Insights into molecular plasticity of choline binding proteins (pneumococcal surface proteins) by SAXS

Phosphocholine moieties decorating the pneumococcal surface are used as a docking station for a family of modular proteins, the so-called choline binding proteins or CBPs. Choline recognition is essential for CBPs function and may also be a determinant for their quaternary structure. There is little knowledge about modular arrangement or oligomeric structures in this family. Therefore, we have used the small angle X-ray scattering (SAXS) technique combined with analytical ultracentrifugation in order to model the three-dimensional envelope of two highly different CBPs: the phage encoded Cpl-1 lysozyme and the pneumococcal phosphorylcholine esterase Pce. Both enzymes have an N-terminal catalytic module and a C-terminal choline-binding module (CBM) that attaches them to the bacterial surface and comprises six and ten sequence repeats in Cpl-1 and Pce, respectively. SAXS experiments have shown an inherent conformational plasticity in Cpl-1 that accounts for the different relative position of these regions in the solution and crystal structures. Dimerization of Cpl-1 upon choline binding has been also visualised for the first time, and monomer-monomer interactions take place through the first CBR where a non-canonical choline binding site has now been identified. This mode of association seems to be independent of the absence or presence of the Cpl-1 catalytic module and reveals that the arrangement of the monomers differs from that previously found in the isolated CBM dimer of pneumococcal LytA amidase. In contrast, Pce displays the same modular disposition in the solution and crystal structures, and remains almost invariant upon choline binding. The present results suggest that protein dimerization and duplication of CBRs may be alternative but not equivalent ways of improving cell wall recognition by CBPs, since they provide different interaction geometries for choline residues present in (lipo)teichoic acids.     

Molecular basis of the antimutagenic activity of the house-cleaning inosine triphosphate pyrophosphatase RdgB from Escherichia coli

Inosine triphosphate pyrophosphatases, which are ubiquitous house-cleaning enzymes, hydrolyze noncanonical nucleoside triphosphates (inosine triphosphate (ITP) and xanthosine triphosphate (XTP)) and prevent the incorporation of hypoxanthine or xanthine into nascent DNA or RNA. Here we present the 1.5-Å-resolution crystal structure of the inosine triphosphate pyrophosphatase RdgB from Escherichia coli in a free state and in complex with a substrate (ITP + Ca^2 +) or a product (inosine monophosphate (IMP)). ITP binding to RdgB induced a large displacement of the α1 helix, closing the enzyme active site. This positions the conserved Lys13 close to the bridging oxygen between the α- and β-phosphates of the substrate, weakening the P_α-O bond. On the other side of the substrate, the conserved Asp69 is proposed to act as a base coordinating the catalytic water molecule. Our data provide insight into the molecular mechanisms of the substrate selectivity and catalysis of RdgB and other ITPases.     

Impact of population structure on genetic diversity of a potential vaccine target in the canine hookworm (Ancylostoma caninum)

Ancylostoma caninum is a globally distributed canine parasitic nematode. To test whether positive selection, population structure, or both affect genetic variation at the candidate vaccine target Ancylostoma secreted protein 1 (asp-1), we have quantified the genetic variation in A. caninum at asp-1 and a mitochondrial gene, cytochrome oxidase subunit 1 (cox-1), using the statistical population analysis tools found in the SNAP Workbench. The mitochondrial gene cox-1 exhibits moderate diversity within 2 North American samples, comparable to the level of variation observed in other parasitic nematodes. The protein coding portion for the C-terminal half of asp-1 shows similar levels of genetic variation in a Wake County, North Carolina, sample as cox-1. Standard tests of neutrality provide little formal evidence for selection acting on this locus, but haplotype networks for 2 of the exon regions have significantly different topologies, consistent with different evolutionary forces shaping variation at either end of a 1.3-kilobase stretch of sequence. Evidence for gene flow among geographically distinct samples suggests that the mobility of hosts of A. caninum is an important contributing factor to the population structure of the parasite.     

