セキセツ チホウ ノウソン ケイザイ チョウサショ ニ オケル デザイン シンコウサク 1930ネンダイ ノ ノウソン コウゲイヒン ヲ メグル ショソウ ノ ナカ デ

学術論

セキセツ チホウ ノウソン ケイザイ チョウサショ ニ オケル デザイン シンコウサク 1930ネンダイ ノ ノウソン コウゲイヒン ヲ メグル ショソウ ノ ナカ デ

学術論

Regulation of colonic epithelial cell homeostasis by mTORC1

Abstract Cell signaling important for homeostatic regulation of colonic epithelial cells (CECs) remains poorly understood. Mammalian target of rapamycin complex 1 (mTORC1), a protein complex that contains the serine-threonine kinase mTOR, mediates signaling that underlies the control of cellular functions such as proliferation and autophagy by various external stimuli. We here show that ablation of tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, specifically in intestinal epithelial cells of mice resulted in increased activity of mTORC1 of, as well as increased proliferative activity of, CECs. Such Tsc2 ablation also reduced the population of Lgr5-positive colonic stem cells and the expression of Wnt target genes in CECs. The stimulatory phosphorylation of the kinase Akt and inhibitory phosphorylation of glycogen synthase kinase 3β were both markedly decreased in the colon of the Tsc2 conditional knockout (CKO) mice. Development of colonic organoids with cryptlike structures was enhanced for Tsc2 CKO mice compared with control mice. Finally, Tsc2 CKO mice manifested increased susceptibility to dextran sulfate sodium–induced colitis. Our results thus suggest that mTORC1 activity promotes the proliferation of, as well as the expression of Wnt target genes in, CECs and thereby contributes to colonic organogenesis and homeostasis

Microstructures of the Porphyrin/Viologen Monolayer on the Clay Surface: Segregation or Integration?

Microstructures of the porphyrin/viologen monolayer on an anionic clay surface (synthetic saponite) were investigated by the observation of photochemical behavior of porphyrin. Fluorescence behaviors of porphyrin–viologen–clay complexes were observed by steady state and time-resolved fluorescence spectroscopy. Although fluorescence of porphyrin was effectively quenched by coadsorbed viologen on the clay surface, a part of the fluorescence of porphyrin was not quenched and remained even at high loading level of viologen. According to time-resolved fluorescence measurement, the decay profile of excited singlet porphyrin can be analyzed by two- or three-component fitting for porphyrin–viologen–clay complexes. These results indicate that porphyrin and viologen adsorb with island (segregation) structure on the clay surface. The size of the island formed by porphyrin was quantitatively estimated for two kinds of porphyrins. It turned out that the porphyrin molecular structure affects the size of the island. It has been believed that electron transfer on a clay surface is inefficient due to segregation of dyes between clay sheets (A. J. Bard et al., <i>J. Phys. Chem.</i> <b>1984</b>, <i>88</i>, 5519). Our results indicate that segregation behavior sensitively depends on the structure of the dye, and it is possible to construct an efficient electron transfer system on the clay surface

Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization

The production of cell-type– and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)–mediated cell-type– and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier–penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed nonspecific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in nonastrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre-DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driven AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target–flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO-dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice