Soil Fertility and Phosphorus Leaching in Irrigated Calcareous Soils of the Mediterranean Region.

To ensure soil quality and soil health, it is necessary to improve fertilization practices while minimizing environmental impacts. The aim of this study was to record the state of the art on soil fertility related to fertilization management (organic and/or mineral) and to detect environmental challenges in highly productive fields. A soil survey was set up in a new irrigated area (c. 20 years old), in the north-eastern part of Spain, which is mainly devoted to double annual crop rotations of cereals and maize. The area also supports an important animal rearing activity. The survey covered 733 ha of calcareous soils, owned by 35 farmers. At each farm, fertilization management was recorded, and soil was analyzed for nutrients and heavy metals. Multivariate analyses were performed. Total N, P, Cu and Zn, and available P, Cu, Zn and Mn soil concentrations were associated to the use of organic amendments. Heavy metals concentrations were below established thresholds. Available P (Olsen-P) was identified as an indicator of the previously adopted fertilization management and of the potential of P leaching towards deeper soil layers. Regression analyses were performed. A displacement of available P from the uppermost layer (0-0.3 m) occurs in the breakpoint of 86 mg P kg-1 soil. Preventative actions might be established from 53 mg P kg-1 soil due to the slowdown in P immobilization. Our results reinforce the importance of setting up P threshold soil levels for best practices of fertilization, as a basis for sustainable agriculture intensification.

Stress-induced vulnerability to develop cocaine addiction depends on cofilin modulation.

Actin dynamics in dendritic spines can be associated with the neurobiological mechanisms supporting the comorbidity between stress exposure and cocaine increase rewards. The actin cytoskeleton remodeling in the nucleus accumbens (NA) has been implicated in the expression of stress-induced cross-sensitization with cocaine. The present study evaluates the involvement of cofilin, a direct regulator of actin dynamics, in the impact of stress on vulnerability to cocaine addiction. We assess whether the neurobiological mechanisms that modulate repeated-cocaine administration also occur in a chronic restraint stress-induced cocaine self-administration model. We also determine if chronic stress induces alterations in dendritic spines through dysregulation of cofilin activity in the NA core. Here, we show that the inhibition of cofilin expression in the NA core using viral short-hairpin RNA is sufficient to prevent the cocaine sensitization induced by chronic stress. The reduced cofilin levels also impede a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor surface expression enhancement and promote the reduction of head diameter in animals pre-exposed to stress after a cocaine challenge in the NA core. Moreover, downregulation of cofilin expression prevents facilitation of the acquisition of cocaine self-administration (SA) in male rats pre-exposed to chronic stress without modifying performance in sucrose SA. These findings reveal a novel, crucial role for cofilin in the neurobiological mechanisms underpinning the comorbidity between stress exposure and addiction-related disorders.

Sex steroid metabolism in benign and malignant intact prostate biopsies: individual profiling of prostate intracrinology.

In vitro studies reveal that androgens, oestrogens, and their metabolites play a crucial role in prostate homeostasis. Most of the studies evaluated intraprostatic hormone metabolism using cell lines or preprocessed specimens. Using an ex vivo model of intact tissue cultures with preserved architecture, we characterized the enzymatic profile of biopsies from patients with benign prostatic hyperplasia (BPH) or cancer (PC), focusing on 17ß-hydroxy-steroid-dehydrogenases (17ß-HSDs) and aromatase activities. Samples from 26 men who underwent prostate needle core biopsies (BPH n = 14; PC n = 12) were incubated with radiolabeled (3)H-testosterone or (3)H-androstenedione. Conversion was evaluated by TLC separation and beta-scanning of extracted supernatants. We identified three major patterns of conversion. The majority of BPHs revealed no active testosterone/oestradiol conversion as opposed to prostate cancer. Conversion correlated with histology and PSA, but not circulating hormones. Highest Gleason scores had a higher androstenedion-to-testosterone conversion and expression of 17ß-HSD-isoenzymes-3/5. Conclusions. We developed an easy tool to profile individual intraprostatic enzymatic activity by characterizing conversion pathways in an intact tissue environment. In fresh biopsies we found that 17ß-HSD-isoenzymes and aromatase activities correlate with biological behaviour allowing for morphofunctional phenotyping of pathology specimens and clinical monitoring of novel enzyme-targeting drugs.

Androgen modulation of pro-inflammatory and anti-inflammatory cytokines during preadipocyte differentiation.

