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Glutathione-S-transferases (GST) catalyze the conjugation of electrophilic compounds to glutathione, thus playing a key role in cell survival and tumor chemoresistance. Cyclopentenone prostaglandins (cyPG) are electrophilic eicosanoids that display potent antiproliferative properties, through multiple mechanisms not completely elucidated. Here we show that the cyPG 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) binds to GSTP1-1 covalently, as demonstrated by mass spectrometry and by the use of biotinylated 15d-PGJ2. Moreover, cyPG inactivate GSTP1-1 irreversibly. The presence of the cyclopentenone moiety is important for these effects. Covalent interactions also occur in cells, in which 15d-PGJ2 binds to endogenous GSTP1-1, irreversibly reduces GST free-thiol content and inhibits GST activity. Protein delivery of GSTP1-1 improves cell survival upon serum deprivation whereas 15d-PGJ2-treated GSTP1-1 displays a reduced protective effect. These results show the first evidence for the formation of stable adducts between cyPG and GSTP1-1 and may offer new perspectives for the development of irreversible GST inhibitors as anticancer agents.

Prostanoids with cyclopentenone structure (cyP) display a potent anti-inflammatory and antiproliferative activity. CyP are reactive compounds, which may modulate cellular functions by multiple mechanisms, including the direct covalent modification of cysteine residues by Michael addition. This interaction displays selectivity since only a subset of cellular proteins is modified by cyP. Several factors have been proposed to influence the selectivity and/or extent of cyP addition to proteins, including determinants related to protein and cyP structure, and levels of cellular thiols, such as glutathione (GSH). Here we have explored the ability of biotinylated cyP analogs to modify several recombinant proteins in vitro, and the influence of GSH in these effects. We have observed that protein modification by cyP is protein- and cyP-selective. Under our conditions, biotinylated 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)-B) was more efficient than biotinylated PGA(1) (PGA(1)-B) at forming adducts with components of the transcription factors NF-kappaB and activator protein-1 (AP-1). However, both biotinylated cyP were nearly equipotent at modifying human GSTP1-1. Interestingly, the presence of GSH differentially modulated the formation of protein-cyP adducts. Under our conditions, GSH reduced the incorporation of cyP into GST, but improved their binding to p50, more intensely in the case of PGA(1)-B. These results evidence the importance of GSH-cyP and/or GSH-protein interactions for the selectivity of protein modification by cyP and suggest a complex role for GSH that may be related to its ability to prevent protein oxidation or induce conformational alterations. This may shed light on the factors involved in the pleiotropic effects of electrophiles with therapeutic potential.

Pathophysiological processes associated with oxidative stress lead to the generation of reactive lipid species. Among them, lipids bearing unsaturated aldehyde or ketone moieties can form covalent adducts with cysteine residues and modulate protein function. Through proteomic techniques we have identified actin as a target for the addition of biotinylated analogs of the cyclopentenone prostaglandins 15-deoxy-Δ 12,14 -PGJ 2 (15d-PGJ 2 ) and PGA 1 in NIH-3T3 fibroblasts. This modification could take place in vitro and mapped to the protein C-terminal end. Other electrophilic lipids, like the isoprostane 8-iso-PGA 1 and 4-hydroxy-2-nonenal, also bound to actin. The C-terminal region of actin is important for monomer–monomer interactions and polymerization. Electron microscopy showed that actin treated with 15d-PGJ 2 or 4-hydroxy-2-nonenal formed filaments which were less abundant and displayed shorter length and altered structure. Streptavidin-gold staining allowed mapping of biotinylated 15d-PGJ 2 at sites of filament disruption. These results shed light on the structural implications of actin modification by lipid electrophiles.

Cyclopentenone prostaglandins (cyPG) with antiinflammatory and antiproliferative properties have been envisaged as leads for the development of therapeutic agents. Because cyPG effects are mediated in part by the formation of covalent adducts with critical signaling proteins, it is important to assess the specificity of this interaction. By using biotinylated derivatives of 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)-B) and PGA(1) (PGA(1)-B) we herein provide novel evidence for the differential selectivity of protein modification by distinct cyPG. The marked quantitative and qualitative differences in the binding of 15d-PGJ(2)-B and PGA(1)-B to cellular proteins were related to a differential reactivity in the presence of glutathione (GSH), both in vitro and in intact cells. Therefore GSH levels may influence not only the intensity but also the specificity of cyPG action.

