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When interfaces utilizing EEG signals are used, these signals can be induced through diverse association techniques. In the present article, differences in recognition performance of the systems related to robot control techniques according to diverse association techniques have been analyzed. For this purpose, elements that can affect the performance other than association techniques were excluded as much as possible, and the focus was placed on the performance analysis per association technique. According to the study results of the present article, the movement association technique based on gestures showed the highest recognition performance in the robot control system for multiple users. In the robot control system for single users, hearing-based phonation association is considered to show the highest recognition performance. In the study results of the present article, superior recognition performance is considered to be derived than the recognition performance displayed by existing systems when applied to the robot control area utilizing EEG signals.

The geographic and biological diversity of China has resulted in the differential adaptation of the eastern honeybee, Apis cerana, to these varied habitats. A. cerana were collected from 14 locations in China. Their genomes were sequenced, and nucleotide polymorphisms were identified at more than 9 million sites. Both STRUCTURE and principal component analysis placed the bees into seven groups. Phylogenomic analysis groups the honeybees into many of the same clusters with high bootstrap values (91%–100%). Populations from Tibet and South Yunnan are sister taxa and together represent the earliest diverging lineage included in this study. We propose that the evolutionary origin of A. cerana in China was in the southern region of Yunnan Province and expanded from there into the southeastern regions and into the northeastern mountain regions. The Cold‐Temperate West Sichuan Plateau and Tropical Diannan populations were compared to identify genes under adaptive selection in these two habitats. Pathway enrichment analysis showing genes under selection, including the Hippo signaling pathway, GABAergic pathway, and trehalose‐phosphate synthase, indicates that most genes under selection pressure are involved in the process of signal transduction and energy metabolism. qRT‐PCR analysis reveals that one gene under selection, the AcVIAAT gene, involved in the GABAergic pathway, is responding to cold temperature stress. Through homologous recombination, we show that the AcVIAAT gene is able to replace the CNAG_01904 gene in the fungus Cryptococcus neoformans and that it makes the fungus less sensitive to conditions of oxidative stress and variations in temperature. Our results contribute to our understanding of the evolutionary origin of A. cerana in China and the molecular basis of environmental adaptation., We identified the Asian honeybee as seven genetic lineages and clarified their genetic relationship and then proposed that the origin of Chinese honeybee in the southern Asian geographic region including south Tibet, south Yunnan, and Himalaya–Hengduan Mountains. We found many genes in response to the abiotic stress were under selection for the genetic population in the regions of higher altitudes. Among these selective genes, based on homologous recombination in C. neoformans, we firstly clarified the gene involved in the pathway of transporting gamma‐aminobutyrate has contribution to tolerate the abiotic stress.

In this chapter we will present a recently developed simulation system using first principle calculations. The first block is based on Geant4 and is devoted to the simulation of the radiation-matter interaction. This toolkit allows to define the photon source type, any possible collimators or filters, the properties of sensing material and the geometrical configuration of the whole system. Simultaneously, the second block calculates the electric and weighting fields through a finite element method given the material properties, the geometry of the electrodes and the applied bias voltage. By combining the interaction positions of each photon and the deposited energy with the information provided by the second block, the charge transport and signal induction can be calculated by solving the equation of motion. In the last step, the spectrum can be reconstructed by applying the operations of the read-out electronics on the simulated current transients. We will present a comparison between simulated and real spectra in a wide range of radiation energies and for detectors with extremely different geometries and dimensions.

