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Early activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death
No full textOxidative stress caused by hydrogen peroxide (H2O2) triggers the hypersensitive response of plants to pathogens. Here, short pulses of H2O2 are shown to cause death of lentil (Lens culinaris) root protoplasts. Dead cells showed DNA fragmentation and ladder formation, typical hallmarks of apoptosis (programmed cell death). DNA damage was evident 12 h after the H2O2 pulse and reached a maximum 12 h later. The commitment of cells to apoptosis caused by H2O2 was characterized by an early increase of lipoxygenase activity, of ultraweak luminescence and of membrane lipid peroxidation, which reached 720, 350 and 300% of controls, respectively, at 6 h after H2O2 treatment. Increased lipoxygenase activity was paralleled by an increase of its protein and mRNA level. Lipoxygenase inhibitors nordihydroguaiaretic acid, eicosatetraynoic acid and plamitoyl ascorbate prevented H2O2-induced DNA fragmentation and ultraweak luminescence, only when added together with H2O2, but not when added 8 h afterwards. Inhibitory anti-lipoxygenase monoclonal antibodies, introduced into the protoplasts by electroporation, protected cells against H2O2-induced apoptosis. On the other hand, lentil lipoxygenase products 9- and 13-hydroperoxy-octadecadienoic acids and their reduced alcohol derivatives were able to force the protoplasts into apoptosis. Altogether, these findings suggest that early activation of lipoxygenase is a key element in the execution of apoptosis induced by oxidative stress in plant cells, in a way surprisingly similar to that observed in animal cells
STRUCTURAL AND FUNCTIONAL PROPERTIES OF LIPOXYGENASE IN SOLUTION
No full textINTRODUCTION: Lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12; LOX-1) belongs to a family of non-heme, non-sulfur iron dioxygenases that take part in the metabolism of polyunsatured fatty acids catalysing their conversion into conjugates hydroperoxides. Soybean LOX-1 has been widely used as a model for studying the functional and structural properties of the homologous family of LOXs. The crystallographic structure of soybean LOX-1 revealed that the protein is organised in two domains (1), a beta-sheet N-terminal domain and a larger, mostly helical C-terminal domain. Mammalian LOXs lack the 30 kDa N-terminal domain present in soybean LOX-1 and other related plant lipoxygenases. Limited proteolysis by tryptic digestion of the native soybean LOX-1 is known to produce a catalytically active 60 kDa fragment, which has been named “mini-LOX” (2). Here, we describe the overall structural characterisation of native soybean LOX-1 and of mini-LOX using the Small Angle X-Ray Scattering (SAXS) approach. The native enzyme and the 60 kDa active fragment were studied both in the presence and in the absence of the inhibitor eicosatetraynoic acid (ETYA) and of glycerol, qualifying the low resolution structures of the enzyme in this different conditions. In parallel, the kinetic properties of the different forms were determined.MATERIALS AND METHODS: Soybean LOX-1 was purified from seeds as described elsewhere (3). Limited proteolysis by tryptic digestion was carried out as reported (2). The isolation of miniLOX was obtained by fast-protein liquid chromatography (FPLC, size exclusion column Superdex-75) using an AKTA Explorer apparatus (Parmacia, Uppsala, Sweden). Dioxygenase activity was calculated as already described (4). SAXS measurements were performed at the synchrotron radiation beam line D24 in the DCI storage ring of LURE (Laboratoire pour l’Utilisation du Rayonnement Electromagnetique, Orsay-Paris). The radius of gyration (Rg) was determined by interpolating the SAXS data on the basis of Guinier approximation I(Q) = I(0) exp(-Rg2Q2/3), where Q = (4sin)/ and I(0) is the scattering intensity at zero scattering angle. The distance distribution function p(r) and the maximum dimension of the protein (Dmax) have been determined using the indirect transform method as implemented in the program GNOM. The theoretical scattering intensities were computed from the atomic coordinates of the crystal structure using the program CRYSOL. The ab initio shape determinations were performed with the dummy atom model (DAM) method (5) using the program DAMMIN. RESULTS: The analysis of the SAXS patterns of the native soybean LOX-1 yielded a value of the radius of gyration, calculated from the Guinier analysis of the scattering intensity, of Rg = 30.0 0.3 Å. Further calculations, using the pair distribution function p(r) of the native enzyme, yielded a value for the maximum dimension of the molecule (Dmax) of 95 5 Å and a value of the radius of gyration of 30.1 0.2 Å, very close to that derived from the Guinier approximation. The comparison between the experimental SAXS data and the theoretical one, obtained from the atomic coordinates of soybean LOX-1 [1F8N.pdb], yields a very good agreement ( =1.747). In parallel to the above-mentioned modelling, ab initio calculations of the overall shape of the protein from the SAXS pattern were performed using the program DAMMIN, no particular conditions of oblateness and symmetry of the particle shape were imposed as constraints. Moreover, the superposition of the solution structure of soybean LOX-1 obtained with the crystal structure clearly shows the strong similarity between the two models. Taken together, these data indicate that the overall shape of the protein does not undergo any significant[...
Structural Stability of Soybean Lipoxygenase-1 in Solution as Probed by Small Angle X-ray Scattering
No full textSoybean 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.[...
Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering
No full textSoybean 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. (c) 2005 Elsevier Ltd. All rights reserved
Acute neuronal injury, excitotoxicity, and the endocannabinoid system
No full textThe 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
Structural Characterization of Soybean Lipoxygenase-1 in Solution: the Interaction with Lipids
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Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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