R26-WntVis reporter mice showing graded response to Wnt signal levels

The canonical Wnt signaling pathway plays a major role in the regulation of embryogenesis and organogenesis, where signal strength-dependent cellular responses are of particular importance. To assess Wnt signal levels in individual cells, and to circumvent the integration site-dependent bias shown in previous Wnt reporter lines, we constructed a new Wnt signal reporter mouse line R26-WntVis. Heptameric TCF/LEF1 binding sequences were combined with a viral minimal promoter to confer a graded response to the reporter depending on Wnt signal strengths. The histone H2B-EGFP fusion protein was chosen as the fluorescent reporter to facilitate single-cell resolution analyses. This WntVis reporter gene was then inserted into the ROSA26 locus in an orientation opposite to that of the endogenous gene. The R26-WntVis allele was introduced into Wnt3a−/− and Wnt3avt/− mutant mouse embryos and compared with wild-type embryos to assess its performance. The R26-WntVis reporter was activated in known Wnt-dependent tissues and responded in a graded fashion to signal intensity. This analysis also indicated that the major Wnt activity early in embryogenesis switched from Wnt3 to Wnt3a around E7.5. The R26-WntVis mouse line will be widely useful for the study of Wnt signal-dependent processes

Activation of tumor suppressor protein PTEN and induction of apoptosis are involved in cAMP-mediated inhibition of cell number in B92 glial cells

金沢大学医薬保健研究域医学系During brain development, cAMP induces morphological changes and inhibits growth effects in several cell types. However, the molecular mechanisms underlying the growth inhibition remain unknown. Tumor suppressor protein phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that inhibits the phosphoinositide 3-kinase (PI3K) pathway. The phosphorylation of Akt, which is one of the key molecules downstream of PI3K, inhibits apoptosis. In this study, we investigated the role of PTEN in cAMP-mediated growth inhibition. B92 rat glial cells were treated with 2 different cAMP stimulatory agents, a phosphodiesterase (PDE) inhibitor and a β-adrenoceptor agonist. Both cAMP stimulatory agents induced marked morphological changes in the cells, decreased cell number, decreased Akt phosphorylation, activated PTEN, cleaved caspase-3, and induced the condensation and fragmentation of nuclei. These results indicate that the cAMP stimulatory agents induced apoptosis. Protein phosphatase inhibitor prevented cAMP-induced dephosphorylation of PTEN and Akt. In addition, cAMP analogs and Epac-selective agonists affected PTEN and Akt activities. These results suggested that cAMP-induced apoptosis may be mediated by PTEN activation and Akt inhibition through protein phosphatase in B92 cells. Our results provide new insight into the role of PTEN in cAMP-induced apoptosis in glial cells. © 2011 Elsevier Ireland Ltd

Campylobacter jejuni感染はT-84細胞におけるCFTR活性化によるCl⁻分泌亢進を抑制する

Campylobacter jejuni causes foodborne disease associated with abdominal pain, gastroenteritis, and diarrhea. These symptoms are induced by bacterial adherence and invasion of host epithelial cells. C. jejuni infection can occur with a low infective dose, suggesting that C. jejuni may have evolved strategies to cope with the bacterial clearance system in the gastrointestinal tract. The mucosa layer is the first line of defense against bacteria. Mucus conditions are maintained by water and anion (especially Cl-) movement. Cystic fibrosis transmembrane conductance regulator (CFTR) is the main Cl- channel transporting Cl- to the lumen. Mutations in CFTR result in dehydrated secreted mucus and bacterial accumulation in the lungs, and recent studies suggest that closely related pathogenic bacteria also may survive in the intestine. However, the relationship between C. jejuni infection and CFTR has been little studied. Here, we used an 125I- efflux assay and measurement of short-circuit current to measure Cl- secretion in C. jejuni-infected T-84 human intestinal epithelial cells. The basic state of Cl- secretion was unchanged by C. jejuni infection, but CFTR activator was observed to induce Cl- secretion suppressed in C. jejuniinfected T-84 cells. The suppression of activated Cl- secretion was bacterial dose-dependent and duration-dependent. A similar result was observed during infection with other C. jejuni strains. The mechanism of suppression may occur by affecting water movement or mucus condition in the intestinal tract. A failure of mucus barrier function may promote bacterial adhesion or invasion of host intestinal epithelial cells, thereby causing bacterial preservation in the host intestinal tract

