Background-Varicella zoster virus (VZV) vasculopathy produces stroke secondary to viral infection of cerebral arteries. Not all patients have rash before cerebral ischemia or stroke. Furthermore, other vasculitides produce similar clinical features and comparable imaging, angiographic, and CSF abnormalities.

A silicon-based device, dubbed a microphysiometer, can be used to detect and monitor the response of cells to a variety of chemical substances, especially ligands for specific plasma membrane receptors. The microphysiometer measures the rate of proton excretion from 10(4) to 10(6) cells. This article gives an overview of experiments currently being carried out with this instrument with emphasis on receptors with seven transmembrane helices and tyrosine kinase receptors. As a scientific instrument, the microphysiometer can be thought of as serving two distinct functions. In terms of detecting specific molecules, selected biological cells in this instrument serve as detectors and amplifiers. The microphysiometer can also investigate cell function and biochemistry. A major application of this instrument may prove to be screening for new receptor ligands. In this respect, the microphysiometer appears to offer significant advantages over other techniques.

Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the 'Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using sixmodule concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/ in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms.

structive sleep apnea syndrome (OSAS) is one of the most important risk factors of cardiovascular disorders. In the treatment of OSAS, nasal continuous positive airway pressure (nCPAP) has been widely used and found to be effective. In the present study, we hypothesized that the hypoxic stress caused by obstructive sleep apnea would increase circulating intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) in untreated OSAS patients compared with an age-matched control group. In addition, we hypothesized that nCPAP may decrease OSAS-induced hypoxic stress and mediators. To examine these hypotheses, we measured circulating ICAM-1 and IL-8 before and after nCPAP therapy in OSAS patients. We observed that nCPAP decreased apnea, desaturation, and the circulating ICAM-1 and IL-8 levels in OSAS patients. The circulating levels of ICAM-1, IL-8, and MCP-1 in untreated OSAS patients were significantly greater than those in the controls. These observations suggest that nCPAP therapy could reduce OSAS-induced hypoxia and generation of inflammatory mediators. Treatment of OSAS using nCPAP can be, therefore, a potential approach to decrease risk of the progression of OSAS-associated disorders. cytokines; cardiovascular disorders; ischemic heart disease; desaturation magnitude; hypoxic stress; intracellular adhesion molecule-1; monocyte chemoattractant protein-1; interleukin-8

Inappropriate elevation of matrix metalloproteinase-9 (MMP9) is reported to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The object of this study was to identify the molecular mechanism underlying this increase of MMP9 expression, and here we show that oxidative stress-dependent reduction of a protein deacetylase, SIRT1, known as a putative antiaging enzyme, causes elevation of MMP9 expression. A sirtuin inhibitor, splitomycin, and SIRT1 knockdown by RNA interference led an increase in MMP9 expression in human monocytic U937 cells and in primary sputum macrophages, which was detected by RT-PCR, Western blot, activity assay, and zymography. In fact, the SIRT1 level was significantly decreased in peripheral lungs of patients with COPD, and this increase was inversely correlated with MMP9 expression and MMP9 promoter activation detected by a chromatin immunoprecipitation assay. H(2)O(2) reduced SIRT1 expression and activity in U937 cells; furthermore, cigarette smoke exposure also caused reduction of SIRT1 expression in lung tissue of A/J mice, with concomitant elevation of MMP9. Intranasal treatment of a selective and novel SIRT1 small molecule activator, SRT2172, blocked the increase of MMP9 expression in the lung as well as pulmonary neutrophilia and the reduction in exercise tolerance. Thus, SIRT1 is a negative regulator of MMP9 expression, and SIRT1 activation is implicated as a novel therapeutic approach to treating chronic inflammatory diseases, in which MMP9 is abundant.

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The downregulation of E-cadherin by Src promotes epithelial to mesenchymal transition and tumorigenesis. However, a simple loss of cell adhesion is not sufficient to explain the diverse developmental roles of Src and metastatic behavior of viral Src-transformed cells. Here, we studied the functions of endogenous and activated forms of Drosophila Src in the context of tracheal epithelial development, during which extensive remodeling of adherens junctions takes place. We show that Src42A is selectively activated in the adherens junctions of epithelia undergoing morphogenesis. Src42A and Src64B are required for tracheal development and to increase the rate of adherens junction turnover. The activation of Src42A caused opposing effects: it reduced the E-cadherin protein level but stimulated transcription of the E-cadherin gene through the activation of Armadillo and TCF. This TCF-dependent pathway was essential for the maintenance of E-cadherin expression and for tissue integrity under conditions of high Src activity. Our data suggest that the two opposing outcomes of Src activation on E-cadherin facilitate the efficient exchange of adherens junctions, demonstrating the key role of Src in the maintenance of epithelial integrity. KEY WORDS:Src, E-cadherin, Armadillo, Drosophila, Trachea, Cancer Development 135, 1355Development 135, -1364Development 135, (2008Development 135, ) doi:10.1242 Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku Kobe 650-0047, Japan. DEVELOPMENT 1356 strain carrying the trachea-specific btl-Gal4 driver and the UAS-GFPmoesin marker. An additional 142 lines lacking the rough eye phenotype were also tested. GS11022 (Src42A) and GS9618 (Src64B) were analyzed for further study. Fly stocks and geneticsWe used strong loss-of-function alleles of Src genes: Src42A 26-1 (Takahashi et al., 2005), Src42A myri (Tateno et al., 2000) and Src64B P1 (Dodson et al., 1998). The following strains were used in this study: trachealess enhancer trap line 1-eve-1 (Perrimon et al., 1991), UAS-wg (Lawrence et al., 1995), UAS-arm S10 (Pai et al., 1997), UAS-TCF⌬N (van de Wetering et al., 1997), UAS-E-cadherin-GFP (Oda and Tsukita, 1999b), UAS-D␣-catenin-GFP (Oda and Tsukita, 1999a), shg-lacZ , UAS-GFP-moesin (Chihara et al., 2003), YF (Tateno et al., 2000), UAS-Src42A-RNAi (NIG Stock Center) and btl-Gal4 (Shiga et al., 1996). Src42A DN was constructed by introducing the K295M mutation at the catalytic center of the kinase domain and was cloned into the pUAST vector (Brand and Perrimon, 1993). Immunostaining and imagingThe following primary antibodies were used: rat anti-Esg (Fuse et al., 1994); mouse anti-tracheal luminal antigen 2A12, mouse anti-Armadillo N27A1 and mouse anti-septin 4C9H4 (Developmental Studies Hybridoma Bank); rabbit anti-␤-galactosidase (Cappel); mouse anti-GFP B-2 and rabbit anti-GFP (MBL); rabbit anti-Src PY418 (Biosource International); rat anti-E-cadherin (DCAD2) (Oda et al., 1994) and rabbit anti-Src42A (Takahashi et al., 2005). Chicken anti-Src42A antibody ...