der 5-Lipoxygenase-Ausschaltung auf den Entzündungsprozess nach Myokardischämie und die Entstehung eines Reperfusionsschadens feststellen. / Leukotrienes are arachidonic acid derivatives that function as important mediators in inflammatory response reactions. To investigate their role in the pathophysiological processes after ischemia and reperfusion of the heart, wildtype mice and 5-LOX-KO-mice that cannot produce leukotrienes were compared using a mouse model of myocardial ischemia and reperfusion. The left anterior coronary artery was ligated for 30 minutes and then reperfused for 24 hours. This yielded no significant difference between wildtype and KO mice with regard to infarct size and amount of TNF-a measured in the heart tissue, even though there was a significantly larger number of infiltrating neutrophiles in the heart tissue of the KO mice. We conclude that there is no effect of the 5-lipoxygenase-knockout on the inflammatory response following myocardial ischemia or on the developing reperfusion injury.

A enzima lipoxigenase através da farinha integral de soja é amplamente utilizada na panificação com objetivos de branqueamento e melhora da reologia. Com base nesse pressuposto, o objetivo inicial deste estudo foi de observar a ação oxidante dessa enzima na presença de dois outros oxidantes (ácido ascórbico e peróxido de benzoíla) nas propriedades reológicas e sensoriais de pães. Sete misturas com diferentes combinações dos três compostos foram elaboradas utilizando-se um delineamento experimental do tipo \"centroide-simplex\", sendo aplicadas à farinha de trigo para o preparo de pães. A enzima apresentou sinergia com o ácido ascórbico na elasticidade da massa, sugerindo uma inter-relação bioquímica entre os dois compostos no fortalecimento da matriz protéica do glúten. A mesma sinergia entre a lipoxigenase da soja e o acido ascórbico foi observada na redução da tonalidade amarela dos pães medida instrumentalmente. A partir desses resultados, buscou-se avaliar a ação da enzima frente a variações tanto na força da farinha como no tempo de fermentação, reproduzindo as condições reais de processamento. Desta vez, um planejamento experimental do tipo Box-Behnken com três fatores (atividade da enzima, força da farinha e tempo de fermentação) em três níveis de variação, foi aplicado no preparo das amostras. Os resultados mostraram que a lipoxigenase da soja (fração 1) é uma enzima oxidante de ação rápida e seu efeito no branqueamento dos carotenóides da farinha de trigo exibiu interação positiva tanto com a força da farinha como o tempo de fermentação. Respostas reológicas e sensoriais decorrentes da ação conjunta de diferentes oxidantes e condições de processamento em pães foram pela primeira vez descritas através de modelos polinomiais com capacidade preditiva. / Soy lipoxygenase enzyme through soy flour is widely used in breadmaking for bleaching and rheological improvement. Based on this fact, the initial objective of this study was to observe the enzymes\' action in the presence of two other oxidants (ascorbic acid and benzoyl peroxide) in the rheological and sensory properties of breads. Seven mixtures with different combinations of the three compounds were prepared using a \"centroid-simplex\" design and applied to wheat flour for breadmaking. The enzyme showed synergism with ascorbic acid in the dough elasticity suggesting a biochemical interrelation between the two compounds in strengthening the gluten protein matrix. The same synergy between soy lipoxygenase and ascorbic acid was observed with the yellow hue reduction of breads measured instrumentally. From these results, the enzyme action was evaluated with wheat strength and proofing time variations reproducing real processing conditions. This time, a Box-Behnken experimental design with three factors (enzyme activity, wheat strength and proofing time), and three variation levels were applied in the sample preparation. The results showed that soy lipoxygenase (type 1) is a fast-acting oxidizing enzyme and its bleaching effect on wheat flour carotenoid exhibited a positive interaction with wheat strength and proofing time. Rheological and sensory results attributed to the combined action of the different oxidants and breadmaking conditions were for the first time described through polynomial models with predictive capacity.

Les lipoxygénases (LOX, EC 1.13.11.12) sont des dioxygénases à fer non héminique très répandues. Chez les végétaux, ces enzymes sont à l’origine d’une voie métabolique impliquée dans de nombreux processus physiologiques, mais aussi dans la réponse à un stress environnemental. La LOX initie la voie en catalysant l’incorporation régiospécifique et stéréospécifique de dioxygène sur le système pentadiénique d’un acide gras libre polyinsaturé (préférentiellement l’acide linoléique ou l’acide linolénique) pour générer un hydroperoxyde d’acide gras.Une lipoxygénase d’olive appelée LOX1, clonée au laboratoire, a été exprimée chez E. coli et purifiée. Elle produit à partir d’acide linoléique des hydroperoxydes de configuration 9S et 13R dans des proportions 2:1. Elle est la seule lipoxygénase végétale décrite à ce jour produisant des hydroperoxydes de configuration R. Les modèles proposés pour expliquer le contrôle de la spécificité réactionnelle des LOX ne s’appliquent pas à la LOX1 d’olive. Afin de mieux comprendre son mécanisme de fonctionnement, un modèle tridimensionnel de la LOX1 d’olive a été construit. La modification par mutagénèse dirigée de deux résidus particuliers, la phénylalanine 277 et la tyrosine 280, a permis d’identifier l’entrée du site actif de la LOX1 d’olive. D’autres résidus particuliers ont été modifiés par mutagénèse dirigée afin d’étudier leur rôle dans le mécanisme catalytique et le contrôle de la spécificité réactionnelle de la LOX1 d’olive. L’analyse globale des résultats obtenus a permis de proposer une première hypothèse quant au fonctionnement de cette enzyme : le substrat pénètrerait dans le site actif de la LOX1 d’olive par son extrémité carboxylate, et serait stabilisé dans le site actif par plusieurs résidus hydrophobes. Un canal pourrait cibler l’oxygène dans le site actif par l’intermédiaire du résidu L579 sur le système pentadiénique du substrat, contrôlant de cette manière la spécificité réactionnelle de la LOX1 d’olive.Par ailleurs, des oxylipines retrouvées chez Arabidopsis, appelées arabidopsides, pourraient être formées par action directe d’une 13-LOX sur des acides gras estérifiés des galactolipides. L’action de la 13-LOX1 de soja, la 9/13-LOX1 d’olive et la 9-LOX de pomme de terre a été testée avec des galactolipides. Une faible activité a été mesurée avec la 13-LOX1 de soja et la 9/13-LOX1 d’olive. Une activité plus importante a été mesurée avec la 9-LOX de pomme de terre. Ces résultats suggèrent que l’action des LOX est possible sur des acides gras estérifiés des galactolipides. / Lipoxygenases (LOXs, EC 1.13.11.12) are widespread dioxygenases containing a non heminic iron atom. In plants, LOXs are at the beginning of a metabolic pathway involved in several physiological processes and in the response to environmental stress. A LOX initiates the pathway, catalyzing a regiospecific and stereospecific insertion of oxygen on the pentadiene system of a free polyunsaturated fatty acid (linoleic or linolenic acid) to form fatty acid hydroperoxides.An olive lipoxygenase called olive LOX1, cloned at laboratory, has been expressed in E. coli strain and purified. Olive LOX1 produces 9S-hydroperoxides of and 13R-hydroperoxides from linoleic acid, in a ratio of 2:1, being the only plant LOX to produce R-hydroperoxides described to date. From the currently known models explaining the control of reactional specificity, none can be applied to olive LOX1. A three-dimensional model has been built by homology modeling to understand the catalytic mechanism of olive LOX1. Site-directed mutagenesis experiments have been used to modify two residues of particular interest, the phenylalanine 277 and the tyrosine 280, allowing us to point the active site entrance near these two residues. Other residues of interest have been modified to study their role in the catalytic mechanism and the reactional specificity of olive LOX1. The results have led us to propose a first hypothesis for the reactional mechanism of this enzyme: the substrate could enter into the active site with its carboxylate-end first, and could be stabilized in the active site by hydrophobic side chains of several residues. A channel could bring oxygen into the active site at a position near the side chain of the leucine 579 residue, this one targeting oxygen onto the pentadiene system of the substrate, controlling by this way the reactional specificity of olive LOX1.LOX are involved in oxylipins synthesis. Arabidopsides are a class of oxylipins found in Arabidopsis that could be produced by action of a 13-LOX on galactolipids, which carry esterified fatty acids. Activity of soybean 13-LOX, olive 9/13-LOX1 and potato 9-LOX has been investigated with galactolipids. A low activity was measured when soybean and olive LOXs were used. Activity was far more important when potato LOX was used. These results suggest that LOX can act on esterified fatty acids, especially galactolipids.

