De novo deletions of SNRPN exon 1 in early human and mouse embryos result in a paternal to maternal imprint switch

Prader-Willi syndrome (PWS) is a neurogenetic disease characterized by infantile hypotonia, gonadal hypoplasia, obsessive behaviour and neonatal feeding difficulties followed by hyperphagia, leading to profound obesity. PWS is due to a lack of paternal genetic information at 15q11-q13 (ref. 2). Five imprinted, paternally expressed genes map to the PWS region, MKRN3 (ref. 3), NDN (ref. 4), NDNL1 (ref. 5), SNRPN (refs 6-8 ) and IPW (ref. 9), as well as two poorly characterized framents designated PAR-1 and PAR-5 (ref. 10). Imprinting of this region involves a bipartite 'imprinting centre' (IC), which overlaps SNRPN (refs 10,11). Deletion of the SNRPN promoter/exon 1 region (the PWS IC element) appears to impair the establishment of the paternal imprint in the male germ line and leads to PWS. Here we report a PWS family in which the father is mosaic for an IC deletion on his paternal chromosome. The deletion chromosome has acquired a maternal methylation imprint in his somatic cells. We have made identical findings in chimaeric mice generated from two independent embryonic stem (ES) cell lines harbouring a similar deletion. Our studies demonstrate that the PWS IC element is not only required for the establishment of the paternal imprint, but also for its postzygotic maintenance.     

Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing

Nicastrin, a transmembrane glycoprotein, forms high molecular weight complexes with presenilin 1 and presenilin 2. Suppression of nicastrin expression in Caenorhabditis elegans embryos induces a subset of notch/glp-1 phenotypes similar to those induced by simultaneous null mutations in both presenilin homologues of C. elegans (sel-12 and hop-1). Nicastrin also binds carboxy-terminal derivatives of beta-amyloid precursor protein (betaAPP), and modulates the production of the amyloid beta-peptide (A beta) from these derivatives. Missense mutations in a conserved hydrophilic domain of nicastrin increase A beta42 and A beta40 peptide secretion. Deletions in this domain inhibit A beta production. Nicastrin and presenilins are therefore likely to be functional components of a multimeric complex necessary for the intramembranous proteolysis of proteins such as Notch/GLP-1 and betaAPP.     

A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.     

The mammalian sodium channel BNC1 is required for normal touch sensation

Of the vertebrate senses, touch is the least understood at the molecular level The ion channels that form the core of the mechanosensory complex and confer touch sensitivity remain unknown. However, the similarity of the brain sodium channel 1 (BNC1) to nematode proteins involved in mechanotransduction indicated that it might be a part of such a mechanosensor. Here we show that disrupting the mouse BNC1 gene markedly reduces the sensitivity of a specific component of mechanosensation: low-threshold rapidly adapting mechanoreceptors. In rodent hairy skin these mechanoreceptors are excited by hair movement. Consistent with this function, we found BNC1 in the lanceolate nerve endings that lie adjacent to and surround the hair follicle. Although BNC1 has been proposed to have a role in pH sensing, the acid-evoked current in cultured sensory neurons and the response of acid-stimulated nociceptors were normal in BNC1 null mice. These data identify the BNC1 channel as essential for the normal detection of light touch and indicate that BNC1 may be a central component of a mechanosensory complex.     

p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours

p73 (ref. 1) has high homology with the tumour suppressor p53 (refs 2-4), as well as with p63, a gene implicated in the maintenance of epithelial stem cells. Despite the localization of the p73 gene to chromosome 1p36.3, a region of frequent aberration in a wide range of human cancers, and the ability of p73 to transactivate p53 target genes, it is unclear whether p73 functions as a tumour suppressor. Here we show that mice functionally deficient for all p73 isoforms exhibit profound defects, including hippocampal dysgenesis, hydrocephalus, chronic infections and inflammation, as well as abnormalities in pheromone sensory pathways. In contrast to p53-deficient mice, however, those lacking p73 show no increased susceptibility to spontaneous tumorigenesis. We report the mechanistic basis of the hippocampal dysgenesis and the loss of pheromone responses, and show that new, potentially dominant-negative, p73 variants are the predominant expression products of this gene in developing and adult tissues. Our data suggest that there is a marked divergence in the physiological functions of the p53 family members, and reveal unique roles for p73 in neurogenesis, sensory pathways and homeostatic control.     