The brain processes information by transmitting signals through highly connected and dynamic networks of neurons. Neurons use specific cellular structures, including axons, dendrites and synapses, and specific molecules, including cell adhesion molecules, ion channels and chemical receptors to form, maintain and communicate among cells in the networks. These cellular and molecular processes take place in environments rich of mechanical cues, thus offering ample opportunities for mechanical regulation of neural development and function. Recent studies have suggested the importance of mechanical cues and their potential regulatory roles in the development and maintenance of these neuronal structures. Also suggested are the importance of mechanical cues and their potential regulatory roles in the interaction and function of molecules mediating the interneuronal communications. In this review, the current understanding is integrated and promising future directions of neuromechanobiology are suggested at the cellular and molecular levels. Several neuronal processes where mechanics likely plays a role are examined and how forces affect ligand binding, conformational change, and signal induction of molecules key to these neuronal processes are indicated, especially at the synapse. The disease relevance of neuromechanobiology as well as therapies and engineering solutions to neurological disorders stemmed from this emergent field of study are also discussed.

Glucagon-like peptide-1 receptor (GLP-1R) agonist-based therapeutics for type 2 diabetes mellitus have attracted worldwide attention. However, there are challenges in the development of small molecule GLP-1R agonists owing to the complexity of ligand recognition and signal induction mechanisms. Here, we attained S6 using virtual screening and fluorescent imaging plate reader (FLIPR)-based calcium assays. The purpose of this study was to identify and characterize S6, a novel small molecule GLP-1R agonist. Data from cellular thermal shift assay (CETSA) and Bio-Layer Interferometry (BLI) indicated that S6 could bind potently with GLP-1R. Radioimmunoassay data showed that S6 potentiated insulin secretion in a glucose-dependent manner and the insulinotropic effect was mediated by GLP-1R. Calcium imaging techniques suggested that S6 elevated the intracellular calcium concentration [(Ca2+)i] by activating GLP-1R. In patch-clamp experiments, we demonstrated that S6 inhibited voltage-dependent K+ (Kv) channels in a GLP-1R-dependent fashion. Besides, S6 significantly prolonged action potential duration but had no effect on voltage-dependent Ca2+ channels. In summary, these findings indicate that S6 stimulates glucose-dependent insulin secretion mainly by acting on GLP-1R, inhibiting Kv channels, increasing (Ca2+)i. This study will provide direction for the screening and development of novel small-molecule agents targeting GLP-1R in the future.

Innate behaviors consist of a succession of genetically-hardwired motor and physiological subprograms that can be coupled to drastic morphogenetic changes. How these integrative responses are orchestrated is not completely understood. Here, we provide insight into these mechanisms by studying pupariation, a multi-step innate behavior of Drosophila larvae that is critical for survival during metamorphosis. We find that the steroid-hormone ecdysone triggers parallel pupariation neuromotor and morphogenetic subprograms, which include the induction of the relaxin-peptide hormone, Dilp8, in the epidermis. Dilp8 acts on six Lgr3-positive thoracic interneurons to couple both subprograms in time and to instruct neuromotor subprogram switching during behavior. Our work reveals that interorgan feedback gates progression between subunits of an innate behavior and points to an ancestral neuromodulatory function of relaxin signaling., Pupariation in Drosophila is triggered by the steroid-hormone ecdysone and requires coordination between associated behavioral and body-reshaping motor subprograms. The authors show that coordination requires ecdysone-dependent Dilp8-Lgr3 signaling between the cuticle epidermis and interneurons.

Robust and precise ratio control of heterogeneous phenotypes within an isogenic population is an essential task, especially in the development and differentiation of a large number of cells such as bacteria, sensory receptors, and blood cells. However, the mechanisms of such ratio control are poorly understood. Here, we employ experimental and mathematical techniques to understand the combined effects of signal induction and gene expression stochasticity on phenotypic multimodality. We identify two strategies to control phenotypic ratios from an initially homogeneous population, suitable roughly to high-noise and low-noise intracellular environments, and we show that both can be used to generate precise fractional differentiation. In noisy gene expression contexts, such as those found in bacteria, induction within the circuit's bistable region is enough to cause noise-induced bimodality within a feasible time frame. However, in less noisy contexts, such as tightly controlled eukaryotic systems, spontaneous state transitions are rare and hence bimodality needs to be induced with a controlled pulse of induction that falls outside the bistable region. Finally, we show that noise levels, system response time, and ratio tuning accuracy impose trade-offs and limitations on both ratio control strategies, which guide the selection of strategy alternatives.

