Multiple Residues in the Second Extracellular Loop Are Critical for M3 Muscarinic Acetylcholine Receptor Activation

Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M3 muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys220 residue, Gln207, Gly211, Arg213, Gly218, Ile222, Phe224, Leu225, and Pro228 were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance

The Importance of Status in Markets: A Market Identity Perspective

[Excerpt] The importance of status in markets is well established. Since Podolny (1993) introduced his status-based model of market competition, more than 160 articles published in top management and sociology journals have explored this area. These articles show that market status affects a broad range of outcomes, including the perceived quality and legitimacy of organizations, the costs of producing a given level of quality, the prices that organizations can charge for specific products, their attractiveness as exchange partners, and their ability to exercise agency. Despite the widespread agreement that status plays an important role in markets, there is less agreement about how to define status. Status has traditionally been defined as a position in a social system that can be ranked among other positions based on relative prestige or social esteem (Weber, 1968; Linton, 1936; Merton, 1957). Following Podolny (1993), however, status has more recently been redefined simply as a signal of quality, thus removing status from its traditional anchoring in the social system. We argue that defining status as a signal of quality unnecessarily limits the explanatory power of status and confuses status with other signals of quality, most notably reputation. We develop instead a framework for studying status in markets that integrates work on status as social positions (Linton, 1936; Merton, 1957) and work on identity as social categories (Hannan, Polos, and Carroll, 2007), thus providing a more comprehensive perspective on status in markets. We seek to accomplish three specific objectives in this chapter. First, we develop a new conceptual framework that integrates theoretical research on status and identity to clarify what we understand by status in markets. Our status-identity framework builds on the definition of status as a position in a social system. However, we add that these positions entail identities that reside in the intersections of the horizontally and vertically differentiated social categories that define the social system. Our status-identity framework builds extensively on the existing research on status and identity because we seek to provide conceptual clarity to existing and future research on status and identity rather than distancing ourselves from that research. We emphasize that our framework must not only help distinguish status from other related theoretical concepts such as reputation and legitimacy, but must also be useful in empirical research by helping to develop theory-driven research agendas. Second, we use our status-identity framework to systematically review existing research on status in markets. Rather than reviewing all market status research, we use our framework to identify the most important theory-driven research questions, and then provide a selective review of how these questions have been addressed in empirical research. Third, having used the status-identity framework to identify important research questions and to evaluate how existing research addresses these questions, we then identify and discuss more systematically the most important areas for future research

Epoxyketone-Based Immunoproteasome Inhibitors

An efficient new route for the preparation of dihydroeponemycin, an active eponemycin derivative, is provided, which includes the synthesis of the intermediate compound, a hydroxymethyl-substituted enone. In addition, a method is provided for synthesizing inhibitors, which includes PI′-modified analogues. These analogues selectively bind to a major immunoproteasome catalytic subunit LMP2 and inactivate its proteolytic activity in a method of treating diseases, including myeloma and other cancers, Huntington\u27s disease and Alzheimer\u27s disease

Study for fast scanning with high axial resolution of Lattice light-sheet microscopy

Department of Biomedical EngineeringResearchers in the optical field have developed light-sheet microscopy to achieve fast scan rates and low photo-toxicity. However, the conventional light-sheet microscopy uses a Gaussian beam or a Bessel beam, so that the resolution in the z-axis direction is not significantly different from the confocal microscope widely used in biology and chemistry. In general, the resolution in the z-axis direction is only about 1/3 of the resolution in the x- and y-directions, although the resolution in the x- and y- direction has been greatly improved for several decades. This is because the side lobe of the light-sheet exists. Lattice light sheet microscopy overcomes these limitations with thin light sheets using 2D optical pattern. The lattice beam produces a sheet with a much narrower core, which provides a higher axial resolution. Sheet-shaped illumination eliminates out-of-focus fluorescence emissions and enables high-speed imaging with low photo-toxicity. The Lattice light-sheet microscope can construct a low fluorescence background grating beam and all excitation light is within a narrow focus depth. Therefore, light that is out of focus is not irradiated onto the sample, so damage by light is limited to the focal plane. We also provide a high-resolution, high-contrast image of 200 to 1000 images per second. In this experiment, we will check the performance of Lattice light-sheet microscope complements the disadvantages of conventional confocal microscopy and light sheet microscopy. A sheet-like pattern could be used to create a much thinner light sheet, and it was confirmed that light above the focal plane was gathered higher than the surrounding area. This focuses the fluorescence emission only on the focal plane to prevent further damage to the sample due to phototoxicity of unfocused light. It was also confirmed that the light was uniformly distributed in the light sheet. In addition, when compared to a single-vessel beam light sheet and a lattice light sheet, it was confirmed that the side lobes actually decreased. The fact that the resolution of the Lattice light-sheet microscopy in the z direction corresponds to the range of about 317 nm to 370 nm was confirmed by photographing a fluorescent bead sample.ope

Histamine and Histamine H4 Receptor Promotes Osteoclastogenesis in Rheumatoid Arthritis.