Modification of the rib operon derived from Bacillus subtilis and its expression in Escherichia coli

A riboflavin operon （rib operon） derived from Bacillus subtilis 368 was modified on structure and the resulting operons were expressed in various strains of Escherichia coli. The results showed that the optimization of the rib operon and the host strain used for expression are two main factors affecting the riboflavin production. Replacing the promoterl and rfn box of the rib operon with a strong constructive promoter spol drastically increased the expression of the rib genes. When E. coli JM109 was used as the host strain, the highest riboflavin production reached 95.3μg/mL （about eight times higher than that of the unmodified r/b operon）. In addition, when tetracycline （20 μg/mL） was used as the selective pressure, compared with the ampicillin resistant transformants, a higher riboflavin yield was obtained in tetracycline resistant host strain.     

Estimation of underdetermined mixing matrix based on support vector machine

In underdetermined blind source separation (BSS), a novel algorithm based on extended support vector machine (SVM) is proposed to estimate the mixing matrix in this paper, including the number of the active sources. Instead of traditional clustering algorithms, it mainly takes the modulus of observations and the number in each direction of arrival, without any prior knowledge about the sources except for sparsity, and it is not sensitive to the initial values. Simulations are given to illustrate availability and robustness of our algorithm.     

Evidence for existence of a close association between CD14 and CXCR4 on monocytic cell line U937

The surface expression of HIV-1 coreceptors (CXCR4 and CCR5) on monocytes can be regulated by the ligand of CD14, and the susceptibility of the cells to HIV-1 is then changed. Our previous study found that monoclonal antibody against CD14 could dramatically inhibit CXCR4-mediated chemotaxis and cell-cell fusion. Based on these studies, we explored potential relationship between CD14 and CXCR4 on monocytic cell line U937. Flow cytometry analysis showed that anti-CXCR4 monoclonal antibody (mAb) 12G5 strongly inhibited binding of the FITC-conjugated anti-CD14 monoclonal antibodies (TUK4 and UCHM1) to U937, while another CXCR4-specific mAb B-R24 did not show any effect on this binding. On the other hand, two anti-CD14 monoclonal antibodies (TUK4 and UCH-M1) obviously inhibited the binding of the PE-conjugated anti-CXCR4 mAb 12G5 to U937 but did not inhibit the binding of mAb 12G5 to CXCR4-transfected 3T3 cells (3T3.T4.CXCR4), which indicates that the blocking of mAb 12G5 binding to CXCR4 by CD14-specific mAbs is not involved in the possibility that CD14-specific mAbs directly bind to CXCR4. These results suggested existence of a close association between CD14 and CXCR4 on monocytic cell line U937.     

Biocompatibility studies of silk fibroin-based artificial nerve grafts in vitro and in vivo

Silk fibroin(SF)has been used extensively in the biomedical field including tissue engineering for the generation of artificial bones,skins or ligaments.We have previously reported on good in vitro biocompatibility of SF fibers with peripheral nerve tissues and cells.In the present study,we developed a novel design of the SF-based artificial nerve graft(SF graft)which was composed of a SF-nerve guidance conduit(NGC)inserted with SF fibers.MTT assay was performed to determine the cytotoxicity of the SF-NGC extract fluid on the cultured L929 cells derived from an immortalized mouse fibroblast cell line.In addition,this SF graft was implanted into adult rats for bridging a 10-mm long sciatic nerve defect.The following-up experiments at initial stage(1-4 week)of nerve regeneration including routine blood tests and histochemical investigation were conducted to evaluate the in vivo biocompatibility of the SF graft with peripheral nerves.The results demonstrated that the SF-NGC graft was biocompatible with the surrounding tissues and cells due to its low inflammatory potential with a grade 0 under the U.S.Pharmacopeia guidelines and it was generally suitable to a certain degree for bridging peripheral nerve defects in virtue of supporting Schwann cell adherence,expansion and migration.Therefore the SF graft is a promising alternative to classical autografts for peripheral nerve repair.