19 Pags.- 3 Tabls.- 5 Figs., [ES] Caracterizar los suelos y conocer sus propiedades fisicoquímicas es fundamental para interpretar los datos de 137Cs y evaluar las implicaciones que el tipo y uso de suelo, agrario, ganadero o forestal, tienen sobre el comportamiento del radioisótopo en el suelo. En este trabajo se han caracterizado suelos representativos de agrosistemas mediterráneos de montaña mediante la apertura de calicatas, el estudio detallado de sus propiedades fisicoquímicas, y el análisis por difracción de rayos X de perfiles de suelo. Asimismo, para evaluar la repercusión del uso del suelo sobre el contenido y distribución del radioisótopo, sus implicaciones sobre los procesos de pérdida de suelo, y la posible transferencia del 137Cs a las plantas, se han muestreado 13 perfiles seccionados de suelos agrícolas y forestales en Calcisoles, Leptosoles, Regosoles, Gypsisoles y Gleysoles. Se observó un amplio rango de variación tanto de las principales propiedades fisicoquímicas de suelo como del contenido de 137Cs, que se relacionó directa y significativamente con la materia orgánica. Los valores de 137Cs más altos se registraron en Leptosoles no cultivados en suelos forestales, en los que se observó una distribución del radioisótopo exponencialmente decreciente con la profundidad, mientras que los Gypsisoles y Regosoles cultivados registraron las concentraciones más bajas, con perfiles homogéneos en profundidad por efecto del laboreo. Los resultados de esta investigación evidencian el efecto del uso y tipo de suelo en la concentración de la actividad del radioisótopo, y son de interés para la correcta aplicación del 137Cs como técnica para cuantificar la pérdida de suelo en agrosistemas mediterráneos., [EN] An accurate characterization of soils and good knowledge of the soil physicochemical properties is the key to understanding 137Cs data and assesses the effect of soil type and different land uses on the behavior of the radionuclide 137Cs in the soils. In this research, representative soils of mountainous Mediterranean agroecosystems have been characterized through the study of soil pits, a detailed determination of physicochemical soil properties and the analysis by X-ray diffraction of soil profiles. Furthermore, to assess the impact of Iand use on the content and distribution of 137Cs, its implications on the soil Ioss processes, and the 137Cs transfer to plants, 13 sectioned soil profiles in agricultural and forestry soils, in Calcisois, Leptosois, Regosois, Gypsisols and Gleysois, have been sampled. The physicochemical soil properties varied widely and also the content of 137Cs, which was significantly positively correlated with organic matter. The highest values of 137Cs were found in uncultivated Leptosols under forest soils, with the highest concentration in the topsoil and a sharp decay with depth, while the lowest values of 137Cs were found in cultivated Gypsisols and Regosols, in which the radionuclide was distributed homogeneously throughout the soil profile by tillage effect. Information gained with this research demonstrated the effect of soil type and land use in the content of 137Cs on the soil, and is of interest for the correct application of the 137Cs technique to quantify soil loss in Mediterranean agroecosystems., Trabajo financiado por el proyecto de la CICYT, EROMED (CGL2O1 1 -25486/BTE).

In the Acknowledgments of our report we unintentionally omitted to include a sentence stating that J.G. holds a postdoctoral fellowship (JAE-Doc) from the Program “Junta para la Ampliacion de Estudios” co-funded by the Fondo Social Europeo (FSE). The authors regret this omission.

Criado-García, Olga et al., Lafora disease (LD), a fatal neurodegenerative disorder characterized by the presence of intracellular inclusions called Lafora bodies (LBs), is caused by loss-of-function mutations in laforin or malin. Previous studies suggested a role of these proteins in the regulation of glycogen biosynthesis, in glycogen dephosphorylation and in the modulation of the intracellular proteolytic systems. However, the contribution of each of these processes to LD pathogenesis is unclear. We have generated a malin-deficient (Epm2b-/-) mouse with a phenotype similar to that of LD patients. By 3-6 months of age, Epm2b-/- mice present neurological and behavioral abnormalities that correlate with a massive presence of LBs in the cortex, hippocampus and cerebellum. Sixteen-day-old Epm2b-/- mice, without detectable LBs, show an impairment of macroautophagy (hereafter called autophagy), which remains compromised in adult animals. These data demonstrate similarities between the Epm2a-/- and Epm2b-/- mice that provide further insights into LD pathogenesis. They illustrate that the dysfunction of autophagy is a consequence of the lack of laforin-malin complexes and a common feature of both mouse models of LD. Because this dysfunction precedes other pathological manifestations, we propose that decreased autophagy plays a primary role in the formation of LBs and it is critical in LD pathogenesis. © The Author 2011. Published by Oxford University Press. All rights reserved., This work was funded by the Spanish Ministerio de Ciencia eInnovacio ́ n (SAF2008-00226 to S.R.C.; BFU2007-61774 andBFU2010-16031toP.B.;BFU2008-00186toE.K.;SAF2008-01907 to P.S.; and SAF2010-18586 to J.M.S.), theRed de Biobancos del FIS (RD09/0076/00011 to C.N.), theFondo de Investigacio ́ n Sanitaria (PI10/02628 to C.N.;PI070023 to M.S.), the Fundacio ́ n Marato TV3 (Ref. 100130to P.S., E.K. and S.R.C.) and the Centro de Investigacio ́ n Bio-me ́dica en Red de Enfermedades Raras (CIBERER).A.M.G.-C. holds a fellowship from the Fundacio ́ n ConchitaRa ́bago

