Protection of the vascular endothelium in experimental situations

One of the factors proposed as mediators of vascular dysfunction observed in diabetes is the increased generation of reactive oxygen species (ROS). This provides support for the use of antioxidants as early and appropriate pharmacological intervention in the development of late diabetic complications. In streptozotocin (STZ)-induced diabetes in rats we observed endothelial dysfuction manifested by reduced endothelium-dependent response to acetylcholine of the superior mesenteric artery (SMA) and aorta, as well as by increased endothelaemia. Changes in endothelium-dependent relaxation of SMA were induced by injury of the nitric oxide radical (·NO)-signalling pathway since the endothelium-derived hyperpolarising factor (EDHF)-component of relaxation was not impaired by diabetes. The endothelial dysfunction was accompanied by decreased ·NO bioavailabity as a consequence of reduced activity of eNOS rather than its reduced expression. The results obtained using the chemiluminiscence method (CL) argue for increased oxidative stress and increased ROS production. The enzyme NAD(P)H-oxidase problably participates in ROS production in the later phases of diabetes. Oxidative stress was also connected with decreased levels of reduced glutathione (GSH) in the early phase of diabetes. After 10 weeks of diabetes, adaptational mechanisms probably took place because GSH levels were not changed compared to controls. Antioxidant properties of SMe1EC2 found in vitro were partly confirmed in vivo. Administration of SMe1EC2 protected endothelial function. It significantly decreased endothelaemia of diabetic rats and improved endothelium-dependent relaxation of arteries, slightly decreased ROS-production and increased bioavailability of ·NO in the aorta. Further studies with higher doses of SMe1EC2 may clarify the mechanism of its endothelium-protective effect in vivo

Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases

We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 Å), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer

The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis

S-D logic-informed customer engagement: Integrative framework, revised fundamental propositions, and application to CRM

Advance online in 2016</p

Maternity testing in a chimerical child

Abstract. Human congenital chimerism is due to the coexistence of two genetically different cell lines either in the whole body or limited to the blood. We present a case of maternity in which the alleged child has ambiguous genitalia, and chimerism was suspected. The maternity was dubious and the father was not available for the study. STR typing of the child revealed the presence of X an Y chromosomes at Amelogenin locus and a double maternal and paternal alleles contribution in certain autosomical loci. We could not exclude maternal relationship between the alleged mother and the child. Molecular analysis performed with highly discriminating STR systems allowed us to clarify the origin of a chimerical individual.

Analysing chemical-induced changes in macroinvertebrate communities in aquatic mesocosm experiments: a comparison of methods

Mesocosm experiments that study the ecological impact of chemicals are often analysed using the multivariate method ‘Principal Response Curves’ (PRCs). Recently, the extension of generalised linear models (GLMs) to multivariate data was introduced as a tool to analyse community data in ecology. Moreover, data aggregation techniques that can be analysed with univariate statistics have been proposed. The aim of this study was to compare their performance. We compiled macroinvertebrate abundance datasets of mesocosm experiments designed for studying the effect of various organic chemicals, mainly pesticides, and re-analysed them. GLMs for multivariate data and selected aggregated endpoints were compared to PRCs regarding their performance and potential to identify affected taxa. In addition, we analysed the inter-replicate variability encountered in the studies. Mesocosm experiments characterised by a higher taxa richness of the community and/or lower taxonomic resolution showed a greater inter-replicate variability, whereas variability decreased the more zero counts were encountered in the samples. GLMs for multivariate data performed equally well as PRCs regarding the community response. However, compared to first axis PRCs, GLMs provided a better indication of individual taxa responding to treatments, as separate models are fitted to each taxon. Data aggregation methods performed considerably poorer compared to PRCs. Multivariate community data, which are generated during mesocosm experiments, should be analysed using multivariate methods to reveal treatment-related community-level responses. GLMs for multivariate data are an alternative to the widely used PRC

Analysing chemical-induced changes in macroinvertebrate communities in aquatic mesocosm experiments: a comparison of methods

Mesocosm experiments that study the ecological impact of chemicals are often analysed using the multivariate method ‘Principal Response Curves’ (PRCs). Recently, the extension of generalised linear models (GLMs) to multivariate data was introduced as a tool to analyse community data in ecology. Moreover, data aggregation techniques that can be analysed with univariate statistics have been proposed. The aim of this study was to compare their performance. We compiled macroinvertebrate abundance datasets of mesocosm experiments designed for studying the effect of various organic chemicals, mainly pesticides, and re-analysed them. GLMs for multivariate data and selected aggregated endpoints were compared to PRCs regarding their performance and potential to identify affected taxa. In addition, we analysed the inter-replicate variability encountered in the studies. Mesocosm experiments characterised by a higher taxa richness of the community and/or lower taxonomic resolution showed a greater inter-replicate variability, whereas variability decreased the more zero counts were encountered in the samples. GLMs for multivariate data performed equally well as PRCs regarding the community response. However, compared to first axis PRCs, GLMs provided a better indication of individual taxa responding to treatments, as separate models are fitted to each taxon. Data aggregation methods performed considerably poorer compared to PRCs. Multivariate community data, which are generated during mesocosm experiments, should be analysed using multivariate methods to reveal treatment-related community-level responses. GLMs for multivariate data are an alternative to the widely used PRC