Histamine H4 receptor (H4R) has immune-modulatory and chemotaxic effects in various immune cells. This study aimed to determine the osteoclastogenic role of H4R in rheumatoid arthritis (RA). The concentration of histamine in synovial fluid (SF) and sera in patients with RA was measured using ELISA. After RA SF and peripheral blood (PB) CD14+ monocytes were treated with histamine, IL-17, IL-21 and IL-22, and a H4R antagonist (JNJ7777120), the gene expression H4R and RANKL was determined by real-time PCR. Osteoclastogenesis was assessed by counting TRAP-positive multinucleated cells in PB CD14+ monocytes cultured with histamine, Th17 cytokines and JNJ7777120. SF and serum concentration of histamine was higher in RA, compared with osteoarthritis and healthy controls. The expression of H4R was increased in PB monocytes in RA patients. Histamine, IL-6, IL-17, IL-21 and IL-22 induced the expression of H4R in monocytes. Histamine, IL-17, and IL-22 stimulated RANKL expression in RA monocytes and JNJ7777120 reduced the RANKL expression. Histamine and Th17 cytokines induced the osteoclast differentiation from monocytes and JNJ7777120 decreased the osteoclastogenesis. H4R mediates RANKL expression and osteoclast differentiation induced by histamine and Th17 cytokines. The blockage of H4R could be a new therapeutic modality for prevention of bone destruction in RA

Collaborative Platforms and Diversifying Partnerships of South-South Cooperation and Triangular Cooperation: Middle Powers’ Struggles for Nation Branding

This study illustrates collaborative platforms and diversifying partnerships for South-South and triangular cooperation in development. The English School’s pluralism-solidarism spectrum is applied as a tool to explain transformative features of the changing international society in times of crisis. The study focuses on the intermediary pluralist-solidarism phase that shows dynamics of middle power coalitions using nation branding and collaborative governance as key strategies. The transitional phase is exemplified by two approaches. One is the bilateral approach to coalition shown through the case of China, whereas the other is the inclusivemultilateral approach demonstrated through the case of South Korea. Implications are given toward relatively loose networks that have the potential to evolve into platforms with institutional grounds, especially for middle powers seeking opportunities in the new normal

Withanolides, Probes and Binding Targets and Methods of Use Thereof

Novel withanolide chemical genetic probes identify the in vivo binding target of withaferin A, which is the intermediate filament type III protein vimentin. In addition, a withanolide-based small molecule screening method screens drug candidates that target intermediate filament type III proteins. The method includes introducing a tagged linker covalently bonded to the withanolide molecule to form a withanolide probe. Better or alternative small molecule compounds as potential drug candidates can be generated based on their likely affinity for the determined binding site in vimentin. The affinity labeled withanolide can also be used to find intermediate filament-associated proteins using chemical proteomics by extracting proteins from cells that were exposed to withanolide-biotin analog. The withanolide probes can be used to monitor expression of vimentin, in tumor samples or other diseased tissues. Withaferin analogs can be used as a treatment for diverse vimentin-associated disorders, such as cancers, angiofibrotic diseases, and chronic inflammation

The Influence Of Job Insecurity On Career Commitment And Attitude In Multinational Corporations

As the perception of lifelong work shifts into lifelong career in the job insecurity market, the career development of employees through professional and competitive career management has become more important than being loyal to a lifelong work. Furthermore, in the case of multinational corporations, such as differentiation from the head office policy, cultural differences in labor relations, and the liquidity of business withdrawal, such a feature has a higher possibility of job insecurity than general companies. Therefore, the purpose of this study is to verify empirically how job insecurity influences career commitment and career attitude through individual, job and career characteristics as intermediation with the members of multinational corporations as objectives. For this purpose, a total of 366 questionnaire data that targeted 27 multinational corporations were collected and analyzed. The result shows that the job insecurity of multinational corporations affects individual characteristic rather than job or career characteristic, and it is confirmed that individual characteristic has an effect on career commitment and career attitude. In the end, multinational corporations, unlike ordinary domestic companies, need active organizational career development program that corresponds to an open corporate culture as well as innovative and open systems and policies that balance both internal and external networking activities in terms of human resource management of corporations

Electrogenic transport and K(+) ion channel expression by the human endolymphatic sac epithelium.

The endolymphatic sac (ES) is a cystic organ that is a part of the inner ear and is connected to the cochlea and vestibule. The ES is thought to be involved in inner ear ion homeostasis and fluid volume regulation for the maintenance of hearing and balance function. Many ion channels, transporters, and exchangers have been identified in the ES luminal epithelium, mainly in animal studies, but there has been no functional study investigating ion transport using human ES tissue. We designed the first functional experiments on electrogenic transport in human ES and investigated the contribution of K(+) channels in the electrogenic transport, which has been rarely identified, even in animal studies, using electrophysiological/pharmacological and molecular biological methods. As a result, we identified functional and molecular evidence for the essential participation of K(+) channels in the electrogenic transport of human ES epithelium. The identified K(+) channels involved in the electrogenic transport were KCNN2, KCNJ14, KCNK2, and KCNK6, and the K(+) transports via those channels are thought to play an important role in the maintenance of the unique ionic milieu of the inner ear fluid