Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing

Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been directly used with an identical protein. Here we applied both EPL and PTS methods to a 140 kDa dimeric multi-domain protein E. coli CheA, and successfully produced the ligated CheA dimer by both approaches. In EPL approach, extensive optimization of the ligation sites and the conditions were required to obtain sufficient amount for an NMR sample of CheA, because CheA contains a dimer forming domain and it was not possible to achieve high reactant concentrations (1–5 mM) of CheA fragments for the ideal EPL condition, thereby resulting in the low yield of segmentally labelled CheA dimer. PTS approach sufficiently produced segmentally labeled ligated CheA in vivo as well as in vitro without extensive optimizations. This is presumably because CheA has self-contained domains connected with long linkers, accommodating a seven-residue mutation without loss of the function, which was introduced by PTS to achieve the high yield. PTS approach was less laborious than EPL approach for the routine preparation of segmentally-isotope labeled CheA dimer. Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10858-012-9628-3) contains supplementary material, which is available to authorized users

Characterization of genetically modified mice for phosphoglycerate mutase, a vitally-essential enzyme in glycolysis

Models de ratolí; Glucòlisi; Diabetis mellitusModelos de ratón; Glucólisis; Diabetes mellitusMouse models; Glycolysis; Diabetes mellitusGlycolytic metabolism is closely involved in physiological homeostasis and pathophysiological states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been reported to exert certain physiological role in vitro, whereas its impact on glucose metabolism in vivo remains unclear. Here, we report the characterization of Pgam1 knockout mice. We observed that homozygous knockout mice of Pgam1 were embryonic lethal. Although we previously reported that both PGAM-1 and -2 affect global glycolytic profile of cancers in vitro, in vivo glucose parameters were less affected both in the heterozygous knockout of Pgam1 and in Pgam2 transgenic mice. Thus, the impact of PGAM on in vivo glucose metabolism is rather complex than expected before.This work was supported in part by grants from the Japan Society for the Promotion of Science (Grants No. 26310103 to HK and No. 15K19283 to HK), and by the Japan Agency for Medical Research and Development (AMED), Core Research for Evolutional Science and Technology (CREST JP17gm0610002h0306 to HK). HK; Hiroshi Kondoh

Phosphorylation-induced conformation of beta(2)-adrenoceptor related to arrestin recruitment revealed by NMR

The C-terminal region of G-protein-coupled receptors (GPCRs), stimulated by agonist binding, is phosphorylated by GPCR kinases, and the phosphorylated GPCRs bind to arrestin, leading to the cellular responses. To understand the mechanism underlying the formation of the phosphorylated GPCR-arrestin complex, we performed NMR analyses of the phosphorylated beta(2)-adrenoceptor (beta(2)AR) and the phosphorylated beta(2)AR-beta-arrestin 1 complex, in the lipid bilayers of nanodisc. Here we show that the phosphorylated C-terminal region adheres to either the intracellular side of the transmembrane region or lipids, and that the phosphorylation of the C-terminal region allosterically alters the conformation around M215(5.54) and M279(6.41), located on transemembrane helices 5 and 6, respectively. In addition, we found that the conformation induced by the phosphorylation is similar to that corresponding to the beta-arrestin-bound state. The phosphorylation-induced structures revealed in this study propose a conserved structural motif of GPCRs that enables beta-arrestin to recognize dozens of GPCRs.Peer reviewe

Study of Sumida River, Part-1; Its COD and EC characteristics from data collected in spring, 2021