BACKGROUND: Macrophages and adipocytes contribute to release of cytokines resulting in the chronic inflammatory profile of the metabolic syndrome. The local increase of proinflammatory cytokines impairs adipogenesis, resulting in formation of dysfunctional adipocytes that are unable to store and handle lipids. The altered lipid fluxes in/from adipocytes affect whole-body metabolism. We investigated the role of androgens on adipocyte-derived proinflammatory and anti-inflammatory cytokines during preadipocyte differentiation. MATERIALS AND METHODS: Various differentiation methods were used to obtain full conversion of 3T3-L1 into mature adipocytes. The degree of adipocyte conversion in the presence/absence of dihydrotestosterone (DHT) was analyzed by measuring intracellular triglycerides (Oil Red O staining). The effects of DHT administration on interleukin 1ß (IL-1ß), IL-2, IL-6, IL-10, IL-12, interferon γ (IFNγ) and tumor necrosis factor α (TNFα) secretion was measured at days 0, 4, 6 and 8 of differentiation using the SearchLight multiplex protein array. RESULTS: DHT regulates a number of cytokines in committed and mature 3T3-L1 adipocytes. IL-1ß and TNFα were readily suppressed at the very early stages of differentiation. IFNγ release was inhibited at day 4, but the effect was no longer detectable on day 8. IL-6 and IL-12 were significantly reduced at day 8 of differentiation. Conversely, the differentiation-dependent increase of IL-2 and IL-10 was further stimulated by DHT since day 0. CONCLUSIONS: We provide evidence that androgens promote an anti-inflammatory profile that parallels the acquisition of a functional adipocyte phenotype. The crosstalk between androgens, adipocyte-derived mediators of inflammation and intracellular lipid fluxes could have profound implications on metabolism of men with obesity and metabolic syndrome.

Pre-receptorial regulation of steroid hormones in bone cells: insights on glucocorticoid-induced osteoporosis.

In the past decades, concern on glucocorticoid-induced osteoporosis has increased with the widespread use of exogenous glucocorticoids (GC). Mature bone-forming cells (osteoblasts) are considered to be the principal site of action of GC in the skeleton. More likely, it is the entire cellular and molecular network surrounding these cells that is targeted by pharmacological doses of GC. Not only osteoblast and osteocyte metabolism, but the whole differentiation of mesenchymal stem cell toward the osteoblast lineage has been proven to be sensitive to GC. The effects of GC on this process are different according to the stage of differentiation of bone cell precursors. The presence of intact GC signalling is crucial for normal bone development and physiology, as opposed to the detrimental effect of high dose exposure. Both the physiological and pharmacological effects of GC are locally modulated by the activity of the 11beta-hydroxysteroid dehydrogenase 1 (HSD1) that acts primarily as a glucocorticoid activator converting the inactive glucocorticoid (cortisone) into the active hormone (cortisol). We reviewed the metabolic and differentiation pathways controlled by GC signalling. These data have been merged with the recent evidences that 11beta-HSD1 exert an important role by regulating the vulnerability of bone cells to GC. The different kinetics of 11beta-HSD1 at various stage of differentiation and the GC-dependency of enzymatic activity have been presented.

A slot blot procedure for the measurement of yessotoxins by a functional assay.

We originally developed a functional assay for the detection of yessotoxins (YTX) based on its capacity to induce dose-dependent changes in cellular levels of two marker proteins, consisting of E-cadherin and an E-cadherin fragment (ECRA100) in epithelial cells. The procedure is time-consuming and we have shortened it by a slot blot format, using antibodies recognizing two different epitopes of E-cadherin (HECD-1 and C20820), thereby discriminating those markers. The best performing membrane under our conditions, in terms of binding capacity and even absorption of proteins, was a positively charged nylon membrane. Treatment of the membrane with 0.5mug of Ab/ml was appropriate for maximal detection of antigens by our slot blot procedure with both HECD-1 and C20820 antibodies. The treatment of cells with YTX, resulting in a relative increase in the cellular levels of ECRA100, led to a dose-dependent increase of the signal detected by Ab HECD-1 without a concomitant increase in the signal detected by Ab C20820 in our slot blot format, and the concentrations of YTX were correlated to both the increase of the signal detected through Ab HECD-1 and to the decrease in the ratio of the signals obtained with the two Abs (C20820 over HECD-1). Upon analyses of extracts from cells treated with shellfish samples, we could detect and quantify YTX in naturally contaminated materials. The slot blot format of our functional assay allows a substantial shortening of its analytical step (about seven hr, as compared to the two working days of the original method), providing YTX measurements that are accurate but show large standard deviations.

Oral administration of yessotoxin stabilizes E-cadherin in mouse colon.