Background: Toll-like receptor (TLR) expression in patients with inflammatory bowel disease is increased when compared with healthy controls. However, the impact of TLR signaling during inflammatory bowel disease is not fully understood. Methods: In this study, we used a murine model of acute phase inflammation in bone marrow chimeric mice to investigate in which cell type TLR2/4 signal induction is important in preventing intestinal inflammation and how intestinal dendritic cells are influenced. Mice were either fed with wild-type bacteria, able to initiate the TLR2/4 signaling cascade, or with mutant strains with impaired signal induction capacity. Results: The induction of the TLR2/4 signal cascade in epithelial cells resulted in inflammation in bone marrow chimeric mice, whereas induction in hematopoietic cells had an opposed function. Furthermore, feeding of wild-type bacteria prevented disease; however, differing signal induction of bacteria had no effect on lamina propria dendritic cell activation. In contrast, functional TLR2/4 signals resulted in increased frequencies of CD103-expressing lamina propria and mesenteric lymph node dendritic cells, which were able to ameliorate disease. Conclusions: The TLR-mediated amelioration of disease, the increase in CD103-expressing cells, and the beneficial function of TLR signal induction in hematopoietic cells indicate that the increased expression of TLRs in patients with inflammatory bowel disease might result in counterregulation of the host and serve in preventing disease.

Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry.

The emergence of low molecular weight kinase inhibitors as "targeted" drugs has led to remarkable advances in the treatment of cancer patients. The clinical benefits of these tumor therapies, however, vary widely in patient populations and with duration of treatment. Intrinsic and acquired resistance against such drugs limits their efficacy. In addition to the well studied mechanisms of resistance based upon drug transport and metabolism, genetic alterations in drug target structures and the activation of compensatory cell signaling have received recent attention. Adaptive responses can be triggered which counteract the initial dependence of tumor cells upon a particular signaling molecule and allow only a transient inhibition of tumor cell growth. These compensating signaling mechanisms are often based upon the relief of repression of regulatory feedback loops. They might involve cell autonomous, intracellular events or they can be mediated via the secretion of growth factor receptor ligands into the tumor microenvironment and signal induction in an auto- or paracrine fashion. The transcription factors Stat3 and Stat5 mediate the biological functions of cytokines, interleukins and growth factors and can be considered as endpoints of multiple signaling pathways. In normal cells this activation is transient and the Stat molecules return to their non-phosphorylated state within a short time period. In tumor cells the balance between activating and de-activating signals is disturbed resulting in the persistent activation of Stat3 or Stat5. The constant activation of Stat3 induces the expression of target genes, which cause the proliferation and survival of cancer cells, as well as their migration and invasive behavior. Activating components of the Jak-Stat pathway have been recognized as potentially valuable drug targets and important principles of compensatory signaling circuit induction during targeted drug treatment have been discovered in the context of kinase inhibition studies in HNSCC cells [1]. The treatment of HNSCC with a specific inhibitor of c-Src, initially resulted in reduced Stat3 and Stat5 activation and subsequently an arrest of cell proliferation and increased apoptosis. However, the inhibition of c-Src only caused a persistent inhibition of Stat5, whereas the inhibition of Stat3 was only transient. The activation of Stat3 was restored within a short time period in the presence of the c-Src inhibitor. This process is mediated through the suppression of P-Stat5 activity and the decrease in the expression of the Stat5 dependent target gene SOCS2, a negative regulator of Jak2. Jak2 activity is enhanced upon SOCS2 downregulation and causes the reactivation of Stat3. A similar observation has been made upon inhibition of Bmx, bone marrow kinase x-linked, activated in the murine glioma cell lines Tu-2449 and Tu-9648. Its inhibition resulted in a transient decrease of P-Stat3 and the induction of a compensatory Stat3 activation mechanism, possibly through the relief of negative feedback inhibition and Jak2 activation. These observations indicate that the inhibition of a single tyrosine kinase might not be sufficient to induce lasting therapeutic effects in cancer patients. Compensatory kinases and pathways might become activated and maintain the growth and survival of tumor cells. The definition of these escape pathways and their preemptive inhibition will suggest effective new combination therapies for cancer.