The Sumida River, a typical urban river in Tokyo, has been recognized as a site for fostering Tokyo’s urbanlife that serves as a venue of leisure and relaxation for its residents. As the Teikyo University of Science(TUS) is located near the Sumida River, students spend a lot of time nearby. Clarifying the characteristics ofSumida River, and teaching it to students, are important not only for enhancing environmental awarenessamong students, but also local environment conservation. In this study, chemical oxygen demand (COD)and electric conductivity (EC) data, major sources of contamination, and the characteristics of the river wereanalyzed and interpreted as follows. 1) Although our COD and EC data showed that Sumida River wasaffected by tidal movement, the river water in our research area (from Otakebashi Bridge to Senju-OhashiBridge) might exhibit backward and forward movements, and it was slightly affected by sea water from theTokyo Bay. 2) Major chemical contaminant sources of investigated area were determined to be the Miyagiwastewater purification facility (WPF) and the Mikawajima WPF at the upstream and downstream sides ofthe TUS, respectively. 3) We presume a two-fraction zone in the Sumida River from our analytical resultsand public data, including a "high COD zone" with an upstream stagnant zone upper the Shirahige Bridge,and a "low COD zone" with a downstream flush zone under the Shirahige Bridge. Raising environmentalawareness concerning the urban river among its residents presumably might contribute to maintaining itsclean and safe environment. We came to conclusion that the importance of educating the citizens aboutkeeping urban rivers environmentally clean and safe for the future generations

Study of Sumida River, Part 2: Analysis of Identification of COD and EC Characteristics in Fall 2021

The Sumida River streamside makes up a part of the Kitasenjyu campus at Teikyo University of Science(TUS). It is essential for university students and faculty members to learn its environmental and chemical characteristics from the viewpoint of environmental education. A water analysis of the Sumida River was conducted in the fall season (Fall 2021) at 10 locations around the Kitasenjyu campus, along with a 24-hour continuous survey in front of the No. 7 building. The fall analysis was performed using the same procedure as the previous analysis in spring (Spring 2021). The conclusions we reached are as follows: 1) Discharge water from the Mikawagima wastewater purification facility (WPF) was found to be the primary source of chemical contamination in the study area, causing an increase in COD (chemical oxygen demand). Upstream, Miyagi WPF was presumed to be the primary source of the chemical contamination. 2) The discharged high COD water from these WPFs was diffused by the tidal movement of Tokyo Bay and then was homogenized in the study area. 3) From our survey on the streaming motion of the Sumida River and public COD data from the Tokyo metropolitan government, we redefined the boundary between high and low COD zones set under the Agastuma Bridge. 4) Urethane foam with photocatalysis material of TiO2 was synthesized as a novel wastewater treatment material. As it displayed good decomposition characteristics oforganic material in tested water, porous and robust materials with TiO2 for continuous outdoor use should be investigated to achieve practical applications shortly

Japan Prosthodontic Society position paper on “occlusal discomfort syndrome”

Purpose: Dentists may encounter patients who present with a sense of a malocclusion but in whom no objective findings can be detected. For the patient who insists that there is occlusal discomfort, in the absence of evidence some dentists elect to perform an occlusal adjustment that not only fails to alleviate symptoms, and may, in fact, exacerbate the discomfort. The patient–dentist relationship is then likely compromised because of a lack of trust. Study selection: In 2011, the Clinical Practice Guidelines Committee of the Japan Prosthodontic Society formulated guidelines for the management of occlusal discomfort. When formulating clinical practice guidelines, the committee bases their recommendations on information derived from scientific evidence. For ‘‘occlusal dysesthesia,’’ however, there are an insufficient number of high-quality papers related to the subject. Therefore, a consensus meeting was convened by the Japan Prosthodontic Society to examine evidence in the Japanese- and English-language literature and generate a multi-center survey to create an appropriate appellation for this condition. Results: As a result of the consensus meeting and survey findings, this condition may be justifiably termed ‘‘occlusal discomfort syndrome.’’ Conclusions: The Japan Prosthodontics Society believes that identification of an umbrella term for occlusal discomfort might serve as a useful guide to formulating clinical practice guidelines in the future. This position paper represents summary findings in the literature combined with the results of a multicenter survey focused on dental occlusal treatment and the condition of patients who present with occlusal discomfort syndrome