Identification of an amino acid sequence in laminin mediating cell attachment, chemotaxis, and receptor binding

We have probed for active sites in the B1 chain of laminin using synthetic peptides comprising certain regions of its amino acid sequence as deduced from cDNA clones. An antibody to a 19-mer from domain III inhibited attachment of HT-1080 and CHO cells to laminin, while the peptide itself was inactive. A nearby peptide (CDPGYIGSR) from domain III with homology to epidermal growth factor was synthesized and found to be one of the principle sites in laminin mediating cell attachment, migration, and receptor binding.     

A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis

Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD⁺ to NADP⁺. In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH.     

Proteomic study on X-irradiation-responsive proteins and ageing: search for responsible proteins for radiation adaptive response

We investigated high- or low-dose irradiation-responsive proteins using proteomics on two-dimensional (2D) PAGE, and the effects of ageing on cell responses to radiation in variously aged rat astrocytes. After 5 Gy irradiation, the relative abundance of peroxiredoxin 2, an antioxidant enzyme, and latexin, an inhibitor of carboxypeptidase, increased. The induction of these proteins was suppressed by ageing, suggesting that the response to high-dose radiation decreased with ageing. The relative abundance of elongation factor 2 (EF-2) fragment increased 3 h and reduced 24 h after 0.1 Gy irradiation. Temporal enhancement of the EF-2 fragment due to low-dose irradiation was suppressed by ageing. Since radiation adaptive response in cultured astrocytes was observed 3 h but not 24 h after 0.1 Gy irradiation and suppressed by ageing as previously reported, alteration of the EF-2 fragment corresponded to the radiation adaptive response. We also examined phospho-protein profiles, resulting in the relative abundance of phospho-EF-1beta and phospho-beta-actin being altered by 0.1 Gy irradiation; however, ageing did not affect the alteration of phospho-EF-1beta and phospho-beta-actin, unlike the EF-2 fragment. The results suggested that the EF-2 fragment was a possible candidate for the protein responsible for the radiation adaptive response in cultured astrocytes.     

A recombinant replication-competent hepatitis C virus expressing Azami-Green, a bright green-emitting fluorescent protein, suitable for visualization of infected cells

The hepatitis C virus (HCV) production system consists of transfecting the human hepatoma cell line Huh7 with genomic HCV RNA (JFH1). To monitor HCV replication by fluorescence microscopy, we constructed a recombinant HCV clone expressing Azami-Green (mAG), a bright green fluorescent protein, by inserting the mAG gene into the nonstructural protein 5A (NS5A) gene; the resultant clone was designated JFH1-hmAG. The Huh-7.5.1 (a subclone of Huh7) cells transfected with JFH1-hmAG RNA were found to produce cytoplasmic NS5A-mAG, as readily visualized by fluorescence microscopy, and infectious virus, as assayed with the culture supernatant, indicating that JFH1-hmAG is infectious and replication-competent. Furthermore, the replication of this virus was inhibited by interferon alpha in a dose-dependent manner. These results suggest that JFH1-hmAG is useful for studying HCV life cycle and the mechanism of interferon’s anti-HCV action and for screening and testing new anti-HCV drugs.     

Bloom's syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress

Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom's syndrome-associated (BLM) helicase, Mus81 nuclease and ataxia telangiectasia mutated and Rad3-related (ATR) kinase to induce transient double-stranded DNA breaks. The induction of transient DNA breaks was accompanied by dissociation of proliferating cell nuclear antigen (PCNA) and DNA polymerase α from replication forks. In cells with functional BLM, Mus81 and ATR, the transient breaks were promptly repaired and DNA continued to replicate at a slow pace in the presence of replication inhibitors. In cells that lacked BLM, Mus81, or ATR, transient breaks did not form, DNA replication did not resume, and exposure to low doses of replication inhibitors was toxic. These observations suggest that BLM helicase, ATR kinase, and Mus81 nuclease are required to convert perturbed replication forks to DNA breaks when cells encounter conditions that decelerate DNA replication, thereby leading to the rapid repair of those breaks and resumption of DNA replication without incurring DNA damage and without activating a cell cycle checkpoint.     

Increased susceptibility to autoimmune gastritis in thymic stromal lymphopoietin receptor-deficient mice

Thymic stromal lymphopoietin (TSLP), mainly produced by epithelial cells, activates a variety of cell types, including dendritic cells, mast cells, T cells, and B cells. It is involved in the pathogenesis of allergic inflammation in the lung, skin, and gastrointestinal tract. In addition, TSLP promotes Th2-type intestinal immunity against helminth infection and regulates Th1-type inflammation in a mouse model of colitis, suggesting that it plays crucial roles in intestinal immune homeostasis. Although autoimmune gastritis (AIG), mediated by inflammatory Th1 responses, develops in the gastric mucosa, it is not clear whether TSLP is involved in regulating these responses in AIG. The aim of this study was to examine the roles of TSLP in the development of AIG. Because BALB/c mice thymectomized 3 d after birth (NTx mice) develop AIG, we used this model to test the role of TSLP in the development of AIG. We found that in AIG-bearing mice, TSLP was expressed in the inflamed stomach and that the serum anti-parietal cell Ab levels in neonatal thymectomized TSLPR-deficient mice (NTx-TSLPR(-/-) mice) were significantly elevated over those in NTx-TSLPR(+/+) mice. In addition, NTx-TSLPR(-/-) mice exhibited an earlier onset of AIG than that observed in NTx-TSLPR(+/+) mice. The rapid development of AIG in NTx-TSLPR(-/-) mice resulted in more aggressive CD4(+) T cell infiltration and more severe loss of parietal and chief cells in the progression phase of AIG, accompanied by enhanced production of IL-12/23p40 and IFN-γ. Taken together, these data suggested that TSLP negatively regulates the development of AIG.     

A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system

We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples.     

Extracellular vesicles released from mesenchymal stromal cells stimulate bone growth in osteogenesis imperfecta

Background

Systemic infusion of mesenchymal stromal cells (MSCs) has been shown to induce acute acceleration of growth velocity in children with osteogenesis imperfecta (OI) despite minimal engraftment of infused MSCs in bones. Using an animal model of OI we have previously shown that MSC infusion stimulates chondrocyte proliferation in the growth plate and that this enhanced proliferation is also observed with infusion of MSC conditioned medium in lieu of MSCs, suggesting that bone growth is due to trophic effects of MSCs. Here we sought to identify the trophic factor secreted by MSCs that mediates this therapeutic activity.

Augmented interleukin 6 production by rat thyrocytes (FRTL5): effect of interleukin 1 beta and thyroid-stimulating hormone

This study shows that rat thyroid follicular (FRTL5) cells can produce interleukin 6 (IL-6) spontaneously and that IL-1 beta enhanced the production of IL-6 dose-dependently. In addition, 10 ng/mL of IL-1 beta in combination with 10 to 1,000 microU/mL of thyroid-stimulating hormone (TSH) synergistically enhanced IL-6 production. It is suggested that IL-6 released from thyroid follicular cells may be a costimulator of autoreactive B and T cells in autoimmune thyroid disease and may constitute another link between the immune and endocrine systems.