Carbohydrate binding specificities and crystal structure of the cholera toxin-like B-subunit from Citrobacter freundii

Enterotoxigenic Escherichia coli and Vibrio cholerae are well known causative agents of severe diarrheal diseases. Both pathogens produce AB₅ toxins, with one enzymatically active A-subunit and a pentamer of receptor-binding B-subunits. The primary receptor for both B-subunits is the GM1 ganglioside (Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer), but the B-subunits from porcine isolates of E. coli also bind neolacto-(Galβ4GlcNAcβ-)terminated glycoconjugates and the B-subunits from human isolates of E. coli (hLTB) have affinity for blood group A type 2-(GalNAcα3(Fucα2)Galβ4GlcNAcβ-)terminated glycoconjugates.     

Application of saturation dye 2D-DIGE proteomics to characterize proteins modulated by oxidized low density lipoprotein treatment of human macrophages

Macrophages are believed to play a crucial role in atherogenesis and atherosclerotic plaque progression, mainly through their role in the accumulation of large amounts of cholesteryl ester and foam cell formation after the uptake into the arterial intima of oxidized LDL (oxLDL) particles known to be proatherogenic. The aim of this study was to use a differential proteomic approach to identify the response of human monocyte-derived macrophages after treatment with oxLDL for 24 h. Mass spectrometry analysis (MALDI-TOF) of 2D-DIGE gels made it possible to identify 9 intracellular and 3 secreted proteins that were up-regulated, 11 intracellular and 1 secreted proteins that were down-regulated, and 2 secreted proteins that were induced. This methodological approach not only confirmed the differential expression levels of proteins known to be regulated by oxLDL in macrophages, such as catalase and pyruvate kinase, but also identified oxLDL modulation of other proteins for the first time, including heat shock proteins (HSP) and Actin cytoskeletal proteins. Semiquantitative Western blot confirmed their role. The HSPs identified included heat shock cognate 71 kDa protein (Hsc70), 75 kDa glucose-regulated protein (GRP75), heat shock 70 kDa protein (Hsp70), and 60 kDa (Hsp60) proteins. These highly conserved intracellular protein chaperones, commonly seen in atherosclerotic plaques, appear to participate in protection against cellular stress. Interestingly, oxLDL also modulated several F-Actin capping proteins involved in Actin polymerization and motility: gelsolin, CapG, and CapZ. In conclusion, we have demonstrated the effects of oxLDL in the modulation of several proteins in human macrophages and established a functional profile of the human macrophage during the atherosclerotic process.     

Boolean models of biosurfactants production in Pseudomonas fluorescens

Cyclolipopeptides (CLPs) are biosurfactants produced by numerous Pseudomonas fluorescens strains. CLP production is known to be regulated at least by the GacA/GacS two-component pathway, but the full regulatory network is yet largely unknown. In the clinical strain MFN1032, CLP production is abolished by a mutation in the phospholipase C gene (plcC) and not restored by plcC complementation. Their production is also subject to phenotypic variation. We used a modelling approach with Boolean networks, which takes into account all these observations concerning CLP production without any assumption on the topology of the considered network. Intensive computation yielded numerous models that satisfy these properties. All models minimizing the number of components point to a bistability in CLP production, which requires the presence of a yet unknown key self-inducible regulator. Furthermore, all suggest that a set of yet unexplained phenotypic variants might also be due to this epigenetic switch. The simplest of these Boolean networks was used to propose a biological regulatory network for CLP production. This modelling approach has allowed a possible regulation to be unravelled and an unusual behaviour of CLP production in P. fluorescens to be explained.     

Cell uptake and radiotoxicity studies of an nuclear localization signal peptide-intercalator conjugate labeled with [99mTc(CO)3]+

A trifunctional bioconjugate consisting of the SV40 nuclear localization signal (NLS) peptide, an aliphatic triamine ligand, and the DNA intercalating pyrene has been synthesized and quantitatively labeled with [(99m)Tc(OH(2))(3)(CO)(3)](+). The radiotoxicity of the resulting nucleus-targeting radiopharmaceutical on B16F1 mouse melanoma cells has been investigated to evaluate the activity of Auger and Coster-Kronig electrons on the viability of cells. We found a dose-dependent significant radiotoxicity of the nucleus-targeting radiopharmaceutical clearly related to the low energy decay of (99m)Tc. These principal results imply a possible therapeutic strategy based on the use of the low-energy Auger electron-emitting (99m)Tc radionuclide attached to nucleus-targeting molecules and comprising an intercalator. Highly efficient DNA targeting vectors could complement the usual role of (99m)Tc in diagnostic applications. The Auger electrons emitted by the (99m)Tc nuclide induce DNA damage leading ultimately, through a mitotic catastrophe pathway, to necrotic cell death. Non-DNA-targeting (99m)Tc complexes display much lower radiotoxicity.     

Association of the Lipoprotein Receptor SCARB1 Common Missense Variant rs4238001 with Incident Coronary Heart Disease

BackgroundPrevious studies in mice and humans have implicated the lipoprotein receptor SCARB1 in association with atherosclerosis and lipid levels. In the current study, we sought to examine association of SCARB1 missense single nucleotide polymorphism (SNP) rs4238001 with incident coronary heart disease (CHD).ConclusionSCARB1 missense rs4238001 is statistically significantly associated with incident CHD across a large population of multiple race/ethnic groups.     

A Soluble Fucose-Specific Lectin from Aspergillus fumigatus Conidia - Structure,Specificity and Possible Role in Fungal Pathogenicity

Aspergillus fumigatus is an important allergen and opportunistic pathogen. Similarly to many other pathogens, it is able to produce lectins that may be involved in the host-pathogen interaction. We focused on the lectin AFL, which was prepared in recombinant form and characterized. Its binding properties were studied using hemagglutination and glycan array analysis. We determined the specificity of the lectin towards l-fucose and fucosylated oligosaccharides, including α1-6 linked core-fucose, which is an important marker for cancerogenesis. Other biologically relevant saccharides such as sialic acid, d-mannose or d-galactose were not bound. Blood group epitopes of the ABH and Lewis systems were recognized, Le^Y being the preferred ligand among others. To provide a correlation between the observed functional characteristics and structural basis, AFL was crystallized in a complex with methyl-α,l-selenofucoside and its structure was solved using the SAD method. Six binding sites, each with different compositions, were identified per monomer and significant differences from the homologous AAL lectin were found. Structure-derived peptides were utilized to prepare anti-AFL polyclonal antibodies, which suggested the presence of AFL on the Aspergillus’ conidia, confirming its expression in vivo. Stimulation of human bronchial cells by AFL led to IL-8 production in a dose-dependent manner. AFL thus probably contributes to the inflammatory response observed upon the exposure of a patient to A. fumigatus. The combination of affinity to human epithelial epitopes, production by conidia and pro-inflammatory activity is remarkable and shows that AFL might be an important virulence factor involved in an early stage of A. fumigatus infection.