Mathematical modelling of human mesenchymal stem cell proliferation and differentiation inside artificial porous scaffolds

We present a mathematical model for the proliferation and differentiation of human mesenchymal stem cells grown inside artificial porous scaffolds under different oxygen concentrations. The values of parameters in the model are determined by comparison of the model solutions to published experimental data, complemented with a sensitivity analysis of the fitted parameters. It is shown that a simple hypothesis whereby the secretion of extra-cellular matrix (ECM) is oxygen dependent and that ECM itself stimulates cell proliferation is sufficient to explain the experimental data, which under conditions of low oxygen reveals increased total cell proliferation, upregulation of the numbers of undifferentiated cells, and extended lag phase. These results may help further to understand how cells proliferate inside artificial materials and are of importance to the field of tissue engineering.     

Lack of micronuclei formation in bone marrow of rats after repeated oral exposure to nickel sulfate hexahydrate

Workplace exposures to mixtures of nickel compounds have been associated with excess respiratory cancer risk. Animal studies with individual nickel compounds indicate that not all nickel substances have the same potency or potential to induce tumors. The bioavailability of nickel ions at critical cellular sites seems to be important to determine the potential of a substance to induce tumors in animals, but much less is understood about the exact nature (genotoxic or non-genotoxic) of the nickel effects. Within many regulatory frameworks (e.g., European Union), substances are classified for mutagenicity based on the available data and this classification will often influence the mode of action assigned to carcinogenic substances and the way in which risk assessment will be conducted. The objective of this study was to evaluate the ability of nickel sulfate hexahydrate to induce micronuclei in polychromatic erythrocytes (PCEs) in rat bone marrow. This study was conducted according to OECD and EU protocol guidelines. In the dose range-finding assays, the maximum tolerated dose was estimated to be 500 mg/kg/day. The doses used in the micronucleus assay were 125, 250, and 500 mg/kg/day. At least 2000 PCEs per animal were analyzed for micronuclei in PCEs. Cytotoxicity was assessed by scoring a minimum of 500 consecutive total polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Nickel sulfate hexahydrate did not induce statistically significant increases in micronucleated PCEs at any dose examined. The negative results in the present study contribute significantly to the weight of evidence evaluation of the mutagenicity (chromosomal level) of nickel substances. These results are consistent with a non-genotoxic mode of action for soluble nickel that could explain the enhancement of cancer risk seen among refinery workers with mixed exposures and its lack of carcinogenicity in animal studies with single exposures.     

Pivotal involvement of Fcgamma receptor IIA in the neutralization of lipopolysaccharide signaling via a potent novel anti-TLR4 monoclonal antibody 15C1

The mammalian Toll-like receptor (TLR) family has evolved to sense pathogens in the environment and protect the host against infection. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria and induces a signaling cascade that, when exaggerated, has been associated with severe sepsis. We have generated a TLR4-specific monoclonal antibody, 15C1, which neutralizes LPS-induced TLR4 activation in a dose-dependent manner. 15C1 potently blocks the effects of LPS on a panel of primary cells and cell lines in vitro. The binding of 15C1 was mapped to an epitope in the second portion of the extracellular region of TLR4, which has been shown previously to be functionally important in the recognition of LPS. Furthermore, we demonstrate a novel mechanism of inhibition, as the effects of 15C1 are partially Fc-dependent, involving the regulatory Fcgamma receptor IIA (CD32A). In addition to introducing 15C1 as a potent clinical candidate for use in the treatment of LPS-mediated indications, our work demonstrates a newly discovered pathway whose manipulation is pivotal in achieving optimal neutralizing benefit.     

Mycobacterium avium subspecies paratuberculosis in Bison (Bison bison) from Northern Canada

Nested polymerase chain reaction (PCR) using the Mycobacterium avium subspecies paratuberculosis (Map)-specific region, locus 251, was used as a screening tool for the detection of Map DNA in fecal samples from northern Canadian bison herds. Further characterization of positive samples (26/835) was performed because Map DNA was found without signs of disease. Strain typing, using PCR-Restriction endonucleas assay (REA), was limited to two samples but revealed that the samples corresponded to a cattle-related strain and a sheep-related strain. Sequencing of part of the IS1311 region from the two samples revealed a unique three base-pair region, which is only found within the northern Canadian bison isolates.