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Supplementary Figure S4: (A,B) Viable AXT cells after the treatment of chemotherapeutic agents. (C) Representative flow cytometric analysis of γ-H2AX abundance in AXT cells as described in Fig.4E. (D) Immunofluorescence analysis of γ-H2AX in AXT cells.

Supplementary Table S1. Sequences of primers and predicted product sizes for real-time RT-PCR analysis

Supplementary Information. Supplementary Materials and Methods and Supplementary Figure Legends

Supplementary movie S1

Chronic constipation and irritable bowel syndrome (IBS) are two of the most common functional gastrointestinal disorders, both of which negatively affect the quality of life of the patients. Since people may have co-existing symptoms of multiple disorders, chronic constipation and IBS with predominant constipation cannot be clearly distinguished. In this cross-sectional study, data were obtained from self-administered questionnaires to assess the prevalence of chronic constipation and IBS, and their overlap, among female undergraduate students in Japan. We also assessed the participants’ mental status, physical activity, lifestyle, and dietary intake, each of which can influence chronic constipation and IBS. These parameters were assessed using a Japanese version of the hospital anxiety and depression scale, and an established semi-quantitative questionnaire available for clinical investigation (FFQg).Among the 131 participants, 9.2% had chronic constipation and 18.0% had IBS, while 2 (1.7%) participants had both constipation and IBS. Increased body mass index and higher dietary intake of protein (energy ratio), fat (energy ratio), cholesterol, animal protein (ratio), animal fat (ratio), and meat and eggs, and decreased carbohydrate energy ratio and vegetable fat ratio, were associated with chronic constipation. A state of anxiety tended to be associated with IBS, but in contrast to the results for chronic constipation, the dietary intake of the nutrients and types of food examined were not associated with IBS, except for the “snacks food group”. The results suggest that overlap between chronic constipation and IBS was not common, and that these two disorders had different clinical characteristics and backgrounds.

Summary: Background: Non-alcoholic steatohepatitis (NASH), a subtype of non-alcoholic fatty liver disease (NAFLD), is a potentially progressive liver disease that can lead to cirrhosis. Obesity increases the risk of NAFLD/NASH, but this disease can also be observed in non-obese individuals. Methods: We investigated the metabolic and histopathological changes in 13 obesity-resistant Slc:Wistar/ST rats fed a high-fat and high-cholesterol (HFC) diet for 9 weeks, and also retrospectively compared the results of 41 Sprague-Dawley (SD) rats that were previously fed with the same protocol to the results of the Slc:Wistar/ST rats. Results: Of the 13 Slc:Wistar/ST rats fed an HFC diet containing 1.25% or 2.5% cholesterol, 11 (84.6%) developed histologically proven NASH without obesity, an increased visceral fat volume, insulin resistance, histopatological severe lobular inflammation and severe hepatic fibrosis. The HFC diets significantly increased the levels of mRNA encoding collagen type I alpha 1 (COL1A1), transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1). The SD rats also developed NASH without obesity, an increased visceral fat volume and insulin resistance, but the metabolic and histopathological effects, such as lower serum adiponectin levels, higher serum leptin levels, histopatological severe lobular inflammation and hepatic fibrosis, seemed to be more pronounced in the SD rats than in the Slc:Wistar/ST rats. Conclusions: These two rat models may reflect the human etiology of NASH that is influenced by dietary factors, and the obesity-resistant Slc:Wistar/ST rat model may be particularly useful for elucidating the pathophysiological mechanism of the so-called “lean NASH”. Keywords: Non-alcoholic fatty liver disease, Obesity, Wistar rat, Sprague-Dawley rat, High-fat diet, Cholesterol

The recapitulation of bone formation via the in vitro generation of bone-like nodules is frequently used to understand bone development. However, current bone-induction techniques are slow and difficult to reproduce. Here, we report the formation of bone-like nodules within ten days, via the use of retinoic acid (RA) to induce the osteogenic differentiation of human induced pluripotent stem cells (hiPSCs) into osteoblast-like and osteocyte-like cells that create human bone tissue when implanted in calvarial defects in mice. We also show that the induction of bone formation depends on cell signalling through the RA receptors RARα and RARβ, which simultaneously activate the BMP (bone morphogenetic protein) and Wnt signalling pathways. Moreover, by using patient-derived hiPSCs, the bone-like nodules recapitulated the osteogenesis-imperfecta phenotype, which was rescued via the correction of disease-causing mutations and partially by an mTOR (mechanistic target of rapamycin) inhibitor. The method of inducing bone nodules may serve as a fast and reproducible model for the study of the formation of both healthy and pathological bone. A fast in vitro model of the formation of bone-like nodules, enabled by the retinoic-acid-mediated induction of the osteogenic differentiation of patient-derived induced pluripotent stem cells, recapitulates the osteogenesis-imperfecta phenotype.

