表紙・目次 『第15回CEReS国際シンポジウム「環境リモートセンシングのこれま での成果とさらなる挑戦」』 2009年12月15日～16日 於千葉大学

Held on 15-16 December, 2009, Chiba Universit

Increased expression of osteogenic marker genes in ActRIIB-Fc-treated calluses at two weeks.

<p>Quantitative real-time PCR analyses of the two-week time point revealed higher expression of essential osteoblast markers osterix, runt-related transcription factor 2 and alkaline phosphatase. Expression of cathepsin K and tartrate resistant acid phosphatase decreased compared to PBS controls which could demonstrate impaired cartilage and bone resorption. Furthermore expression of Sox9 was also lower compared to controls. Expression levels of Sclerostin and Dkk-1, negative regulators of Wnt-signaling, were also lower in ActRIIB-Fc-treated mice. Expression of Smad1/5/8 target genes Id1 and Id3 were decreased as well. * = p<0.05, ** = p<0.01. n = 7 for PBS groups and 8 for ActRIIB-Fc groups.</p

Immunohistochemical analyses of the calluses.

<p>Trends towards decreased number of cathepsin K+ cells in ActRIIB-Fc-treated mice were seen at both two and four weeks (A). The number of Runx2 positive cells was slightly increased at four weeks compared to PBS controls (p = 0.346) (B). There were no changes in the number of p-Smad1+ cells at either two or four weeks (C) but trends of increased number of p-Smad2+ cells were observed at two (p = 0.056) and four weeks (p = 0.209) due to ActRIIB-Fc-treatment (D). n = 8–9 for PBS 2 weeks, n = 10 for ActRIIB-Fc 2 weeks, n = 5–8 for PBS 4 weeks and n = 5–8 for ActRIIB-Fc 4 weeks.</p

ActRIIB-Fc robustly accelerated fracture healing and callus mineralization.

<p>(A-D) Representative radiographic and micro-computed cross-sectional images of the fracture callus of the PBS- and ActRIIB-Fc-treated mice at two and four weeks. There were no significant differences in bone structure between the groups at the two-week time point. (E-I) At four weeks, ActRIIB-Fc treatment resulted in greater increases bone volume/tissue volume, trabecular numbers and volumetric bone mineral density as well as decreased trabecular separation and structural model index-* = p<0.05, ** = p<0.01, *** = p<0.001. n = 8 for PBS 2 weeks, 7 for ActRIIB-Fc 2 weeks, 8 for PBS 4 weeks and 6 for ActRIIB-Fc 4 weeks.</p

Changes in body weight compared to the start of the experiment (%).

<p>Changes in body weight compared to the start of the experiment (%).</p

Treatment with ActRIIB-Fc improves mechanical strength of calluses.

<p>ActRIIB-Fc increases callus strength in the four week groups compared to PBS controls in terms of (A) maximum load, (B) stiffness and (C) bending strength. No significant changes were noted between the two week groups. * = p<0.05 n = 6 for PBS 2 weeks, 8 for ActRIIB-Fc 2 weeks, 5 for PBS 4 weeks and 7 for ActRIIB-Fc 4 weeks.</p

Histological analysis of the calluses.

<p>Representative hematoxylin and eosin stained histological images of fracture calluses at four weeks. (A and C) Overview image of the callus (in black and white in order to distinguish the newly formed trabecular bone more easily) and (B and D) larger magnification of the trabecular bone within the callus at four weeks. Black arrows pinpoint newly formed trabeculae. At the two week time point, ActRIIB-Fc treatment caused increased (E) woven bone volume and (F) cartilage volume compared to PBS controls resulting in increased (G) mineralized tissue per tissue volume. At the four-week time point ActRIIB-Fc treatment greatly enhanced (H) bone volume/tissue volume and (J) trabecular numbers and decreased their (I) separation * = p<0.05 ** = p<0.01, *** = p<0.001. n = 6 for PBS 2 weeks, 6 for ActRIIB-Fc 2 weeks, 7 for PBS 4 weeks and 7 for ActRIIB-Fc 4 weeks.</p

Overexpression of cathepsin K in mice decreases collagen deposition and lung resistance in response to bleomycin-induced pulmonary fibrosis-2

