A Possible Contribution of Altered Cathepsin B Expression to the Development of Skin Sclerosis and Vasculopathy in Systemic Sclerosis

Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and 19 normal controls. Since the deficiency of transcription factor Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1+/− and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming growth factor-β (TGF-β) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal fibroblasts, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1+/− mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal fibroblasts from early dcSSc, CTSB expression was decreased compared with normal fibroblasts and significantly reversed by TGF-β1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc

Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His(−), Met(−), Lys(−), Trp(−), Leu(−), Arg(−), and Ura(−) auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose

Species habitat associations in an old-growth beech forest community organised by landslide disturbances

<p>In order to understand the processes that govern tree species’ spatial associations with habitats in landslide-affected forest communities, we investigated the habitat associations of six major tree species in an old-growth beech forest community located on a steep flank slope where landslides had occurred. All stems ≥ 5cm in diameter at breast height were mapped on a 1.14 ha polygonal plot and the topographic conditions (slope inclination and convexity), canopy state, and forest floor vegetation (i.e. dwarf bamboo and fern cover) were assessed. Most stems of <i>Fagus crenata</i> and <i>Acer japonicum</i> as well as many stems of <i>Magnolia obovata</i> belonging to the canopy layer were located on sites with low fern cover, whereas many stems of <i>M. obovata</i> below the canopy layer were associated with sites having high dwarf bamboo cover. The locations of the two <i>Acer</i> species belonging to the canopy and lowest layers coincided with sites having convex topography. Stems of <i>Aesculus turbinata</i> below the canopy layer were generally found on sites with gentle slopes, whereas most stems of <i>Quercus crispula</i> coincided with steeply sloping sites. While the results demonstrate that steep slopes created as landslide headscarps provide suitable habitats for species with less shade tolerance, it should be noted that there were few consistent patterns of habitat association across the layers. Our results suggest that unpredictable sporadic landslide disturbances with varying intensities and spatial scales, and their recursiveness, are at least partly responsible for habitat association patterns observed in the forest community.</p