Background Nifedipine has been reported to cause impairment of insulin sensitivity. But recently a controlled-released formulation of nifedipine (nifedipine-GITS) has been reported that it could improve insulin sensitivity. Methods We evaluated insulin sensitivity in two groups of essential hypertensive subjects before and after treatment with either long-acting nifedipine (nifedipine-CR, Adalat CR tablets; Bayer Yakuhin, Osaka, Japan) ( n = 10) or metoprolol ( n = 9). Insulin sensitivity was evaluated from the steady-state plasma glucose (SSPG) level measured at the steady-state insulin level (20 to 30 μU/mL) using a modification of the SSPG method previously reported. Results The SSPG was initially high, but was significantly reduced by nifedipine-CR treatment (from 133 ± 14 mg/dL to 95 ± 8 mg/dL). However, SSPG was not significantly altered by treatment in the metoprolol group (from 103 ± 15 mg/dL to 119 ± 12 mg/dL). Conclusions Our results indicate that the long-acting nifedipine (nifedipine-CR) is associated with improved insulin sensitivity.

Insulin resistance and hyperinsulinemia have been observed in over 70% of the nonobese, nondiabetic subjects with essential hypertension (HT). Alpha-1 blockers, ACE-antagonists, long-acting Ca blockers including nifedipine CR, some form of β-blockers, tilisolor, which is reported to increase blood flow, improve insulin sensitivity when blood pressure is better controlled. Decrease of serum potassium during insulin sensitivity test and intraplatelet free Ca 2+ concentration is positively and negatively correlated with insulin sensitivity, respectively. Blood pressure is correlated with insulin resistance, which is also observed in secondary HT. The resistance is correlated with salt sensitivity as well as impaired nocturnal fall of blood pressure. These suggest the possible association of insulin resistance with altered intracellular cation metabolism. Insulin resistance and associated hyperinsulinemia have been observed in effort as well as vasospastic angina pectoris (VSAP), atherothrombotic cerebral infarction, and in ASO without obesity, HT, or diabetes, suggesting the resistance resulting from endothelial dysfunction. Insulin resistance has been observed in heart failure and is correlated with angiotensin II. Resistance is also observed in hypertrophic cardiomyopathy and is partially correlated with TNF-α. These results indicate that insulin resistance seem to be multifactorial. An effort to normalize insulin sensitivity is crucial to eliminate multiple risk factors as well as to prevent the progression of atherosclerotic vascular lesions.

Osteosarcoma is a malignant bone tumor in children and adolescents characterized by intrinsic therapeutic resistance. The IGF2 is expressed at elevated levels in osteosarcoma after treatment with chemotherapy, prompting an examination of its functional contributions to resistance. We found that continuous exposure to IGF2 or insulin in the absence of serum created a dormant growth state in osteosarcoma cells that conferred resistance to various chemotherapeutic drugs in vitro. Mechanistic investigations revealed that this dormant state correlated with downregulation of downstream signaling by the IGF1 receptor, heightened cell survival, enhanced autophagy, and the presence of extracellular glutamine. Notably, inhibiting autophagy or depleting glutamine was sufficient to increase chemotherapeutic sensitivity in osteosarcoma xenografts in mice. Clinically, we confirmed that IGF expression levels were elevated in human osteosarcoma specimens from patients who received chemotherapy. Together, our results suggest that activation of IGF or insulin signaling preserves the survival of osteosarcoma cells under chemotherapeutic stress, providing a drug-resistant population that may engender minimal residual disease. Attenuating this survival mechanism may help overcome therapeutic resistance in osteosarcoma. Cancer Res; 74(22); 6531–41. ©2014 AACR.

Histone deacetylase (HDAC) inhibitors can induce transcriptional activation of a number of genes and induce cellular differentiation as histone acetylation levels increase. Although these inhibitors induce apoptosis in several cell lines, the precise mechanism by which they do so remains obscure. This study shows that HDAC inhibitors, sodium butyrate and trichostatin A (TSA), abrogate interleukin (IL)-2–mediated gene expression in IL-2–dependent cells. The HDAC inhibitors readily induced apoptosis in IL-2–dependent ILT-Mat cells and BAF-B03 transfectants expressing the IL-2 receptor βc chain, whereas they induced far less apoptosis in cytokine-independent K562 cells. However, these inhibitors similarly increased acetylation levels of histones in both cells. Although histone hyperacetylation is believed to lead to transcriptional activation, the results showed an abrogation of IL-2–mediated induction of c-myc,bag-1, and LC-PTP gene expression. This observed abrogation of gene expression occurred prior to phosphatidylserine externalization, a process that occurs in early apoptotic cells. Considering the biologic role played by IL-2–mediated gene expression in cell survival, these data suggest that its abrogation may contribute to the apoptotic process induced by HDAC inhibitors.

