NMR Line Shape Analysis of a Multi-state Ligand Binding Mechanism in Chitosanase

Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases

アイヌ民俗図資料の見方

公開研究会「人びとの暮らしと生業─『日本近世生活絵引』作成への問題点をさぐる─」を振り返ってA Look Back at the Workshop on Obtaining Historical Information from PicturesHow to Use Pictorial Materials in Historical Studies

Differentiation of human embryonic stem cells and human induced pluripotent stem cells into steroid-producing cells.

Although there have been reports of the differentiation of mesenchymal stem cells and mouse embryonic stem (ES) cells into steroid-producing cells, the differentiation of human ES/induced pluripotent stem (iPS) cells into steroid-producing cells has not been reported. The purpose of our present study was to establish a method for inducing differentiation of human ES/iPS cells into steroid-producing cells. The first approach we tried was embryoid body formation and further culture on adherent plates. The resultant differentiated cells expressed mRNA encoding the steroidogenic enzymes steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, CYP17A1, and CYP19, and secreted progesterone was detected in the cell medium. However, expression of human chorionic gonadotropin was also detected, suggesting the differentiated cells were trophoblast like. We next tried a multistep approach. As a first step, human ES/iPS cells were induced to differentiate into the mesodermal lineage. After 7 d of differentiation induced by 6-bromoindirubin-3'-oxime (a glycogen synthase kinase-3β inhibitor), the human ES/iPS cells had differentiated into fetal liver kinase-1- and platelet derived growth factor receptor-α-expressing mesodermal lineage cells. As a second step, plasmid DNA encoding steroidogenic factor-1, a master regulator of steroidogenesis, was introduced into these mesodermal cells. The forced expression of steroidogenic factor-1 and subsequent addition of 8-bromoadenosine 3',5'-cyclic monophosphate induced the mesodermal cells to differentiate into the steroidogenic cell lineage, and expression of CYP21A2 and CYP11B1, in addition to steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, CYP11A1, and CYP17A1, was detected. Moreover, secreted cortisol was detected in the medium, but human chorionic gonadotropin was not. These findings indicate that the steroid-producing cells obtained through the described multistep method are not trophoblast like; instead, they exhibit characteristics of adrenal cortical cells

Abundant Lipid and Protein Components of Drusen

Drusen are extracellular lesions characteristic of aging and age-related maculopathy, a major retinal disease of the elderly. We determined the relative proportions of lipids and proteins in drusen capped with retinal pigment epithelium (RPE) and in RPE isolated from non-macular regions of 36 human retinas with grossly normal maculas obtained <6 hr after death.Druse pellets were examined by light and electron microscopy. Component proteins were extracted using novel methods for preserved tissues, separated, subjected to tryptic digestion and LC-MS(MS)(2) analysis using an ion trap mass spectrometer, and identified with reference to databases. Lipid classes were separated using thin layer chromatography and quantified by densitometry. Major druse components were esterified cholesterol (EC), phosphatidylcholine (PC), and protein (37.5+/-13.7, 36.9+/-12.9, and 43.0+/-11.5 ng/druse, respectively). Lipid-containing particles (median diameter, 77 nm) occupied 37-44% of druse volume. Major proteins include vitronectin, complement component 9, apoE, and clusterin, previously seen in drusen, and ATP synthase subunit beta, scavenger receptor B2, and retinol dehydrogenase 5, previously seen in RPE. Drusen and RPE had similar protein profiles, with higher intensities and greater variability in drusen. C8, part of the complement membrane attack complex, was localized in drusen by immunofluorescence.At least 40% of druse content is comprised by lipids dominated by EC and PC, 2 components that are potentially accounted for by just one pathway, the secretion of lipoproteins by RPE. Manipulating genes encoding apolipoprotein pathways would be a fruitful approach to producing drusen with high EC content in laboratory animals. Therapies that directly mitigate drusen should prepare for the substantial volume of neutral lipids. The catalog of major druse proteins is nearing completion

The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase

This research was originally published in Journal of Biological Chemistry. Ken-ichi Kosami, Izuru Ohki, Minoru Nagano, Kyoko Furuita, Toshihiko Sugiki, Yoji Kawano, Tsutomu Kawasaki, Toshimichi Fujiwara, Atsushi Nakagawa, Ko Shimamoto and Chojiro Kojima. The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase. Journal of Biological Chemistry. 2014; 289, 28569-28578. © the American Society for Biochemistry and Molecular Biology

Perubahan Harga Lahan dalam Kaitannya dengan Pembangunan Pertanian di Pedesaan Lampung

IndonesianDalam pembangunan pertanian diperlukan empat faktor penggerak yaitu sumberdaya lahan, sumberdaya manusia, teknologi dan kelembagaan. Keempat faktor diatas saling terkait satu sama lain, sehingga bila salahsatu faktor diatas mengalami hambatan sulit tercapai sasaran yang diinginkan. Pesatnya laju pembangunan beberapa tahun terakhir, menyebabkan sumberdaya lahan terasa semakin terbatas. Hal ini disebabkan oleh terjadinya Perubahan fungsi lahan untuk kepentingan pembangunan itu sendiri. Bertitik tolak dari permasalahan diatas, sumberdaya lahan khususnya lahan pertanian dapat merupakan permasalahan pada masa mendatang. Sumberdaya lahan untuk pertanian akan merupakan suatu komoditi langka dan mempunyai nilai yang tinggi. Kondisi seperti ini akan banyak membawa dampak, baik terhadap nilai lahan, kelembagaan pertanian dan lain sebagainya. Prubahan-Perubahan yang terjadi sudah tentu akan mempengaruhi pembangunan pertanian pada masa mendatang

Morphological study of tooth development in podoplanin-deficient mice.

Podoplanin is a mucin-type highly O-glycosylated glycoprotein identified in several somatyic cells: podocytes, alveolar epithelial cells, lymphatic endothelial cells, lymph node stromal fibroblastic reticular cells, osteocytes, odontoblasts, mesothelial cells, glia cells, and others. It has been reported that podoplanin-RhoA interaction induces cytoskeleton relaxation and cell process stretching in fibroblastic cells and osteocytes, and that podoplanin plays a critical role in type I alveolar cell differentiation. It appears that podoplanin plays a number of different roles in contributing to cell functioning and growth by signaling. However, little is known about the functions of podoplanin in the somatic cells of the adult organism because an absence of podoplanin is lethal at birth by the respiratory failure. In this report, we investigated the tooth germ development in podoplanin-knockout mice, and the dentin formation in podoplanin-conditional knockout mice having neural crest-derived cells with deficiency in podoplanin by the Wnt1 promoter and enhancer-driven Cre recombinase: Wnt1-Cre;PdpnΔ/Δmice. In the Wnt1-Cre;PdpnΔ/Δmice, the tooth and alveolar bone showed no morphological abnormalities and grow normally, indicating that podoplanin is not critical in the development of the tooth and bone.福岡歯科大学2017年