Résumé Certaines bactéries de la rhizosphère (PGPR, rhizobactéries promotrices de la croissance des plantes) exercent un effet bénéfique sur la croissance des plantes en stimulant des mécanismes de défense inductibles chez lhôte, rendant celle-ci moins susceptible vis-à-vis dune infection ultérieure par un agent pathogène. Ce phénomène appelé résistance systémique induite (ISR) a été mis en évidence chez plusieurs plantes pour lutter contre une gamme relativement large de pathogènes fongiques, bactériens ou viraux. Cependant, les bases moléculaires des mécanismes de défense proprement dits stimulés lors de lISR restent assez méconnues malgré les nombreux travaux réalisés cette dernière décennie. Dans ce contexte, des souches de PGPR (Bacillus subtilis et Pseudomonas putida) capables de protéger certaines plantes via linduction de lISR sont étudiées depuis plusieurs années au laboratoire. Dans le cadre de cette thèse de doctorat, nous avons étudié l´effet protecteur de ces bactéries dans deux pathosystèmes différents, tomate/Botrytis cinerea et concombre/Colletotrichum lagenarium. Nos résultats ont montré la capacité de P. putida BTP1 à induire l´ISR chez la tomate et le concombre sur base de la réduction des symptômes de maladie observée et sur base de la séparation spatiale des deux agents, bénéfique au niveau racinaire et pathogène au niveau foliaire. Cette résistance a été clairement associée avec la stimulation de la voie des oxylipines chez la tomate. Linduction de cette voie métabolique a dabord été mise en évidence biochimiquement par une augmentation des activités lipoxygénase et lipide hydroperoxydase dans les feuilles des plantes traitées avec Pseudomonas. De plus, au niveau moléculaire, les analyses par northern blot nous ont permis d´identifier un nouvel isoforme de gène Lox chez la tomate qui est exprimé différentiellement chez les plants traités par la bactérie. Ce gène dénommé LoxF montre une homologie de 82% avec un des cinq isoformes connus chez cette plante, LoxC. LoxF est essentiellement surexprimé dans les feuilles de tomates pré-inoculées avec BTP1 suite à l´infection par le pathogène. Il en est de même pour lactivité enzymatique correspondante de la lipoxygénase qui naugmente significativement chez les plants traités par rapport aux témoins quaprès infection par Botrytis. Ces résultats sous-entendent un phénomène de « priming » ou « mise en alerte » étroitement associé avec linduction de résistance systémique chez les plantes. La reconnaissance de la bactérie au niveau racinaire met en alerte la plante sans que des bouleversements majeurs au niveau métabolique ou génétique ne soient observés. Lhôte réagit alors plus fortement et plus rapidement pour mettre en uvre ses mécanismes de défense une fois le pathogène perçu. Par biotest sur TLC et analyses HPLC, CPG et LC-MS, nous avons mis en évidence laccumulation dune molécule dans les feuilles de tomate prétraitées avec BTP1. Cette molécule semble être de nature apolaire mais non phénolique et ne correspond pas aux phytoalexines connues de la tomate. Des études complémentaires doivent être réalisées pour lidentifier chimiquement mais sa cinétique daccumulation dans les tissus de la plante est étroitement associée à celle de la stimulation de la lipoxygénase. Elle pourrait donc dériver de cette voie métabolique. Des réactions de défense similaires ont été observées suite au traitement avec Bacillus subtilis chez la tomate et ces essais ont permis de mettre en évidence le rôle des lipopeptides produits en tant quéliciteurs impliqués dans linduction du phénomène de lISR par cette souche. Par contre, bien que les deux souches soient capables dinduire la résistance chez le concombre, aucune accumulation claire de phytoalexines ni de stimulation significative de la voie de la lipoxygénase nont pu y être associées chez cette plante. Globalement, nos résultats suggèrent que les voies métaboliques activées dans le cadre de lISR varient en fonction de lespèce végétale même si le microorganisme inducteur est identique. Abstract Some plant growth promoting rhizobacteria (PGPR) are able to stimulate inducible defence mechanisms that render the host plant less susceptible to a subsequent pathogen attack. This phenomenon called induced systemic resistance (ISR) can occur in several plant species against a wide range of bacterial, viral and fungal pathogens. Despite extensive work carried out this last decade, many aspects of the molecular basis underlying this rhizobacteria-mediated ISR remain unclear. In this context, Bacillus subtilis and Pseudomonas putida