The 1.9-A crystal structure of the noncollagenous (NC1) domain of human placenta collagen IV shows stabilization via a novel type of covalent Met-Lys cross-link

Triple-helical collagen IV protomers associate through their N- and C-termini forming a three-dimensional network, which provides basement membranes with an anchoring scaffold and mechanical strength. The noncollagenous (NC1) domain of the C-terminal junction between two adjacent collagen IV protomers from human placenta was crystallized and its 1.9-A structure was solved by multiple anomalous diffraction (MAD) phasing. This hexameric NC1 particle is composed of two trimeric caps, which interact through a large planar interface. Each cap is formed by two alpha 1 fragments and one alpha 2 fragment with a similar previously uncharacterized fold, segmentally arranged around an axial tunnel. Each monomer chain folds into two structurally very similar subdomains, which each contain a finger-like hairpin loop that inserts into a six-stranded beta-sheet of the neighboring subdomain of the same or the adjacent chain. Thus each trimer forms a quite regular, but nonclassical, sixfold propeller. The trimer-trimer interaction is further stabilized by a previously uncharacterized type of covalent cross-link between the side chains of a Met and a Lys residue of the alpha 1 and alpha 2 chains from opposite trimers, explaining previous findings of nonreducible cross-links in NC1. This structure provides insights into NC1-related diseases such as Goodpasture and Alport syndromes.     

Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes

Conditional mutagenesis is a powerful tool to analyze gene functions in mammalian cells. The site-specific recombinase Cre can be used to recombine loxP-modified alleles under temporal and spatial control. However, the efficient delivery of biologically active Cre recombinase to living cells represents a limiting factor. In this study we compared the potential of a hydrophobic peptide modified from Kaposi fibroblast growth factor with a basic peptide derived from HIV-TAT to promote cellular uptake of recombinant Cre. We present the production and characterization of a Cre protein that enters mammalian cells and subsequently performs recombination with high efficiency in a time- and concentration-dependent manner. Histidine-tagged Cre recombinase transduced inefficiently unless fused to a nuclear localization signal (NLS). Fusion of NLS-Cre to the fibroblast growth factor transduction peptide did not improve the transducibility, whereas fusion with the TAT peptide significantly enhanced cellular uptake and subsequent recombination. More than 95% recombination efficiency in fibroblast cells, as well as murine embryonic stem cells, was achieved after His-TAT-NLS-Cre transduction. Efficient recombination could also be obtained in primary splenocytes ex vivo. We expect that application of His-TAT-NLS-Cre, which can be produced readily in large quantities from a bacterial source, will expand our abilities to manipulate mammalian genomes.     

Evidence for a NOD2-independent susceptibility locus for inflammatory bowel disease on chromosome 16p

Heritable predisposition to inflammatory bowel disease (IBD) has been demonstrated by epidemiological and genetic analysis. Linkage of IBD to broad regions of chromosome 16 has been established by analysis of multiple populations. NOD2, located on proximal 16q, was recently identified as an IBD gene. As the linkage regions on chromosome 16 are large, we have investigated the possibility that NOD2 is not the only IBD gene located on this chromosome. A high-density experiment using 39 microsatellite markers was performed to identify additional regions of association, and to indicate areas of interest for further investigation. A triple-peaked configuration of the linkage curve with peak logarithm of odds (lod) scores of 2.7, 3.2, and 3.1 was observed on proximal 16p, proximal 16q, and central 16q, respectively. The cohort was stratified by coding individuals carrying the NOD2 single nucleotide polymorphism (SNP)8 and SNP13 "unknown." Significance at the central peak, corresponding to the genomic location of NOD2, then decreased from 3.2 to 1.2. The maximal lod scores on the proximal p-arm (lod = 2.1) and central q-arm (lod = 2.6) changed only moderately. An exploratory association analysis (TRANSMIT) yielded a strong lead at D16S3068 (P = 0.00028). The region around this marker was further investigated by using anonymous SNPs. An associated haplotype containing three SNPs was identified (peak significance P = 0.00027, IBD phenotype). On stratification based on NOD2 genotype, this significance increased to P = 0.0001. These results confirm the importance of NOD2 and provide evidence for a second IBD gene located on chromosome 16p.     

Cognitive impairment correlates with hypothalamo-pituitary-adrenal axis dysregulation in multiple sclerosis

Hypothalamo-pituitary-adrenal (HPA) dysregulation has recently been demonstrated in multiple sclerosis (MS) by means of combined dexamethasone corticotropin-releasing hormone (Dex-CRH) suppression tests. Authors found a correlation with course of disease and to a lesser extent with depressive symptoms. In this study, we aimed to further evaluate whether HPA disturbances in MS are correlated with cognitive impairment, disability status, and fatigue. Dex-CRH tests were performed in a total of 40 patients and 11 healthy controls. Concomitantly, cognitive impairment was evaluated using the symbol digit modalities test and fatigue was assessed by different fatigue severity scales.When comparing patient subpopulations to healthy subjects, Dex-CRH stimulation tests indicated an HPA hyperactivity in primary and secondary progressive MS, while relapsing-remitting patients had response patterns similar to controls. However, results were only significant for one of the six analysed parameters, i.e. area under the curve calculations of ACTH stimulation. Within the patient sample, clear-cut differences emerged between groups of different cognitive impairment, being significant for all ACTH response parameters. Our results suggest an HPA hyperactivation related to increased cognitive impairment. Indicators of HPA axis activation further correlated substantially with neurologic disability, but only moderately with duration of disease and even less with depressive symptoms and fatigue. We conclude that the observed dysregulation is more likely a secondary effect of the extent of brain damage rather than primarily involved in the pathogenesis of MS.     

Down-regulation of retroviral transgene expression during differentiation of progenitor-derived dendritic cells

OBJECTIVE: Hematopoietic progenitor cells are a promising source for generation of genetically modified dendritic cells. A prerequisite for using these cells in therapeutic approaches is stable vector-mediated transgene expression during and after cell maturation. We investigated the expression of enhanced green fluorescence protein (EGFP) mediated by retroviral vectors in dendritic cells and other hematopoietic cells differentiated in vitro. MATERIAL AND METHODS: CD34(+) cells were efficiently transduced with retroviral vector constructs known to mediate different expression levels due to distinct cis-acting elements. EGFP(+) cells were purified by cell sorting and differentiated to monocytes, granulocytes, dendritic cells, and erythrocytes. Coexpression of EGFP and cell type-specific markers was analyzed by flow cytometry. RESULTS: Transgene expression from various retroviral vectors was silenced exclusively in dendritic cells, but not in other mature myeloid cells. Loss of EGFP was most pronounced in cells initially displaying low expression levels. This was confirmed by using a retroviral vector coding for a variant of EGFP with significantly reduced half-life. In contrast, a majority of dendritic cells showed stable expression when a self-inactivating retroviral construct using an internal cytomegalovirus promotor was used. CONCLUSIONS: We suggest that expression from the retroviral long terminal repeat is silenced during dendritic cell differentiation in vitro. High levels of stable transgene product in progenitor cells may mask a loss of expression. An improvement of retroviral vectors mediating stable transgenic expression is necessary for therapeutic approaches using gene-modified dendritic cells.