It has been suggested that circulating concentrations of hepatocyte growth factor (HGF) are increased in individuals with vascular endothelial damage, such as in hypertensive patients and subjects with atherosclerosis. Because the influence of genetic variation of HGF has not been examined, we identified single nucleotide polymorphisms (SNPs) in the HGF gene, and investigated the association between these SNPs and blood pressure or carotid atherosclerosis in the Japanese general population. We identified 21 SNPs in the HGF gene by direct sequencing in a test population of 32 Japanese subjects. Among them, considering allele frequency and linkage disequilibrium, three SNPs, C-1652T in the promoter, T43839A in intron 8, and T44222C in intron 9, were genotyped in 2412 members of the Japanese general population randomly selected from the residents in Suita city. None of the three SNPs were significantly associated with blood pressure. After adjusting for age, smoking habits, consumption of alcohol, and the presence of diabetes mellitus and dyslipidemia, female subjects with the T allele of T43839A had more severe carotid atherosclerosis compared to individuals with the A allele. This study provides the first evidence that HGF may be a candidate susceptibility loci that affects the progression of atherosclerosis in Japanese subjects.

TitleTransfection and Transformation Systems for Pock-Forming andThiostrepton-Producing Streptomyces azureusAuthor(s) Ogata, Seiya; Koyama-Miyoshi, Yuko; Hayashida, ShinsakuCitation九州大学農学部紀要 ¦¦ 29(4) ¦¦ p179-188; Journal of the Faculty ofAgriculture, Kyushu University ¦¦ 29(4) ¦¦ p179-188Issue Date 1985-06URL http://hdl.handle.net/2324/23804Right

Description des conditions de transfection par l'intermediaire de protoplastes de S. azureus et de l'ADN du phage SA+1, et du clonage du gene his

As part of a search for a new staining agent with higher selectivity for particular metals, we examined the staining properties of thiazolylazo-benzoic acid derivatives for histochemical staining of metals in rat liver tissue. Thiazolylazodimethylaminobenzoic acid (TAMB) stained only two metals, palladium(Pd) and copper(Cu), among a variety of metals. The results obtained revealed that the TAMB method was superior to the conventional p-dimethylaminobenzylidenerhodanine method with regard to selectivity for various metals when staining for Pd in histochemical practice. The differentiation of Pd from Cu was achieved by using masking agents, pyrophosphate or tripolyphosphate.

The ionic partial molar volumes at infinite dilution, V∞(ion), were determined for several dicarboxylate ions, disulfonate ions, tartrate ions, and [Co(gly)(en)2]2+ (gly=glycinate ion and en=ethylenediamine) from density measurements at 25°C. The values of V∞(ion) obtained for the divalent anions generally increased with their van der Waals volumes. Significant differences in V∞(ion) were observed between geometrical isomers with dicarboxylate and disulfonate ions and were attributed to those in both of electrostriction volume and the void space volume around the anions. The former volume was decreased and the latter was increased with increasing distance between two negatively charged groups in the anions. A relatively strong electrostriction behavior of the tartrate ions, compared to the dicarboxylate ions, suggested the presence of an extra electrostriction caused by hydroxyl groups. The electrostriction strength of [Co(gly)(en)2]2+ was significantly large compared to that of [Ni(en)3]2+, rather close ...

The ion-association constants (K) of [Fe(phen)3]2+ with o- and m-benzenedisulfonate, 2,6- and 2,7-naphthalenedisulfonate ions, determined by conductivity measurements at 0-50 °C, were considerably larger than the electrostatic prediction: K(25°C)=99±3, 204±5, 1021±29, and 792±6 dm3mol−1, respectively. Their hydrophobic behavior was apparently similar to that of alkanesulfonate ions. However, the significant enthalpy contribution was found to the hydrophobic ion association.

The purpose of this study was to examine the relationship between clinical findings and subgingival relationship between clinical findings and subgingival microbial flora in periodontitis at the first medical examination and after initial preparation. The results obtained were as follows: 1. Clinical findings with the exception of plaque index showed improvement after initial preparation in comparison with the first medical examination. 2. In phase contrast microscopy, both total bacteria and incidence of spirochetes and motile rods decreased after initial preparation in comparison with the first medical examination. 3. Clinical findings with the exception of plaque index were related to the total bacteria and proportional distribution of spirochetes and motile rods in periodontal pockets, observed in phase contrast microscopy. 4. Total bacteria and proportional distribution of black-pigmented Bacteroides in periodontal pockets decreased after initial preparation in comparison with the first medical examination.