国語科教育における中国語教材導入の可能性と意義 -中国語版『はらぺこあおむし』の授業実践を通して-

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SIRT1 inactivation inhibits Wnt and promotes p53 a pathway.

<p><b>A)</b> Representative photomicrographs showing β-catenin immunohistochemical staining of intestinal sections from APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice. Scale bar indicates 50 µm length. Polyps from both groups demonstrate intense cytoplasmic and nuclear staining for β-catenin. B) Bar graph with frequencies of crypts with indicated number of cells with nuclear β-catenin staining in normal appearing mucosa of APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice. The average number of β-catenin positive cells per crypt is reduced in APC^+/min SIRT1^−/− mice compared to APC^+/min SIRT1^+/+ animals 2.1±1.2 vs 3.2±1.3 (p = 1.5×10⁻⁵, at least 100 crypts per genotype were scored). C) Representative photomicrographs of normal appearing mucosa from APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice demonstrating a reduced number of basal cells with nuclear β-catenin in APC^+/min SIRT1^−/− animals. Arrows are directed toward representative basal cells with nuclear β-catenin. Scale bar indicates 25 µm length. D) Inhibition of SIRT1 with EX527 (2 µM) and cambinol (50 µM) reduces activity of the TCF/LEF driven firefly luciferase reporter (TOP FLASH) transiently transfected into SW480 cells. TOP FLASH luciferase reporter contains minimal promoter along with three TCF binding sites, which have been mutated in FOP FLASH reporters. Bars represent means ± SEM of relative firefly luciferase activity normalized to renilla luciferase activity from the thymidine kinase promoter-driven renilla reporter that was co-transfected with TOP FLASH and FOP FLASH reporters. Each transfection is carried out in quadruplicate. **p<0.01. E) SW480 cells with shRNA-mediated downregulation of SIRT1 (top: western blot for SIRT1 and actin) exhibit reduced activity of the transiently transfected TCF/LEF driven firefly luciferase reporter (TOP FLASH). Bars represent means ± SEM of relative firefly luciferase activity normalized to renilla luciferase activity from the thymidine kinase promoter-driven renilla reporter that was co-transfected with TOP FLASH and FOP FLASH reporters. Each transfection is carried out in quadruplicate. *p<0.05. F) Immunoblot for acetyl-p53, p53 and actin from cells treated with etoposide, SIRT1 inhibitor EX527 or the combination of the two drugs. Inhibition of SIRT1 leads to p53 hyperacetylation in Hct116 colon cancer cell line. Hyperacetylation of p53 is observed in cells treated with EX527 and a combination of EX527 and etoposide. Etoposide alone modestly induces p53 acetylation.</p

Enterocyte-specific SIRT1 deletion increases the rate of apoptosis in the intestinal tumors of APC^+/min mice.

<p>(A) A representative western blot showing expression of the wild-type SIRT1 protein in the intestinal epithelium of APC^+/min SIRT1^+/+ mice (first lane) and a truncated version in the epithelium of APC^+/min SIRT1^−/− mice (second lane). Liver cells of the APC^+/min SIRT1^−/− mice (third lane), as well as other tissues (not shown) express the wild-type protein. Pan-actin immunostaining served as a loading control. (B) Representative photographs of unfixed small intestines (distal segments) showing similar polyp number for APC^+/min SIRT1^+/+ (right) and APC^+/min SIRT1^−/− (left) mice. Scale bar indicates 5 mm length. (C) Representative photomicrographs of typical polyps from the two groups of mice, stained with hematoxylin and eosin. Scale bar indicates 100 µm length. (D) Representative photomicrographs and a bar graph showing Ki-67 immunohistochemical staining of polyp sections from APC^+/min SIRT1^+/+ (left) and APC^+/min SIRT1^−/− (middle) mice. Scale bar indicates 100 µm length. Proliferation index for polyps from APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice (right), expressed as a fraction of Ki-67 positive cells within each polyp. Bars represent means ± SEM, n = 20 polyps per group. No statistically significant difference was observed. (E) Representative photomicrographs and a bar graph showing activated (cleaved) caspase-3 immunohistochemical staining of polyp sections from APC^+/min SIRT1^+/+ (left) and APC^+/min SIRT1^−/− (middle) mice. Scale bar indicates 100 µm length. Absolute numbers of apoptotic (caspase-3 positive) cells per high power field (400 x) for polyps from SIRT1^+/+ and SIRT1^−/− mice (right). Bars represent means ± SEM, n = 25 polyps per group. ***p<0.001.</p

Phenotypic Optimization of Urea–Thiophene Carboxamides To Yield Potent, Well Tolerated, and Orally Active Protective Agents against Aminoglycoside-Induced Hearing Loss

Hearing loss is a major public health concern with no pharmaceutical intervention for hearing protection or restoration. Using zebrafish neuromast hair cells, a robust model for mammalian auditory and vestibular hair cells, we identified a urea–thiophene carboxamide, <b>1</b> (ORC-001), as protective against aminoglycoside antibiotic (AGA)-induced hair cell death. The 50% protection (HC₅₀) concentration conferred by <b>1</b> is 3.2 μM with protection against 200 μM neomycin approaching 100%. Compound <b>1</b> was sufficiently safe and drug-like to validate otoprotection in an <i>in vivo</i> rat hearing loss model. We explored the structure–activity relationship (SAR) of this compound series to improve otoprotective potency, improve pharmacokinetic properties and eliminate off-target activity. We present the optimization of <b>1</b> to yield <b>90</b> (ORC-13661). Compound <b>90</b> protects mechanosensory hair cells with HC₅₀ of 120 nM and demonstrates 100% protection in the zebrafish assay and superior physiochemical, pharmacokinetic, and toxicologic properties, as well as complete <i>in vivo</i> protection in rats