Knecht, Erwin et al., Lafora disease (LD), a fatal neurodegenerative disorder characterized by intracellular inclusions called Lafora bodies (LBs), is caused by recessive loss-of-function mutations in the genes encoding either laforin or malin. Previous studies suggested a role of these proteins in regulating glycogen biosynthesis, in glycogen dephosphorylation and in the modulation of intracellular proteolytic systems. However, the contribution of each of these processes to LD pathogenesis is unclear. Here we review our recent finding that dysfunction of autophagy is a common feature of both laforin- and malin-deficient mice, preceding other pathological manifestations. We propose that autophagy plays a primary role in LD pathogenesis and is a potential target for its treatment. © 2012 Landes Bioscience., Spanish Ministry of Science and Innovation (SAF2008-00226 to SRdeC, BFU2010-16031 to PB; BFU2008-00186, SAF2008-01907, SAF2010-18586); the Red de Biobancos del FIS (RD09/0076/00011); the Fondo de Investigación Sanitaria (PI10/02628, PI070023); the Fundación Marato TV3 (Ref.100130); the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER); Fundación Conchita Rábago

11 páginas, 6 figuras -- PAGS nros. 4161-4171, Cyclopentenone prostaglandins (cyPG) are reactive eicosanoids that may display anti-inflammatory and antiproliferative actions, possibly offering therapeutic potential. Here we report the identification of members of the aldo-keto reductase (AKR) family as selective targets of the cyPG prostaglandin A1 (PGA1). AKR enzymes metabolize aldehydes and drugs containing carbonyl groups and are involved in inflammation and tumorigenesis. Thus, these enzymes represent a class of targets to develop small molecule inhibitors with therapeutic activity. Molecular modeling studies pointed to the covalent binding of PGA1 to Cys299, close to the active site of AKR, with His111 and Tyr49, which are highly conserved in the AKR family, playing a role in PGA1 orientation. Among AKR enzymes, AKR1B10 is considered as a tumor marker and contributes to tumor development and chemoresistance. We validated the direct modification of AKR1B10 by biotinylated PGA1 (PGA1-B) in cells, and confirmed that mutation of Cys299 abolishes PGA1-B incorporation, whereas substitution of His111 or Tyr49 reduced the interaction. Modification of AKR1B10 by PGA1 correlated with loss of enzymatic activity and both effects were increased by depletion of cellular glutathione. Moreover, in lung cancer cells PGA1 reduced tumorigenic potential and increased accumulation of the AKR substrate doxorubicin, potentiating cell-cycle arrest induced by this hemotherapeutic agent. Our findings define PGA1 as a new AKR inhibitor and they offer a framework to develop compounds that could counteract cancer chemoresistance, This work was supported by grants SAF2009-11642 from MiCInn and RETICS RD07/0064/0007 from ISCIII to D. Pérez-Sala and SAF2006-12713-C02-02 (CICYT) and S-BIO/0214/2006 (CAM) to F. Gago. J. Gayarre was the recipient of fellowships from CSIC (I3P), EMBO, and FEBS (short term fellowships). C. Coderch was supported by a MiCInn FPU (AP2007-01225) fellowship. Part of this work was undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme. Feedback from COST Action CM1001 is appreciated. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact

