Direct Dynamic Protein-Affinity Selection Mass-Spectrometry

A new methodology is described enabling the affinity screening of potential ligands towards the human estrogen receptor alpha ligand binding domain (ERα-LBD). In-solution incubation is performed of the analyte and the His-tagged ERα-LBD. The bound complex is immobilized on a nickel-loaded protein-affinity selection column, where after the unbound fraction is removed. The immobilized protein–ligand complex is exposed to a decreased pH value and an increased organic modifier concentration releasing the ligand for MS detection, and precipitating the proteins on a filter positioned between the affinity column and the mass spectrometer. The trapping column can be regenerated for reuse at least 70 times. The advantages of the methodology over existing methodologies are the absence of a pre-concentration as well as a chromatographic separation step, resulting in a significantly shorter analysis time compared to previously described procedures, and in addition, allowing the determination of solutes with unfavorable chromatographic properties. The overall analysis time now can be reduced about 250% to approximately 6 min. Replacing the filters after every measurement results in an intra-day standard deviation of 14.8% and an inter-day standard deviation of 21.3%

Determination of Vitamin A and its Metabolites in Rat Testis: Possible Involvement of Vitamin A in Testicular Toxicity Caused by Molinate

This study was conducted to evaluate the effect of molinate on retinoids homeostasis in rat testis. Molinate was administrated to male Sprague–Dawley rats (200 mg kg−1 in corn oil, ip). Retinoid measurements were made at 6, 12, 48 and 168 h time points after administration. Testis levels of retinoic acid decreased (32 %) in a statistically significant manner at the 12 and 48 h time points. However, retinol and retinaldehyde were not significantly affected by molinate. These results suggest that molinate affects retinoic acid synthesis in testis and could contribute to understanding the molecular mechanism of molinate involved testicular toxicity

Interleukin-10 enhances the intestinal epithelial barrier in the presence of corticosteroids through p38 MAPK activity in Caco-2 monolayers : a possible mechanism for steroid responsiveness in ulcerative colitis

Altres ajuts: 2012 Spanish Gastroenterological Association i CIBER G0034Glucocorticosteroids are the first line therapy for moderate-severe flare-ups of ulcerative colitis. Despite that, up to 60% of patients do not respond adequately to steroid treatment. Previously, we reported that low IL-10 mRNA levels in intestine are associated with a poor response to glucocorticoids in active Crohn's disease. Here, we test whether IL-10 can favour the response to glucocorticoids by improving the TNFα-induced intestinal barrier damage (assessed by transepithelial electrical resistance) in Caco-2 monolayers, and their possible implications on glucocorticoid responsiveness in active ulcerative colitis. We show that the association of IL-10 and glucocorticoids improves the integrity of TNFα-treated Caco-2 cells and that p38 MAPK plays a key role. In vitro, IL-10 facilitates the nuclear translocation of p38 MAPK-phosphorylated thereby modulating glucocorticoids-receptor-α, IL-10-receptor-α and desmoglein-2 expression. In glucocorticoids-refractory patients, p38 MAPK phosphorylation and membrane desmoglein-2 expression are reduced in colonic epithelial cells. These results suggest that p38 MAPK-mediated synergism between IL-10 and glucocorticoids improves desmosome straightness contributing to the recovery of intestinal epithelium and reducing luminal antigens contact with lamina propria in ulcerative colitis. This study highlights the link between the intestinal epithelium in glucocorticoids-response in ulcerative colitis

Stream denitrification across biomes and its response to anthropogenic nitrate loading

Author Posting. © The Author(s), 2008. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature 452 (2008): 202-205, doi:10.1038/nature06686.Worldwide, anthropogenic addition of bioavailable nitrogen (N) to the biosphere is increasing and terrestrial ecosystems are becoming increasingly N saturated, causing more bioavailable N to enter groundwater and surface waters. Large-scale N budgets show that an average of about 20-25% of the N added to the biosphere is exported from rivers to the ocean or inland basins, indicating substantial sinks for N must exist in the landscape. Streams and rivers may be important sinks for bioavailable N owing to their hydrologic connections with terrestrial systems, high rates of biological activity, and streambed sediment environments that favor microbial denitrification. Here, using data from 15N tracer experiments replicated across 72 streams and 8 regions representing several biomes, we show that total biotic uptake and denitrification of nitrate increase with stream nitrate concentration, but that the efficiency of biotic uptake and denitrification declines as concentration increases, reducing the proportion of instream nitrate that is removed from transport. Total uptake of nitrate was related to ecosystem photosynthesis and denitrification was related to ecosystem respiration. Additionally, we use a stream network model to demonstrate that excess nitrate in streams elicits a disproportionate increase in the fraction of nitrate that is exported to receiving waters and reduces the relative role of small versus large streams as nitrate sinks.Funding for this research was provided by the National Science Foundation

Recent developments in protein–ligand affinity mass spectrometry

This review provides an overview of direct and indirect technologies to screen protein–ligand interactions with mass spectrometry. These technologies have as a key feature the selection or affinity purification of ligands in mixtures prior to detection. Specific fields of interest for these technologies are metabolic profiling of bioactive metabolites, natural extract screening, and the screening of libraries for bioactives, such as parallel synthesis libraries and small combichem libraries. The review addresses the principles of each of the methods discussed, with a focus on developments in recent years, and the applicability of the methods to lead generation and development in drug discovery