Nized and BAL cells (A, B), freshly isolated type II alveolar epithelial cells (A, C), lung homogenate (A, D), and concentrated BAL fluid supernatant (E) were prepared for analysis of cath K mRNA by real-time RT-PCR (A) and western blotting (B-E). (A), data are given as fold change in cathepsin K mRNA of bleomycin-challenged cath K tg mice compared to wild-type mice. (F) Gelatin zymography employing concentrated BAL fluid protein from mock- or bleomycin-treated wild-type mice and cath K tg mice. Data are shown as mean ± SD of n = 3 determinations. (B-F), data represent at least n = 3–5 independent analyses. * indicates < 0.05 versus bleomycin-treated wild-type mice at day 14 post-treatment.<p><b>Copyright information:</b></p><p>Taken from "Overexpression of cathepsin K in mice decreases collagen deposition and lung resistance in response to bleomycin-induced pulmonary fibrosis"</p><p>http://respiratory-research.com/content/9/1/54</p><p>Respiratory Research 2008;9(1):54-54.</p><p>Published online 18 Jul 2008</p><p>PMCID:PMC2490691.</p><p></p

Zfp521 is a suppressor of adipogenesis in vitro.

<p>(A) C3H10T1/2 cells were differentiated and RNA isolated at the indicated time points. Gene expression of <i>Zfp521</i> and <i>Pparg</i> was measured by Q-PCR and normalized to cyclophilin. Data shown as mean of three biological replicates. (B) Protein lysates isolated during 3T3-L1 adipogenesis were subjected to western blotting with anti-Zfp521 antibody. (C, left) Zfp521 mRNA expression was measured in fractionated subcutaneous and epididymal fat tissue taken from wild-type mice (SV, stromal-vascular fraction; AD, adipocytes). (C, right) SV of epididymal fat tissue from Zfp423^GFP transgenic mice was sorted with GFP antibody and plated. After washing away floating cells, Zfp521 mRNA expression was measured in GFP− and GFP+ cells. (D–G) C3H10T1/2 cells were retrovirally transduced with Zfp521, empty vector, shRNA specific forZfp521 (sh521), or a scrambled hairpin (Scr). Overexpression and knock-down were confirmed by immunoblotting of Zfp521 prior to differentiation in the boxed insert. Cells were differentiated with DMI or DMI plus rosiglitazone (DMIR) and stained with oil red-O (D, F) and adipocyte markers were determined by Q-PCR (E, G) on day 8. Data presented as mean ± SD, <i>n</i> = 3, *<i>p</i><0.05.</p

Zfp521 inhibits Ebf1 transcriptional activity through physical interaction.

<p>(A) 3T3-L1 preadipocytes were harvested and endogenous Ebf1 was immunoprecipitated using anti-Ebf1 beads and blotted with normal goat-IgG or anti-Zfp521. (B–D) 3T3-L1 preadipocytes were co-transfected with vectors expressing Flag-Zfp521, Myc-Ebf1, and various reporter plasmids containing the <i>mb1</i>-promoter (B), <i>Sncg</i>-promoter (C), or <i>Cebpa</i>-promoter (D). At 24 h after transfection, luciferase activity was normalized to β-galactosidase activity. Data presented as mean ± SD, <i>n</i> = 4, *<i>p</i><0.05. (E) 3T3-L1 preadipocytes were stably transduced with Flag-Ebf1 or Flag-Zfp521. ChIP assay was performed on 3T3-L1 cells that were treated with DMI for 1 h using anti-Flag antibody or an IgG control using PCR primers directed at regions of the Cebpa containing the putative Ebf sites. (F, G) Immortalized <i>Zfp521^+/+</i> and <i>Zfp521^−/−</i> MEFs were transduced with a retrovirus expressing Ebf1, Zfp521, or empty vector and differentiated prior to staining with oil red-O after 8 d (F) and adipocyte gene expression was measured by Q-PCR (G). (H, I) C3H10T1/2 cells were transduced with a retrovirus expressing Zfp521WT, Zfp521ΔZF27-30, Zfp521Δ13aa, or empty pMSCV vector. Cells were differentiated with DMIR and stained with oil red-O and gene expression was measured on day 6. (J) Zfp521 and Ebf1 were expressed in C3H10T1/2 cells alone or in combination, and expression of Zfp521, Ebf1, and Zfp423 was determined by Q-PCR. (K) Zfp521, Zfp521Δ27-30, or pMSCV was expressed in C3H10T1/2 cells and expression of Zfp521, Ebf1, and Zfp423 was determined by Q-PCR. Data presented as mean ± SD, <i>n</i> = 3, *<i>p</i><0.05.</p