Biochemical characterization of the recombinant human Nogo-A ectodomain

Nogo-A is a physiologically relevant inhibitor of neuronal growth and regeneration in the myelin of the adult human central nervous system and has attracted considerable attention as a molecular target for the treatment of spinal cord injuries. To gain insight into the structural and functional properties of the large extramembrane region that is characteristic for the Nogo-A splice form of this member of the Reticulon family of membrane proteins, we cloned and expressed the region comprising residues 334-966 as a soluble homogeneous protein in the periplasm of Escherichia coli. SDS/PAGE, under nonreducing conditions, and a systematic substitution analysis of all six Cys residues of Nogo-A indicated that this domain forms two structural disulfide bonds among Cys residues 424, 464, 559 and 597, whereas the Cys residues at positions 699 and 912 seem to be dispensable for folding. The occurrence of a hot spot for host cell proteases and a limited proteolysis experiment suggest that the N-terminal region of Nogo-A up to residue 373 is structurally disordered. Although analytical gel permeation chromatography revealed a large apparent molecular size for the recombinant Nogo-A fragment, indicating oligomer formation, data from analytical ultracentrifugation and dynamic light scattering support a stable monomeric quaternary structure. Notably, the CD spectrum is indicative of a high content of random coil, such that Nogo-A exhibits--at least in part--features of a natively unfolded protein. Nevertheless, the protein fragment identified in this study, as well as its biochemical analysis, provide a promising starting point for future investigations to track down globular subdomains and functionally important regions as well as putative receptor-binding sites therein.     

A comparative study of S/MAR prediction tools

Background  

S/MARs are regions of the DNA that are attached to the nuclear matrix. These regions are known to affect substantially the expression of genes. The computer prediction of S/MARs is a highly significant task which could contribute to our understanding of chromatin organisation in eukaryotic cells, the number and distribution of boundary elements, and the understanding of gene regulation in eukaryotic cells. However, while a number of S/MAR predictors have been proposed, their accuracy has so far not come under scrutiny.     

Application of mass spectrometry technologies for the discovery of low-molecular weight modulators of enzymes and protein-protein interactions

In recent years, mass spectrometry has gained widespread use as an assay and screening technology in drug discovery because it enables sensitive, label-free detection of low-molecular weight modulators of biomolecules as well as sensitive and accurate detection of high-molecular weight modifications of biomolecules. Electrospray and matrix-assisted laser desorption ionization are the most widely used ionization techniques to identify chemical compounds interfering with enzymatic function, receptor-ligand binding or molecules modulating a protein-protein interaction of interest. Mass spectrometry based techniques are no longer restricted to screening in biochemical assay systems but have now become also applicable to imaging of biomolecules and chemical compounds in cell-based assay systems and even in highly complex tissue sections.     

Engrafting fetal liver cells into multiple tissues of healthy adult mice without the use of immunosuppressants

We have shown the fetal liver cell engraftments into multiple tissues of adult healthy mice, achieved without suppressing the animals’ immune systems. Fetal cells from the livers of male C57Bl/6J Black lineage mice at day 13 to 15 of gestation were injected intravenously into female adult CC57W/MY White mice. The grafting was evaluated by Y-chromosome-specific PCR, cytometric analysis of fluorescently stained donor cells, and histological analysis. All the methods consistently showed the presence of multiple engraftments randomly distributed through the various organs of the recipients. After 60 days, the grafts still constituted 0.1 to 2.75% of the tissues. The grafted cells did not change their appearance in any of the organs except the brain, where they became enlarged. Inflammatory reactions were not detected in any of the histological preparations. The frequency of engraftments was higher in the liver, indicating that similarity between the donor and recipient cells facilitates engraftment. The high inherent plasticity of fetal liver cells underlies their ability to integrate into healthy recipient organs, which can be governed by environmental conditions and connections with neighboring cells rather than by the initial cellular developmental programs. The fact that fetal liver cells can be grafted into multiple tissues of healthy animals indicates that they can be used to replace the natural loss of cells in adult organisms.     

Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus

In mice, seminal fluid elicits an inflammation-like response in the female genital tract that activates immune adaptations to advance the likelihood of conception and pregnancy. In this study, we examined whether similar changes in leukocyte and cytokine parameters occur in the human cervix in response to the male partner's seminal fluid. After a period of abstinence in proven-fertile women, duplicate sets of biopsies were taken from the ectocervix in the periovulatory period and again 48 h later, 12 h after unprotected vaginal coitus, vaginal coitus with use of a condom, or no coitus. A substantial influx of CD45(+) cells mainly comprising CD14(+) macrophages and CD1a(+) dendritic cells expressing CD11a and MHC class II was evident in both the stratified epithelium and deeper stromal tissue after coitus. CD3(+)CD8(+)CD45RO(+) T cells were also abundant and increased after coitus. Leukocyte recruitment did not occur without coitus or with condom-protected coitus. An accompanying increase in CSF2, IL6, IL8, and IL1A expression was detected by quantitative RT-PCR, and microarray analysis showed genes linked with inflammation, immune response, and related pathways are induced by seminal fluid in cervical tissues. We conclude that seminal fluid introduced at intercourse elicits expression of proinflammatory cytokines and chemokines, and a robust recruitment of macrophages, dendritic cells, and memory T cells. The leukocyte and cytokine environment induced in the cervix by seminal fluid appears competent to initiate adaptations in the female immune response that promote fertility. This response is also relevant to transmission of sexually transmitted pathogens and potentially, susceptibility to cervical metaplasia.     

IFN-γ Production by Allogeneic Foxp3+ Regulatory T Cells Is Essential for Preventing Experimental Graft-versus-Host Disease

It is emerging that CD4(+)Foxp3(+) regulatory T (Treg) cells can produce the proinflammatory cytokine IFN-γ when stimulated in a Th1 cytokine environment. In this study, we report that Foxp3(+) Treg cells readily produced IFN-γ in vivo in a highly inflammatory model of graft-versus-host disease (GVHD) and during a Th1-dominated immune response to intracellular bacteria. Moreover, stimulation in vitro via TCR in the presence of IL-12 alone was sufficient to induce IFN-γ production by Treg cells in a dose-dependent manner. Transfer of donor Treg cells can prevent lethal GVHD; therefore, we used this model as a robust readout for in vivo Treg function. Interestingly, >50% of allogeneic donor, but not residual recipient Foxp3(+) Treg cells produced IFN-γ after transplantation, suggesting that this cytokine production was alloantigen specific. These IFN-γ producers were stable Foxp3(+) Treg cells because methylation analysis of the Foxp3 gene locus of transferred and reisolated Treg cells during GVHD showed a fully demethylated Treg-specific-demethylated region. Next, we addressed whether IFN-γ production was supporting or rather impairing the immunosuppressive function of Treg cells during GVHD. Blocking of IFN-γ with specific mAb completely abolished the beneficial effect of donor Treg cells. We could further show that only wild-type Treg cells, but not Treg cells from IFN-γ-deficient donor mice, prevented GVHD. This indicated that Treg cell-intrinsic IFN-γ production was required for their protective function. In conclusion, our data show that IFN-γ produced by Foxp3(+) Treg cells has essential immune-regulatory functions that are required for prevention of experimental GVHD.     

MyD88 Drives the IFN-β Response to Lactobacillus acidophilus in Dendritic Cells through a Mechanism Involving IRF1, IRF3, and IRF7

Type I IFNs are induced by pathogens to protect the host from infection and boost the immune response. We have recently demonstrated that this IFN response is not restricted to pathogens, as the Gram-positive bacterium Lactobacillus acidophilus, a natural inhabitant of the intestine, induces high levels of IFN-β in dendritic cells. In the current study, we investigate the intracellular pathways involved in IFN-β upon stimulation of dendritic cells with L. acidophilus and reveal that this IFN-β induction requires phagosomal uptake and processing but bypasses the endosomal receptors TLR7 and TLR9. The IFN-β production is fully dependent on the TIR adapter molecule MyD88, partly dependent on IFN regulatory factor (IRF)1, but independent of the TIR domain-containing adapter inducing IFN-β MyD88 adapter-like, IRF and IRF7. However, our results suggest that IRF3 and IRF7 have complementary roles in IFN-β signaling. The IFN-β production is strongly impaired by inhibitors of spleen tyrosine kinase (Syk) and PI3K. Our results indicate that L. acidophilus induces IFN-β independently of the receptors typically used by bacteria, as it requires MyD88, Syk, and PI3K signaling and phagosomal processing to activate IRF1 and IRF3/IRF7 and thereby the release of IFN-β.     

Whole-Genome Sequence of Livestock-Associated ST398 Methicillin-Resistant Staphylococcus aureus Isolated from Humans in Canada

Despite reports of high colonization rates of ST398 livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among pigs and pig farmers, the incidence of LA-MRSA infection in the general population in Canada appears to be rare in comparison to that in some European countries. In this study, the complete genome sequence of a Canadian representative LA-MRSA isolate (08BA02176) from a human postoperative surgical site infection was acquired and compared to the sequenced genome of an LA-MRSA isolate (S0385) from Europe to identify genetic traits that may explain differences in the success of these particular strains in some locales.