Emerging trade-offs - impact of photoprotectants (PsbS, xanthophylls, and vitamin E) on oxylipins as regulators of development and defense.

This review summarizes evidence for a mechanistic link between plant photoprotection and the synthesis of oxylipin hormones as regulators of development and defense. Knockout mutants of Arabidopsis, deficient in various key components of the chloroplast photoprotection system, consistently produced greater concentrations of the hormone jasmonic acid or its precursor 12- oxo-phytodienoic acid (OPDA), both members of the oxylipin messenger family. Characterized plants include several mutants deficient in PsbS (an intrinsic chlorophyll-binding protein of photosystem II) or pigments (zeaxanthin and/or lutein) required for photoprotective thermal dissipation of excess excitation energy in the chloroplast and a mutant deficient in reactive oxygen detoxification via the antioxidant vitamin E (tocopherol). Evidence is also presented that certain plant defenses against herbivores or pathogens are elevated for these mutants. This evidence furthermore indicates that wild-type Arabidopsis plants possess less than maximal defenses against herbivores or pathogens, and suggest that plant lines with superior defenses against abiotic stress may have lower biotic defenses. The implications of this apparent trade-off between abiotic and biotic plant defenses for plant ecology as well as for plant breeding/engineering are explored, and the need for research further addressing this important issue is highlighted.

Characterization of oxylipins and dioxygenase genes in the asexual fungus Aspergillus niger.

BACKGROUND: Aspergillus niger is an ascomycetous fungus that is known to reproduce through asexual spores, only. Interestingly, recent genome analysis of A. niger has revealed the presence of a full complement of functional genes related to sexual reproduction 1. An example of such genes are the dioxygenase genes which in Aspergillus nidulans, have been shown to be connected to oxylipin production and regulation of both sexual and asexual sporulation 234. Nevertheless, the presence of sex related genes alone does not confirm sexual sporulation in A. niger. RESULTS: The current study shows experimentally that A. niger produces the oxylipins 8,11-dihydroxy octadecadienoic acid (8,11-diHOD), 5,8-dihydroxy octadecadienoic acid (5,8-diHOD), lactonized 5,8-diHOD, 8-hydroxy octadecadienoic acid (8-HOD), 10-hydroxy octadecadienoic acid (10-HOD), small amounts of 8-hydroxy octadecamonoenoic acid (8-HOM), 9-hydroxy octadecadienoic acid (9-HOD) and 13-hydroxy octadecadienoic acid (13-HOD). Importantly, this study shows that the A. niger genome contains three putative dioxygenase genes, ppoA, ppoC and ppoD. Expression analysis confirmed that all three genes are indeed expressed under the conditions tested. CONCLUSION: A. niger produces the same oxylipins and has similar dioxygenase genes as A. nidulans. Their presence could point towards the existence of sexual reproduction in A. niger or a broader role for the gene products in physiology, than just sexual development.

Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering.

Soybean lipoxygenase-1 (LOX-1) is used widely as a model for studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1 is organized in a beta-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect of substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations.

Conversion of linoleic acid into novel oxylipins by the mushroom Agaricus bisporus.

Oxylipins are associated with important processes of the fungal life cycle, such as spore formation. Here, we report the formation of FA metabolites in Agaricus bisporus. Incubation of a crude extract of lamellae with linoleic acid (18:2) led to the extensive formation of two oxylipins. They were identified as 8(R)-hydroxy-9Z,12Z-octadecadienoic acid (8-HOD) and 8(R),11 (S)-dihydroxy-9Z,12Z-octadecadienoic acid (8,11-diHOD) by using RP-HPLC, GC-MS, IR, GC-MS analysis of diastereomeric derivatives, and 1H NMR and 13C NMR spectroscopy. Neither compound has been reported before in A. bisporus. Oleic (18:1), alpha-linolenic (18:3n-3), and gamma-linolenic (18:3n-6) acids were converted into their 8-hydroxy derivatives as well, and 18:3n-3 was further metabolized to its 8,11-diol derivative. Reactions with [U-13C]18:2 demonstrated that the compounds 8-HOD and 8,11-diHOD were formed from exogenously supplied 18:2. When [U-13C]8-HOD was supplied, it was not converted into 8,11-diHOD, indicating that it was not an intermediate in the formation of 8,11-diHOD. When a crude extract of A. bisporus was incubated under an atmosphere of 16O2/18O2, the two hydroxyl groups of 8,11-diHOD contained either two 180 atoms or two 60 atoms. Species that contained one of each isotope could not be detected. We propose that the formation of the 8,11-dihydroxy compounds occurs through either an 8,11-endoperoxy, an 8-peroxo free radical, or an 8-hydroperoxy intermediate. In the latter case, the reaction should be catalyzed by dioxygenase with novel specificity.

In vivo excitotoxicity induced by ouabain, a Na+/K+-ATPase inhibitor.

The susceptibility of immature rat brain to neurotoxicity of N-methyl-D-aspartate (NMDA) has provided a widely used paradigm to study excitotoxicity relevant to acute neurodegenerative diseases such as cerebral ischemia. In this study, excitotoxicity was induced via injection of ouabain (1 mM/0.5 microL), a Na+/K+ -ATPase-inhibitor, into neonatal rat brain and compared with NMDA injection. The aim of the study was to induce excitotoxicity secondary to cellular membrane depolarization, thereby more closely mimicking the pathophysiologic processes of ischemia-induced brain injury where NMDA-receptor overstimulation by glutamate follows, not precedes, membrane depolarization. Na+/K+ -ATPase-inhibition caused an acute, 40% +/- 8% decrease of the apparent diffusion coefficient (ADC) of water, as measured using diffusion-weighted magnetic resonance imaging (MRI), and resulted in infarctlike lesions as measured using T2-weighted MRI and histology up to 2 weeks later. Localized one- and two-dimensional 1H-magnetic resonance spectroscopy (MRS) demonstrated that the early excitotoxic diffusion changes were not accompanied by an overall metabolic disturbance. Furthermore, 31P-MRS demonstrated that energy depletion is not a prerequisite for ADC decrease or excitotoxic cell death. Treatment with the NMDA-antagonist MK-801 (1 mg/kg) attenuated the volume of tissue exhibiting a decreased ADC (P < 0.005), demonstrating that the ouabain-induced injury is indeed excitotoxic in nature. The authors argue that, compared with NMDA-injection, ouabain-induced excitotoxicity elicits more appropriate glutamate-receptor overstimulation and is better suited to detect relevant neuroprotection in that it is more sensitive to attenuation of synaptic glutamate levels.

Acute neuronal injury, excitotoxicity, and the endocannabinoid system.

The endocannabinoid system is a valuable target for drug discovery, because it is involved in the regulation of many cellular and physiological functions. The endocannabinoid system constitutes the endogenous lipids anandamide, 2-arachidonoylglycerol and noladin ether, and the cannabinoid CB1 and CB2 receptors as well as the proteins for their inactivation. It is thought that (endo)cannabinoid-based drugs may potentially be useful to reduce the effects of neurodegeneration. This paper reviews recent developments in the endocannabinoid system and its involvement in neuroprotection. Exogenous (endo)cannabinoids have been shown to exert neuroprotection in a variety of in vitro and in vivo models of neuronal injury via different mechanisms, such as prevention of excitotoxicity by CB1-mediated inhibition of glutamatergic transmission, reduction of calcium influx, and subsequent inhibition of deleterious cascades, TNF-alpha formation, and anti-oxidant activity. It has been suggested that the release of endogenous endocannabinoids during neuronal injury might be a protective response. However, several observations indicate that the role of the endocannabinoid system as a general endogenous protection system is questionable. The data are critically reviewed and possible explanations are given.

Oxygenated metabolites of anandamide and 2-arachidonoylglycerol: conformational analysis and interaction with cannabinoid receptors, membrane transporter, and fatty acid amide hydrolase.

This study was aimed at finding structural requirements for the interaction of the acyl chain of endocannabinoids with cannabinoid receptors, membrane transporter protein, and fatty acid amide hydrolase (FAAH). To this end, the flexibility of the acyl chain was restricted by introduction of an 1-hydroxy-2Z,4E-pentadiene system in anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) at various positions using different lipoxygenases. This brought about selectivity and attenuated the binding potency of AEA and 2-AG. Although the displacement constants were modest, 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoyl-N-(2-hydroxyethyl)amine was found to bind selectively to the CB(1) receptor, whereas its 1-arachidonoyl-sn-glycerol analogue and 13(S)-hydroxy-octadeca-9Z,11E-dienoyl-N-(2-hydroxyethyl)amine could selectively bind to the CB(2) receptor. 11(S)-Hydroxy-eicosa-5Z,8Z,12E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did not bind to either receptor, whereas 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did bind to both CB receptors with an affinity similar to that of AEA. All oxygenated anandamide derivatives were good inhibitors of FAAH (low micromolar K(i)) but were ineffective on the AEA transporter. 2-AG rapidly isomerizes into 1(3)-arachidonoyl-sn-glycerol. Both 1- and 3-arachidonoyl-sn-glycerol did not bind to either CB receptor and did not interfere with AEA transport. Thus, after it is isomerized, 2-AG is inactivated, thereby decreasing effective concentrations of 2-AG. Analysis of (1)H NMR spectra revealed that chloroform did not induce notably different conformations in the acyl chain of 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid as compared with water. Molecular dynamics (MD) simulations of AEA and its analogues in the presence of explicit water molecules revealed that a tightly folded conformation of the acyl chain is not the only requirement for CB(1) binding. Structural details of the C(2)-C(15) loop, such as an sp(2) carbon at position 11, are necessary for receptor binding. The MD simulations may suggest that the average orientations of the pentyl tail of AEA and 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine are different from that of the low-affinity, inactive ligands.

