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Background: Maltogenic amylases that are known to date form dimers to perform hydrolysis. Results: The structure of maltogenic amylase from Staphylothermus showed a novel domain at the N terminus associated with the active site. Conclusion:Staphylothermus amylase has all of its substrate-binding structural components in a single monomer. Significance: This is the first report of the newly observed domain arrangement adopted by hyperthermophilic archaic maltogenic amylase.

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Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O2) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation.

Gap junctions (GJs) are intercellular channels composed of connexins. Cellular molecules smaller than 1 kDa can diffuse through GJs by a process termed gap junctional intercellular communication (GJIC), which plays essential roles in various pathological and physiological conditions. Gambogic acid (GA), a major component of a natural yellow dye, has been used as traditional medicine and has been reported to have various therapeutic effects, including an anti-cancer effect. In this study, two different GJ assay methods showed that GA and its analogs inhibited GJIC. The inhibition was rapidly reversible and was not mediated by changes in surface expression or S368 phosphorylation of Cx43, cellular calcium concentration, or redox state. We also developed an assay system to measure the intercellular communication induced by Cx40, Cx30, and Cx43. Dihydrogambogic acid (D-GA) potently inhibited GJIC by Cx40 (IC50 = 5.1 μM), whereas the IC50 value of carbenoxolone, which is known as a broad spectrum GJIC inhibitor, was 105.2 μM. Thus, D-GA can act as a pharmacological tool for the inhibition of Cx40.