To better understand the role of nitric oxide (NO) in mango fruit ripening, fruit that was pretreated with salicylic acid (SA) and NO was stored at 25 °C for 11 days. Fruit firmness, ethylene production, total soluble solids (TSS), titratable acidity (TA) and vitamin C content were monitored. The expression of six genes involved in ethylene biosynthesis and signalling, namely, MiACO, MiACS, MiETR1, MiERS1, MiEIN2 and MiERF, was investigated using real-time qPCR. The results showed that the treatment caused significant inhibition of the firmness decrease and ethylene output, maintained relatively high levels of TSS and vitamin C, and delayed the decrease in the TA. Furthermore, MiACO and MiERS1 were significantly downregulated, whereas MiETR1 and MiEIN2 were upregulated, and MiACS and MiERF exhibited fluctuating expression patterns. The findings of the study suggested that MiACO, MiACS, MiETR1, MiERS1, MiEIN2 and MiERF might participate in the SA and NO signal induction pathways during ripening.

The RIG-I-like receptors (RLRs)—RIG-I, MDA5, and LGP2—detect intracellular pathogenic RNA and elicit an antiviral immune response during viral infection. The protein architecture of the RLR family consists of multiple functional domains, including N-terminal Caspase Activation and Recruitment Domains (CARDs) for signaling initiation, a central RNA helicase core, and a C-terminal domain for RNA sensing. With these specialized sensing-and-responding modules, RLRs are able to selectively bind non-self RNA species and trigger downstream signaling events leading to interferon production. This article summarizes the recent progress toward defining the precise mechanisms of RNA recognition and subsequent signal induction by RLRs. Accepted version

Bidirectional intercellular signaling is an essential feature of multicellular organisms, and the engineering of complex biological systems will require multiple pathways for intercellular signaling with minimal crosstalk. Natural quorum‐sensing systems provide components for cell communication, but their use is often constrained by signal crosstalk. We have established new orthogonal systems for cell–cell communication using acyl homoserine lactone signaling systems. Quantitative measurements in contexts of differing receiver protein expression allowed us to separate different types of crosstalk between 3‐oxo‐C6‐ and 3‐oxo‐C12‐homoserine lactones, cognate receiver proteins, and DNA promoters. Mutating promoter sequences minimized interactions with heterologous receiver proteins. We used experimental data to parameterize a computational model for signal crosstalk and to estimate the effect of receiver protein levels on signal crosstalk. We used this model to predict optimal expression levels for receiver proteins, to create an effective two‐channel cell communication device. Establishment of a novel spatial assay allowed measurement of interactions between geometrically constrained cell populations via these diffusible signals. We built relay devices capable of long‐range signal propagation mediated by cycles of signal induction, communication and response by discrete cell populations. This work demonstrates the ability to systematically reduce crosstalk within intercellular signaling systems and to use these systems to engineer complex spatiotemporal patterning in cell populations.