Biosynthesis of endocannabinoids and their modes of action in neurodegenerative diseases.

Endocannabinoids are thought to function as retrograde messengers, which modulate neurotransmitter release by activating presynaptic cannabinoid receptors. Anandamide and 2-arachidonoylglycerol (2-AG) are the two best studied endogenous lipids which can act as endocannabinoids. Together with the proteins responsible for their biosynthesis, inactivation and the cannabinoid receptors, these lipids constitute the endocannabinoid system. This system is proposed to be involved in various neurodegenerative diseases such as Parkinson's and Huntington's diseases as well as Multiple Sclerosis. It has been demonstrated that the endocannabinoid system can protect neurons against glutamate excitotoxicity and acute neuronal damage in both in vitro and in vivo models. In this paper we review the data concerning the involvement of the endocannabinoid system in neurodegenerative diseases in which neuronal cell death may be elicited by excitotoxicity. We focus on the biosynthesis of endocannabinoids and on their modes of action in animal models of these neurodegenerative diseases.

Development of a biocatalytic process for the production of c6-aldehydes from vegetable oils by soybean lipoxygenase and recombinant hydroperoxide lyase.

Volatile C6- and C9-aldehydes and alcohols are widely used as food flavors to reconstitute the "fresh green" odor of fruits and vegetables lost during processing. To meet the high demand for natural flavors, an efficient, cheap, and versatile biocatalytic process was developed to produce C6-aldehydes on a large scale. Vegetable oils were converted by soybean lipoxygenase and recombinant hydroperoxide lyase into hexanal and (2E)- or (3Z)-hexenal. In contrast to plant extracts, generally used as enzyme sources, high molar conversions were obtained with recombinant hydroperoxide lyase (50% for hexanal and 26% for hexenal formation), and no side products were formed. Furthermore, recombinant hydroperoxide lyase lacks isomerase activity, allowing production of (3Z)-hexenal, which could not be obtained in previously described processes. Recombinant hydroperoxide lyase is stable and can be stored at 4 degrees C for 1 month without significant loss of activity.

N-acylethanolamines are metabolized by lipoxygenase and amidohydrolase in competing pathways during cottonseed imbibition.

Saturated and unsaturated N-acylethanolamines (NAEs) occur in desiccated seeds primarily as 16C and 18C species with N-palmitoylethanolamine and N-linoleoylethanolamine (NAE 18:2) being most abundant. Here, we examined the metabolic fate of NAEs in vitro and in vivo in imbibed cotton (Gossypium hirsutum) seeds. When synthetic [1-(14)C]N-palmitoylethanolamine was used as a substrate, free fatty acids (FFA) were produced by extracts of imbibed cottonseeds. When synthetic [1-(14)C]NAE 18:2 was used as a substrate, FFA and an additional lipid product(s) were formed. On the basis of polarity, we presumed that the unidentified lipid was a product of the lipoxygenase (LOX) pathway and that inclusion of the characteristic LOX inhibitors nordihydroguaiaretic acid and eicosatetraynoic acid reduced its formation in vitro and in vivo. The conversion of NAE 18:2 in imbibed cottonseed extracts to 12-oxo-13-hydroxy-N-(9Z)-octadecanoylethanolamine was confirmed by gas chromatography-mass spectrometry, indicating the presence of 13-LOX and 13-allene oxide synthase, which metabolized NAE 18:2. Cell fractionation studies showed that the NAE amidohydrolase, responsible for FFA production, was associated mostly with microsomes, whereas LOX, responsible for NAE 18:2-oxylipin production, was distributed in cytosol-enriched fractions and microsomes. The highest activity toward NAE by amidohydrolase was observed 4 to 8 h after imbibition and by LOX 8 h after imbibition. Our results collectively indicate that two pathways exist for NAE metabolism during seed imbibition: one to hydrolyze NAEs in a manner similar to the inactivation of endocannabinoid mediators in animal systems and the other to form novel NAE-derived oxylipins. The rapid depletion of NAEs by these pathways continues to point to a role for NAE metabolites in seed germination.