The GABAA/benzodiazepine receptor has been solubilized from membrane preparations of bovine cerebral cortex and has been reconstituted, in a functionally active form, into phospholipid vesicles. In preliminary experiments, the receptor was labeled with the photoactive benzodiazepine [3H]flunitrazepam prior to solubilization. A peptide of apparent molecular weight 53,500 was specifically labeled by this method, and this was used as a marker for the receptor during the reconstitution procedures. The labeled protein was solubilized with approximately 40% efficiency by 1% beta-octyl glucoside. Reconstitution was achieved by mixing the solubilized proteins with a 4:1 mixture of soybean asolectin and bovine brain phospholipids, followed by chromatography on Sephadex G-50-80 to remove detergent. The incorporation of the GABAA receptor into membrane vesicles has been verified by sucrose gradient centrifugation in which the [3H]-flunitrazepam-labeled peptide comigrated with [14C]phosphatidylcholine used as a lipid marker. Vesicles prepared without labeled markers retained the ability to bind both [3H]flunitrazepam and the GABA analogue [3H]muscimol. Furthermore, the binding parameters were very similar to those measured using native membrane preparations. A novel fluorescence technique has been used to measure chloride transport mediated by the GABAA receptor in reconstituted vesicles. Chloride influx was rapidly stimulated in the presence of micromolar concentrations of muscimol and was blocked by preincubation of the membranes with muscimol (desensitization). Flux was also blocked by pretreatment with the competitive GABAA receptor blocker bicuculline or with the noncompetitive GABAA receptor antagonist picrotoxin.
Gibberellin (GA) is a phytohormone that regulates various developmental processes during the plant life cycle. In this study, we identify a new GA agonist, diphegaractin, using a wheat cell-free based drug screening system with grape GA receptor. A GA-dependent interaction assay system using GA receptors and DELLA proteins from Vitis vinifera was constructed using AlphaScreen technology and cell-free produced proteins. From the chemical compound library, diphegaractin was found to enhance the interactions between GA receptors and DELLA proteins from grape in vitro. In grapes, we found that diphegaractin induces elongation of the bunch and increases the sugar concentration of grape berries. Furthermore, diphegaractin shows GA-like activity, including promotion of root elongation in lettuce and Arabidopsis, as well as reducing peel pigmentation and suppressing peel puffing in citrus fruit. To the best of our knowledge, this study is the first to successfully identify a GA receptor agonist showing GA-like activity in agricultural plants using an in vitro molecular-targeted drug screening system.
The focus of this paper is the Keijō toshi kōsōzu (Illustrated Plan for Keijō City), which includes drawings of planned Government-General facilities within Gyeongbokgung Palace. These illustrations offer rich clues about the Government-General's plans to renovate Gyeongbokgung during the period of Japanese colonial rule over Korea. This paper will highlight aspects of the Keijō toshi kōsōzu that show the due consideration had not been given, and suggest that the designs reflect the ideas of their creator.
This article examines the Illustrated Plan for Keij� (Seoul) City discovered in materials previously held by Kuratomi Y�zabur�, in terms of its designer, period of production, and significance in the context of urban planning. Based on an examination of the illustrated buildings, it can be inferred that the plan was drafted sometime between November 1912 and August 1913. Furthermore, based on the design of the new Government-General building and the signature, it is possible to confirm that the German architect Georg de Lalande drafted the plans. Much like the plans concentrating Tokyo government buildings in one area, this plan took as its main axis the line running from the palace to Nandaemun station, and planned for the government offices belonging to the Government-General, city hall, and the post office to run along this axis. In line with the trends of contemporaneous Keij� city plans, this design laid out a road grid that utilized existing roads and waterfront spaces utilizing existing water systems.
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