Distinct ROS signaling pathways initiated by singlet oxygen (1O2) or superoxide and hydrogen peroxide have been attributed to either cell death or acclimation, respectively. Recent studies have revealed that more complex antagonistic and synergistic relationships exist within and between these pathways. As specific chloroplastic ROS signals are difficult to study, rapid systemic signaling experiments using localized high light (HL) stress or ROS treatments were used in this study to uncouple signals required for direct HL and ROS perception and distal systemic acquired acclimation (SAA). A qPCR approach was chosen to determine local perception and distal signal reception. Analysis of a thylakoidal ascorbate peroxidase mutant (tapx), the 1O2-retrograde signaling double mutant (ex1/ex2), and an apoplastic signaling double mutant (rbohD/F) revealed that tAPX and EXECUTER 1 are required for both HL and systemic acclimation stress perception. Apoplastic membrane-localized RBOHs were required for systemic spread of the signal but not for local signal induction in directly stressed tissues. Endogenous ROS treatments revealed a very strong systemic response induced by a localized 1 h induction of 1O2 using the conditional flu mutant. A qPCR time course of 1O2 induced systemic marker genes in directly and indirectly connected leaves revealed a direct vascular connection component of both immediate and longer term SAA signaling responses. These results reveal the importance of an EXECUTER-dependent 1O2 retrograde signal for both local and long distance RBOH-dependent acclimation signaling that is distinct from other HL signaling pathways, and that direct vascular connections have a role in spatial-temporal SAA induction.

The induction and regulation network of tumor cell apoptosis is built by integrating several seperated pathway of signal transduction and analyzing the properties and process. Three local area regulation centers such as caspase family, Bcl-2 family and Cyt C protein are found and the mechanisms of signal induction and regulation of every center are discussed.

Water oxidation in photosystem II is catalyzed by the CaMn(4) cluster. The electrons extracted from the CaMn(4) cluster are transferred to P(680)(+) via the redox-active tyrosine residue D1-Tyr161 (Y(Z)). The oxidation of Y(Z) is coupled to a deprotonation creating the neutral radical Y(Z)(*). Light-induced oxidation of Y(Z) is possible down to extreme temperatures. This can be observed as a split EPR signal from Y(Z)(*) in a magnetic interaction with the CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active PSII. Here we have used the split S(0) EPR signal to study the mechanism of Y(Z) oxidation at 5 K in the S(0) state. The state of the hydrogen bond between Y(Z) and its proposed hydrogen bond partner D1-His190 is investigated by varying the pH. The split S(0) EPR signal was induced by illumination at 5 K between pH 3.9 and pH 9.0. Maximum signal intensity was observed between pH 6 and pH 7. On both the acidic and alkaline sides the signal intensity decreased with the apparent pK(a)s (pK(app)) approximately 4.8 and approximately 7.9, respectively. The illumination protocol used to induce the split S(0) EPR signal also induces a mixed radical signal in the g approximately 2 region. One part of this signal decays with similar kinetics as the split S(0) EPR signal ( approximately 3 min, at 5 K) and is easily distinguished from a stable radical originating from Car/Chl. We suggest that this fast-decaying radical originates from Y(Z)(*). The pH dependence of the light-induced fast-decaying radical was measured in the same pH range as for the split S(0) EPR signal. The pK(app) for the light-induced fast-decaying radical was identical at acidic pH ( approximately 4.8). At alkaline pH the behavior was more complex. Between pH 6.6 and pH 7.7 the signal decreased with pK(app) approximately 7.2. However, above pH 7.7 the induction of the radical species was pH independent. We compare our results with the pH dependence of the split S(1) EPR signal induced at 5 K and the S(0) --> S(1) and S(1) --> S(2) transitions at room temperature. The result allows mechanistic conclusions concerning differences between the hydrogen bond pattern around Y(Z) in the S(0) and S(1) states.

