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We challenged the stabilization of a G‐protein coupled receptor (GPCR) in the active state solely by multiple amino‐acid mutations without the agonist binding. For many GPCRs, the free energy of the active state is higher than that of the inactive state. When the inactive state is stabilized through the lowering of its free energy, the apparent midpoint temperature of thermal denaturation Tm exhibits a significant increase. However, this is not always the case for the stabilization of the active state. We constructed a modified version of our methodology combining statistical thermodynamics and evolutionary molecular engineering, which was recently developed for the inactive state. First, several residues to be mutated are determined using our statistical‐thermodynamics theory. Second, a gene (mutant) library is constructed using Escherichia coli cells to efficiently explore most of the mutational space. Third, for the mutant screening, the mutants prepared in accordance with the library are expressed in engineered Saccharomyces cerevisiae YB14 cells which can grow only when a GPCR mutant stabilized in the active state has signaling function. For the adenosine A2A receptor tested, the methodology enabled us to sort out two triple mutants and a double mutant. It was experimentally corroborated that all the mutants exhibit much higher binding affinity for G protein than the wild type. Analyses indicated that the mutations make the structural characteristics shift toward those of the active state. However, only slight increases in Tm resulted from the mutations, suggesting the unsuitability of Tm to the stability measure for the active state. [ABSTRACT FROM AUTHOR]

During embryonic development, cells differentiate in a coordinated manner, aligning their fate decisions and differentiation stages with those of surrounding cells. However, little is known about the mechanisms that regulate this synchrony. Here we show that cells in close proximity synchronize their differentiation stages and cellular phenotypes with each other via extracellular vesicle (EV)‐mediated cellular communication. We previously established a mouse embryonic stem cell (ESC) line harbouring an inducible constitutively active protein kinase A (CA‐PKA) gene and found that the ESCs rapidly differentiated into mesoderm after PKA activation. In the present study, we performed a co‐culture of Control‐ESCs and PKA‐ESCs, finding that both ESC types rapidly differentiated in synchrony even when PKA was activated only in PKA‐ESCs, a phenomenon we named 'Phenotypic Synchrony of Cells (PSyC)'. We further demonstrated PSyC was mediated by EVs containing miR‐132. PKA‐ESC‐derived EVs and miR‐132‐containing artificial nano‐vesicles similarly enhanced mesoderm and cardiomyocyte differentiation in ESCs and ex vivo embryos, respectively. PSyC is a new form of cell‐cell communication mediated by the EV regulation of neighbouring cells and could be broadly involved in tissue development and homeostasis. [ABSTRACT FROM AUTHOR]

Many plasma membrane (PM) functions depend on the cholesterol concentration in the PM in strikingly nonlinear, cooperative ways: fully functional in the presence of physiological cholesterol levels (35~45 mol%), and nonfunctional below 25 mol% cholesterol; namely, still in the presence of high concentrations of cholesterol. This suggests the involvement of cholesterol‐based complexes/domains formed cooperatively. In this review, by examining the results obtained by using fluorescent lipid analogs and avoiding the trap of circular logic, often found in the raft literature, we point out the fundamental similarities of liquid‐ordered (Lo)‐phase domains in giant unilamellar vesicles, Lo‐phase‐like domains formed at lower temperatures in giant PM vesicles, and detergent‐resistant membranes: these domains are formed by cooperative interactions of cholesterol, saturated acyl chains, and unsaturated acyl chains, in the presence of >25 mol% cholesterol. The literature contains evidence, indicating that the domains formed by the same basic cooperative molecular interactions exist and play essential roles in signal transduction in the PM. Therefore, as a working definition, we propose that raft domains in the PM are liquid‐like molecular complexes/domains formed by cooperative interactions of cholesterol with saturated acyl chains as well as unsaturated acyl chains, due to saturated acyl chains' weak multiple accommodating interactions with cholesterol and cholesterol's low miscibility with unsaturated acyl chains and TM proteins. Molecules move within raft domains and exchange with those in the bulk PM. We provide a logically established collection of fluorescent lipid probes that preferentially partition into raft and non‐raft domains, as defined here, in the PM. [ABSTRACT FROM AUTHOR]

Early elevation of factor IX level in japanese brothers with Haemophilia B Leyden who are carrying c. -35 g > a mutations in the promoter region of F9 Haemophilia B Leyden may be overlooked in cases of mild haemophilia B. Hence, there is a need to recognise that haemophilia B Leyden is also found in Japanese. Keywords: c. -35 G > A; early elevation; factor IX; Haemophilia B Leyden; Japanese EN c. -35 G > A early elevation factor IX Haemophilia B Leyden Japanese e510 e512 3 07/21/21 20210701 NES 210701 INTRODUCTION Haemophilia B Leyden is a rare subtype of haemophilia B, which is caused by mutations within a 40-basepair region called Leyden-specific region in the I F9 i promoter. [Extracted from the article]

Cholesterol distribution and dynamics in the plasma membrane ( PM) are poorly understood. The recent development of Bodipy488-conjugated cholesterol molecule (Bdp-Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent-molecule imaging. Surprisingly, in the intact PM, Bdp-Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient ( D) of 3.4 µm2/second, which was ∼10 times greater than that of non-raft phospholipid molecules (0.33 µm2/second), despite Bdp-Chol's probable association with raft domains. Furthermore, Bdp-Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp-Chol and Cy3-conjugated dioleoylphosphatidylethanolamine (Cy3-DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm2/second, respectively), indicating that the actin-based membrane skeleton reduces the D of Bdp-Chol only by a factor of ∼2 from that in the blebbed PM, whereas it reduces the D of Cy3- DOPE by a factor of ∼20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin-based membrane-skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp-Chol passing through the compartment boundaries, once it enters the boundary, is ∼10× greater than that of Cy3- DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane-skeleton-induced compartments and are contained within them. [ABSTRACT FROM AUTHOR]

Abstract: Single-molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano-meso-scales (2–200nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time-dependent inhomogeneous two-dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions. [Copyright &y& Elsevier]

The communications environment is currently well organized. Still, it is not easy to handle requirements for urgent communications in a disaster. In a large-scale disaster, it is highly probable that communications functions will be hampered, making access to communication links itself difficult. There is a need for studies directed at assuring robustness and reliability in the emergency communications system. This paper proposes an access protocol for wireless networks in which the centralized control hierarchical network and the horizontal connection ad hoc network are combined. The routing method in which a terminal that cannot directly communicate with the base station (BS) sends urgent data to the BS through neighboring terminals, and an access control protocol for multihopping, are described. In the proposed scheme, the terminal monitors communications in neighboring nodes and dynamically generates a route based on the number of hops to the BS. Evaluations show that the percentage of terminals that can reach the BS by multihopping involving a few hops is significantly improved. It is also shown that the terminal knows the number of hops to the BS in the proposed method, which makes it possible to predict the throughput and the delay time, which is a property suited to urgent communications. © 2005 Wiley Periodicals, Inc. Electron Comm Jpn Pt 1, 88(7): 52–64, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecja.20141 [ABSTRACT FROM AUTHOR]