strains able to protect plants via induction of ISR have been studied for several years in our laboratory. In this thesis, we first aimed at evaluating the protective effect of these strains in different pathosystems and second at identifying the associated defence mechanisms. Our results showed the capacity of P. putida BTP1 to induce ISR in the tomato/Botrytis cinerea and cucumber/Colletotrichum lagenarium pathosystems on the basis of reduction of disease symptoms and on the basis of the spatial separation of the two agents, beneficial at the root level and pathogenic at the leaf level. This resistance was clearly associated the stimulation of the oxylipin pathway in tomato. The induction of this metabolic pathway was evidenced biochemically by an increase in the lipoxygenase and lipid hydroperoxydase activities in the leaves of plants treated with Pseudomonas compared to controls. Moreover, at the molecular level, northern blot analyses enabled us to identify a new isoforme of Lox gene in tomato which is expressed differentially in plants treated with the bacterium. This gene called LoxF shows a homology of 82% with one of the five isoformes known in this plant, LoxC. LoxF is mainly expressed in tomato leaves pre-inoculated with BTP1 after infection by pathogen. The same applies for the lipoxygenase enzyme activity which increases significantly in treated plants only after infection by Botrytis. These results imply a phenomenon of "priming" closely associated with the systemic resistance induction in plants. The recognition of the bacterium at the root level primes the plant but in most cases major changes at the metabolic or genetic level are not observed. The host then reacts more strongly and more quickly to express defence mechanisms once the pathogen is perceived. In addition, by combining the use of TLC-based biotests and analytical methods such as HPLC, CPG and LC-MS analyses, we highlighted the accumulation of a molecule in BTP1-pretreated tomato leaves. This molecule seems to be of a non-polar nature but not phenolic and does not correspond to any phytoalexin known in tomato. Complementary studies must be carried out to identify its structure but its kinetic of accumulation in the plant tissues is closely associated with the stimulation of the lipoxygenase enzyme. It could thus derive from this metabolic pathway. Similar defence reactions were observed in tomato following treatment with Bacillus subtilis and through these tests, we also highlighted the role of some lipopeptides produced by this strain as elicitors responsible for the induction of the ISR phenomenon. On the other hand, although the two strains are able to induce resistance in cucumber, no clear accumulation of phytoalexins nor of significant stimulation of the lipoxygenase pathway could be associated with disease reduction in this plant. All together, our results suggest that the metabolic pathways activated during ISR vary in function of the plant and pathogen species even if the inducing micro-organism is identical.

Lipoxygenase (LOX)-derived oxylipins are known to play critical roles in defense against herbivores and pathogens. The objective of this study was to determine the biochemical, molecular and physiological roles of a specific maize lipoxygenase gene, ZmLOX10, with special emphasis on LOX10-derived oxylipins in plant-insect and plant-pathogen interactions. To achieve this goal, independent mutant alleles were generated and genetically advanced to create near-isogenic mutant and wild-type lines suitable for functional analysis. Here we provide genetic evidence that LOX10 is the sole LOX isoform in maize required for the biosynthesis of green leafy volatiles (GLV) in leaves and show that LOX10- mediated GLVs play a significant role in direct and indirect defense responses to insects through their regulation of jasmonic acid and volatile organic compound production. Contrary to the defensive role of LOX10 in plant-insect interactions, tests for susceptibility to fungal pathogens suggest that LOX10-mediated GLVs may contribute to the development of disease symptoms to the economically important maize pathogens, Aspergillus flavus and Colletotrichum graminicola. Specifically, LOX10-derived GLVs may facilitate aflatoxin accumulation in response to A. flavus infection and may play a positive role in anthracnose leaf blight and stalk rot caused by C. graminicola. Collectively, our results suggest that metabolites derived from GLV-regulated pathways have a significant impact on molecular plant-herbivore and plant-pathogen interactions.