Enteropathogenic Escherichia coli (EPEC) mimic a ligand-receptor interaction to induce 'pedestal-like' pseudopodia on mammalian cells, providing a tractable system to study tyrosine kinase signalling to the actin cytoskeleton. EPEC delivers its own receptor (Tir), which is engaged by a bacterial surface ligand (intimin). When Tir delivery and activity are uncoupled, intimin-induced Tir clustering stimulates Tir(Y474) phosphorylation by the Src-family kinase (SFK) c-Fyn, triggering actin polymerization and pedestal formation. How c-Fyn specifically targets Tir and is regulated remains unknown. We show that clustering transfers Tir into cholesterol-rich detergent-resistant microdomains (DRMs), a signal prompting transient c-Fyn accumulation at bacterial adhesion sites. Co-clustering of Tir(Y474) and c-Fyn in DRMs rapidly stimulates robust kinase activation both by induced c-Fyn(Y531) dephosphorylation to unlock the inactive state and by reciprocal c-Fyn(Y417) autophosphorylation to promote activity. After signal induction, c-Fyn dissipates and the resting state restored by Csk-dependent phosphorylation of c-Fyn(Y531). These data illustrate a sophisticated mechanism evolved by a pathogen effector to reversibly regulate SFKs, and resolve early interactions at a model receptor initiating tyrosine kinase signalling.

beta-Galactosidase (beta-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) beta-d-galactopyranoside (DDAOG), can be cleaved by beta-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a beta-gal activity assay method. The DDAO signal was stable for at least 18h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the beta-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The beta-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The beta-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of beta-gal systems currently in use.

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice., Author Summary N-linked glycosylation is a post-translational modification occurring on membrane proteins such as G protein-coupled receptors (GPCR). Smoothened (Smo) is a GPCR that functions as the signal transducer of the Hedgehog (Hh) pathway. We used a mutagenesis approach to assess the role of N-glycans in Smo signaling in two genetic models for Hh pathway activity, Drosophila and mouse. In doing so, we discovered a divergence in glycan function between them. We mapped an essential N-glycan acceptor site that when lost in Drosophila, triggered ER retention, altered Smo protein stability and blunted its signaling capacity. Conversely, ER exit of the murine protein was unaffected by glycan loss, as was its ability to traffic and induce a G protein-independent signal to activate Hh target genes. However, the ability of vertebrate Smo to induce a distinct G protein-dependent signal was lost. This suggests that N-linked glycosylation may influence signal bias of vertebrate Smo to favor one signal output over the other. We therefore propose that the role of this conserved post-translational modification may have been repurposed from governing Smo ER exit in the fly to influencing effector route selection in vertebrates.

Summary: Polarized T cells are mobile cells optimized for migration, receptor scanning, and signaling. When in contact with antigen-presenting cells (APCs), polarized T cells can develop a spectrum of biophysical interaction modes ranging from adhesive sticking to dynamic crawling. Both static and dynamic contacts support sustained triggering of the T-cell receptor (TCR), leading to signal induction, T blast formation, and proliferation. In dynamic interactions, T cells crawl across the surface of the APC at speeds of 2–6 µm/min and simultaneously establish an asymmetric tight yet mobile junction plane, representing a dynamic immunological synapse. In dynamic synapses three functional compartments of the polarized T cell are in close contact with the APC surface, i.e. leading edge, cell body and uropod. Through its mobility, the asymmetric junction is topographically suited for receptor scanning and engagement at the leading edge, retrograde receptor movement along the junction, and exit from the uropod. Herein we develop a model on scanning encounters between T cells and APCs that includes the simultaneous engagement of T-cell leading edge and uropod and implicates a serial receptor triggering mode in cell-cell recognition.

Cell-cell interactions mediated by Notch and its ligands are known to effect many cell fate decisions in both invertebrates and vertebrates. However, the mechanisms involved in ligand induced Notch activation are unknown. Recently it was shown that, in at least some cases, endocytosis of the extracellular domain of Notch and ligand by the signaling cell is required for signal induction in the receptive cell. These results imply that soluble ligands (ligand extracellular domains) although capable of binding Notch would be unlikely to activate it. To test the potential activity of soluble Notch ligands, we generated monomeric and dimeric forms of the Notch ligand Delta-1 by fusing the extracellular domain to either a series of myc epitopes (Delta-1(ext-myc)) or to the Fc portion of human IgG-1 (Delta-1(ext-IgG)), respectively. Notch activation, assayed by inhibition of differentiation in C2 myoblasts and by HES1 transactivation in U20S cells, occurred when either Delta-1(ext-myc) or Delta-1(ext-IgG) were first immobilized on the plastic surface. However, Notch was not activated by either monomeric or dimeric ligand in solution (non-immobilized). Furthermore, both non-immobilized Delta-1(ext-myc) and Delta-1(ext-IgG) blocked the effect of immobilized Delta. These results indicate that Delta-1 extracellular domain must be immobilized to induce Notch activation in C2 or U20S cells and that non-immobilized Delta-1 extracellular domain is inhibitory to Notch function. These results imply that ligand stabilization may be essential for Notch activation.