YTX has been shown to disrupt the E-cadherin-catenin system in cultured epithelial cells, raising some concern that ingestion of seafood contaminated by YTX might favour tumour spreading and metastasis formation in vivo. In order to probe whether YTX might affect cadherin systems in vivo, we have set up a study involving repeated oral dosing of the toxin in mice (1mg/kg/day, for 7 days) and analysis of E-cadherin and N-cadherin in tissue extracts obtained at the end of the dosing scheme, as well as 1 and 3 months after YTX administration. We found that the E-cadherin pools obtained from lung and kidney were not altered by YTX in any of our experimental conditions. Extracts from mouse colon contained intact E-cadherin and an E-cadherin fragment of about 90 kDa (ECRA(90)), displaying a molecular alteration resembling that caused by YTX in cultured cells. We found that the relative proportion of ECRA(90), as compared to intact E-cadherin, was higher in colon extracts from control mice than from YTX-treated animals, indicating that oral administration of YTX to mice stabilizes E-cadherin of mouse colon. No significant difference could be detected in samples prepared from colons obtained 30 or 90 days after termination of YTX treatment. Oral administration of YTX to mice did not lead to a significant increase in the fragments of E-cadherin detectable in serum, neither it altered the N-cadherin pool of mouse heart. Electron microscopy analysis showed no substantial ultrastructural differences between controls and YTX-treated mice. Our findings show that ingestion of food contaminated by YTX poses a low risk of disruption of the E-cadherin system in vivo.

Probing the influence of cis-trans isomers on model lipid membrane fluidity using cis-parinaric acid and a stop-flow technique.

Stop-flow experiments exploiting the fluorescence of cis-parinaric acid in monounsaturated lipid vesicles allow the model membrane behaviour, notably the membrane fluidity, to be correlated to the cis:trans lipid ratios.

Comparison of phosphatidylcholine vesicle properties related to geometrical isomerism.

Glycerophosphatidylcholine containing trans-unsaturated fatty acid residues was prepared by reaction of the corresponding naturally occurring cis lipid with photochemically generated thiyl radicals. This modified lipid was chosen as the simplest model for gaining some insights of the complex scenario of membrane formation, in connection with the role of lipid geometry and the predominance of cis lipids in eukaryotic cells. The critical aggregation concentration for the spontaneous formation of vesicles was determined for cis and trans isomers with cis-parinaric acid used as a fluorescent probe and it was found to be similar for both lipids. Vesicle dimensions were investigated by light scattering and electron microscopy, and the type of fatty acid residues influenced the vesicle diameter, with a decrease along the series cis > trans > saturated. Fluorescence measurement of dye release from trans and cis vesicles showed also a different permeability. A picture emerged of the geometrical isomer preference in cells as a process driven by natural selection during the life evolution of different organisms, both in terms of compartment dimensions and membrane functionality.

Functional assay to measure yessotoxins in contaminated mussel samples.

Yessotoxin (YTX) treatment of MCF-7 cells results in the accumulation of a 100-kDa fragment of E-cadherin (ECRA(100)) without a parallel loss of the intact protein in cytosoluble extracts. As a consequence, concentration-dependent increases in the total immunoreactivity detectable by anti-E-cadherin antibodies relative to controls (RTI) and in the relative immunoreactivity of ECRA(100) (RI) are observed. These responses have been exploited to develop a functional assay to measure YTX in samples from contaminated mussels by a three-step procedure, consisting of (i) treatment of MCF-7 cells with YTX standard in the concentration range 0-1nM and of unknown samples; (ii) preparation of cellular extracts, fractionation of proteins by polyacrylamide gel electrophoresis under denaturing conditions, and immunoblotting with anti-E-cadherin antibodies, followed by densitometric analyses of autoradiographies and calculation of RI of ECRA(100) and of RTI of the samples; and (iii) interpolation of the YTX concentrations in unknown samples on standard curves, by the RI of ECRA(100) and the RTI of the samples. The procedure has been used to measure yessotoxins in contaminated mussel samples, and the results obtained show that this functional assay is very sensitive (limit of detection of about 100ng equivalent YTX/g of digestive gland), and robust, as (i) it is insensitive to matrix effects in the range of toxin concentrations relevant for risk assessment to protect humans from exposure to YTX, (ii) calculations are based on a molecular parameter (the RI of ECRA(100)) which is not affected by errors in sample preparation, (iii) it can be performed by the use of antibodies commercially available from different companies, and (iv) it does not show an absolute need of calibration by a pure standard within each assay.