The cyclopentenone prostaglandin (cyPG) PGA 1 displays potent anti-proliferative and anti-inflammatory effects. Therefore, PGA 1 derivatives are being studied as therapeutic agents. One major mechanism for cyPG action is the modification of protein cysteine residues, the nature of the modified proteins being highly dependent on the structure of the cyPG. Biotinylated cyPGs may aid in the proteomic identification of cyPG targets of therapeutic interest. However, for the identified targets to be relevant it is critical to assess whether biotinylated cyPGs retain the desired biological activity. Here we have explored the anti-inflammatory, anti-proliferative and cell stress-inducing effects of a biotinylated analog of PGA 1 (PGA 1 -biotinamide, PGA 1 -B), to establish its validity to identify cyPG–protein interactions of potential therapeutic interest. PGA 1 and PGA 1 -B displayed similar effects on cell viability, Hsp70 and heme oxygenase-1 induction and pro-inflammatory gene inhibition. Remarkably, PGA 1 -B did not activate PPAR. Therefore, this biotinylated analog can be useful to identify PPAR-independent effects of cyPGs. Protein modification and subcellular distribution of PGA 1 -B targets were cell-type-dependent. Through proteomic and biochemical approaches we have identified a novel set of PGA 1 -B targets including proteins involved in stress response, protein synthesis, cytoskeletal regulation and carbohydrate metabolism. Moreover, the modification of several of the targets identified could be reproduced in vitro. These results unveil novel interactions of PGA 1 that will contribute to delineate the mechanisms for the anti-proliferative and metabolic actions of this cyPG.

11 páginas, 4 figuras, 2 tablas -- PAGS nros. 474-1484, Prostaglandins with cyclopentenone structure (cyPG) display potent antiproliferative actions that have elicited their study as potential anticancer agents. Several natural and synthetic analogs of the cyPG prostaglandin A1 (PGA1) have proven antitumoral efficacy in cancer cell lines and animal models. In addition, PGA1 has been used as an inhibitor of transcription factor NF-κB-mediated processes, including inflammatory gene expression and viral replication. An important determinant for these effects is the ability of cyPG to form Michael adducts with free thiol groups. The chemical nature of this interaction implies that PGA1 could covalently modify cysteine residues in a large number of cellular proteins potentially involved in its beneficial effects. However, only a few targets of PGA1 have been identified. In previous work, we have observed that a biotinylated analog of PGA1 that retains the cyclopentenone moiety (PGA1-B) binds to multiple targets in fibroblasts. Here, we have addressed the identification of these targets through a proteomic approach. Cell fractionation followed by avidin affinity chromatography yielded a fraction enriched in proteins modified by PGA1-B. Analysis of this fraction by SDS-PAGE and LC-MS/MS allowed the identification of the chaperone Hsp90, elongation and initiation factors for protein synthesis and cytoskeletal proteins including actin, tubulin and vimentin. Furthermore, we have characterized the modification of vimentin both in vitro and in intact cells. Our observations indicate that cysteine 328 is the main site for PGA1 addition. These results may contribute to a better understanding of the mechanism of action of PGA1 and the potential of cyPG-based therapeutic strategies, This work was supported by grants SAF2006-03489 (Ministerio de Educación y Ciencia) and 0179/1 (Fundación La Caixa) to D.P.-S. J.G. is the recipient of a pre-doctoral fellowship from the I3P Program (C.S.I.C., Fondo Social Europeo). B. G. is the recipient of a fellowship from the FPI program (Ministerio de Educación y Ciencia). The stay of J.G. at the laboratory of J.T. was supported by EMBO and FEBS short term fellowships. The technical assistance of M.J. Carrasco is gratefully acknowledged

10 Pags.- 4 Tabls.- 2 Figs., A modified irrigation project model (first developed and tested in its original form by Tanji in 1977) is presented which evaluates the impacts of present as well as future alternative land and water resource management on the volume and salt concentration in the irrigation return flow. Modifications were necessary to fit more closely into the prevailing hydrologic and salinity conditions in the Ebro River Basin (Spain), and include the initial and final stored soil water, rim inflows from adjacent hydrologic systems and contributions of gypsum and soluble salts to salt loadings. The model (which requires a total of 25 input variables) was calibrated in a 4,000 ha irrigation project where the salinity problem is not serious (average EC e is 2.2 dS/m), although gypsum is present in large quantities. Results show that model calibration and verification were, respectively, within −2% and +13% of measured surface irrigation return flows (volume and TDS, total dissolved solids) from the 1982 (calibration) and 1983 (verification) hydrological years. The performed sensitivity analysis shows that the key parameter for minimizing salt loading (at present one ha yields about 20 t of salt per year) in irrigation return flows is an increase in water application efficiency (WAE), which presently is only 48%. This increase in WAE will not lead to a substantial rise in the TDS of return flows because of gypsiferous character of these soils., The purpose of this paper is to describe modifications of an irrigation project hydrosalinity model (Tanji 1977) introduced to adapt it more closely to the hydrologic and salinity conditions prevailing in the Ebro River Basin, and to present their calibratio/a and validation steps obtained with data gathered in the Violada Irrigation District (Faci et al. 1985). This model has been programmed in BASIC language on a small Hewlett-Packard computer (HP-85) with 32 K of random access memory (RAM)., This work was supported by a grant from the Spanish-USA Joint Committee and by the CAICYT (Spain).