Met Kinase Inhibitor E7050 Reverses Three Different Mechanisms of Hepatocyte Growth Factor-induced Tyrosine Kinase Inhibitor Resistance in EGFR Mutant Lung Cancer

PURPOSE: Hepatocyte growth factor (HGF) induces resistance to reversible and irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutant lung cancer cells by activating Met and the downstream PI3K/Akt pathway. Moreover, continuous exposure to HGF accelerates the emergence of EGFR-TKI-resistant clones. We assayed whether a new Met kinase inhibitor, E7050, which is currently being evaluated in clinical trials, could overcome these three mechanisms of resistance to EGFR-TKIs. EXPERIMENTAL DESIGN: The effects of E7050 on HGF-induced resistance to reversible (gefitinib), irreversible (BIBW2992), and mutant-selective (WZ4002) EGFR-TKIs were determined using the EGFR-mutant human lung cancer cell lines PC-9 and HCC827 with an exon 19 deletion, and H1975 with an T790M secondary mutation. PC-9 cells were mixed with HGF-producing fibroblasts, MRC-5 cells, and subcutaneously inoculated into SCID mice and the therapeutic effects of E7050 plus gefitinib were assayed. RESULTS: E7050 circumvented resistance to all of the reversible, irreversible, and mutant-selective EGFR-TKIs induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines, by blocking the Met/Gab1/PI3K/Akt pathway in vitro. E7050 also prevented the emergence of gefitinib-resistant HCC827 cells induced by continuous exposure to HGF. In the in vivo model, E7050 plus gefitinib resulted in marked regression of tumor growth associated with inhibition of Akt phosphorylation in cancer cells. CONCLUSIONS: A new Met kinase inhibitor, E7050, reverses the three HGF-induced mechanisms of gefitinib resistance, suggesting that E7050 may overcome HGF-induced resistance to gefitinib and next-generation EGFR-TKIs

Yokukansan Inhibits Neuronal Death during ER Stress by Regulating the Unfolded Protein Response

Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death.We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein.Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs.Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD

遠隔地小規模校での学習支援連携の定着への課題 : 藤女子大学と厚田中学校による2年間の取り組みを振り返って

本論では、今年度で二年目を終えた藤女子大学の学生による石狩市立厚田中学校での学習支援(スクール・アシスタント・ティチャー:SAT)事業の現状と課題、そして将来的な展望を報告する。厚田中学校は、藤女子大学花川校舎から車で50分ほどの海岸部に位置する全校生徒22名の小規模校である。そこで主に教職課程を履修している大学生が、中学校教員や保護者と協力しつつ、数学や家庭科などの授業、学校行事の面で、生徒と触れ合い、多様な学習支援を行なっている。本論は、大学・中学校側のSAT担当教員だけではなく、実際に学習支援に参加した学生の視点から、今年度を振り返り、来年への展望を述べている。現状分析としては、1. 学校行事(学校祭・餅つき大会・卒業式)への参加、2. 地域との関わり(ピザ教室)、3. 学生主体の連絡調整が促進されたことが、今年度の成果といえる。その一方で、依然として、遠隔地域での学習支援という特色上、1. 厚田への交通手段、それに伴う2. 学生の時間の確保が課題として残った。しかし、来年度(2013年)は、厚田中学校での学習支援を経験し三年目の学生も4年生として在籍するため、SAT事業の継続性・発展性を視野に入れた、彼女たちの集大成に期待したい。なお、今年度のSAT事業は、石狩市教育委員会の予算と共に、藤女子大学QOL研究所からの補助金を通して、運営された。This paper aims to report the current conditions and future prospects of learning support partnerships (School Assistant Teacher: SAT) between Atsuta Junior High School and Fuji Women\u27s University. This program has been implemented by Ishikari City board of education with the university students. In this program almost ten students who want to become a teacher has involved in learning support for junior high school students especially in the subject of mathematics. This year is the second year of implementation of this program. Comparing to last year,our project promote university students the involvement in school events, relation with community and guardian of students,and university student\u27s autonomy in terms of coordination of this program. On the other hand we should challenge some problems such as transportation to Atsuta and obtaining the number of students to maintain the learning support in junior high school