Colorectal carcinoma (CRC) is often lethal when invasion and/or metastasis occur. 15-lipoxygenase-1 (15-LO-1), an enzyme of the inflammatory eicosanoid pathway, oxidatively metabolizes linoleic acid and its expression is repressed in CRC. In the present study, the hypothesis that the lack of 15-LO-1 expression in CRC cells may contribute to the tumorigenesis was investigated. Therefore 15-LO-1 was introduced to colon cancer cell line HT-29 that does not have detectable levels of the 15-LO-1. The HT-29 cells were transiently transfected with the eukaryotic expression vector pcDNA3.1-15-LO-1 and the effects of 15-LO-1 expression on the proliferation, apoptosis as wells as metastatic potential of the cells were investigated. Cellular proliferation was analyzed by MTT assay and the apoptotic potential of 15-LO-1 was evaluated by acridine orange, floating cell ratio and caspase-3 assays as well as expression levels of the antiapoptotic protein XIAP. Cellular migration and invasion were investigated by Boyden chamber migration and Matrigel invasion assay.The data indicates that 15-LO-1 expression significantly decreased cell proliferation and increased apoptosis. In addition, a significant reduction was observed in migratory and invasive capacity 15-LO-1 expression also significantly reduced the expression of metastasis associated 1 protein (MTA-1). Taken together we propose that 15-LO-1 expression in CRC can inhibit colon cancer cell growth through induction of apoptotic cell death and may contribute to the inhibition of metastatic capacity in vitro which may be exploited for therapeutic purposes.

Lipoxygenases (LOX) catalyze regio- and stereospecific oxygenation of polyunsaturated fatty acids to hydroperoxides. These hydroperoxides are further metabolized to leukotrienes and lipoxins in mammals, and are involved in asthma and inflammation. LOX of animals and plants contain iron as catalytic metal (FeLOX). Filamentous fungi use both FeLOX, and manganese containing LOX (MnLOX). The role of LOX in fungi is still not known. This thesis focuses on expression of novel MnLOX, analyses of their reaction mechanism and products by HPLC-MS/MS, protein crystallization and analysis of the first MnLOX structure.   MnLOX from G. graminis, M. salvinii, M. oryzae, F. oxysporum and C. gloeosporioides were expressed in Pichia pastoris, purified and characterized by HPLC-MS/MS. All MnLOX catalyzes suprafacial hydrogen abstraction and oxygen insertion. Replacement of one Ile to Phe in the active site of MnLOX of G. graminis could switch the mechanism from suprafacial to mainly antarafacial. MnLOX of F. oxysporum was interesting since it catalyzes oxygenation of linoleic acid to 11R- instead of the more common 11S-hydroperoxides. This feature could be attributed to a single Ser/Phe exchange in the active site.   We found that Gg-MnLOX utilizes hydrogen tunneling in the reaction mechanism, but was slightly more temperature dependent than soybean FeLOX. It is an intriguing question why some fungal LOX use manganese and not iron as catalytic metal and whether the large redox potential of Mn2+/Mn3+ (1.5 V) can be tuned close to that of Fe2+/Fe3+ (0.77 V) for redox cycling and catalysis. We present crystallization conditions for two MnLOX, and the 2.07 Å crystal structure of MnLOX from M. oryzae, solved using sulfur and manganese single anomalous dispersion (SAD). The structure reveals a similar metal coordinating sphere as FeLOX but the metal ligand Asn473 was positioned on a short loop instead of a helix and formed interactions with a conserved Gln. This feature could be essential for the use of manganese as catalytic metal in LOX. We found three Phe residues that likely facilitate the suprafacial hydrogen abstraction and oxygen insertion for MnLOX. These findings provide new insight into the unique reaction mechanism of MnLOX.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.