Cognate interactions of naive T cells with antigen-presenting dendritic cells require physical cell–cell contacts leading to signal induction and T cell activation. Using a three-dimensional collagen matrix videomicroscopy model for ovalbumin peptide-specific activation of murine and oxidative mitogenesis of human T cells, we show that T cells maintain vigorous migration upon cognate interactions to DC (dendritic cell), continuously crawl across the DC surface, and rapidly detach (median within 6–12 min). These dynamic and short-lived encounters favor sequential contacts with the same or other DC and trigger calcium influx, upregulation of activation markers, T blast formation, and proliferation. We conclude that a tissue environment supports the accumulation of sequential signals, implicating a numeric or "digital" control mechanism for an ongoing primary immune response.

Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extracellular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis. Several other functions have been suggested for this protein, and its up-regulation is associated with various disease states related to calcification, including arterial plaque formation and the formation of kidney stones. Although expression of this gene has been demonstrated in multiple tissues, its regulation is not well understood. Our previous studies on the roles of the retinoblastoma protein (pRB) and p300/CBP in the regulation of osteoblast differentiation revealed a link between osteopontin induction and the synthesis of alkaline phosphatase. In this paper, we describe results specifically linking induction of osteopontin to the enzymatic activity of alkaline phosphatase in the medium, which results in the generation of free phosphate. This elevation of free phosphate in the medium is sufficient to signal induction of osteopontin RNA and protein. The strong and specific induction of osteopontin in direct response to increased phosphate levels provides a mechanism to explain how expression of this product is normally regulated in bone and suggests how it may become up-regulated in damaged tissue.

The NF-kappaB precursor p105 has dual functions: cytoplasmic retention of attached NF-kappaB proteins and generation of p50 by processing. It is poorly understood whether these activities of p105 are responsive to signalling processes that are known to activate NF-kappaB p50-p65. We propose a model that p105 is inducibly degraded, and that its degradation liberates sequestered NF-kappaB subunits, including its processing product p50. p50 homodimers are specifically bound by the transcription activator Bcl-3. We show that TNFalpha, IL-1beta or phorbolester (PMA) trigger rapid formation of Bcl-3-p50 complexes with the same kinetics as activation of p50-p65 complexes. TNF-alpha-induced Bcl-3-p50 formation requires proteasome activity, but is independent of p50-p65 released from IkappaBalpha, indicating a pathway that involves p105 proteolysis. The IkappaB kinases IKKalpha and IKKbeta physically interact with p105 and inducibly phosphorylate three C-terminal serines. p105 is degraded upon TNF-alpha stimulation, but only when the IKK phospho-acceptor sites are intact. Furthermore, a p105 mutant, lacking the IKK phosphorylation sites, acts as a super-repressor of IKK-induced NF-kappaB transcriptional activity. Thus, the known NF-kappaB stimuli not only cause nuclear accumulation of p50-p65 heterodimers but also of Bcl-3-p50 and perhaps further transcription activator complexes which are formed upon IKK-mediated p105 degradation.

Illuminating a photosystem II sample at low temperatures (here 5-10 K) yields so-called split signals detectable with continuous wave-electron paramagnetic resonance (CW-EPR). These signals reflect the oxidized, deprotonated radical of D1-Tyr161 (YZ(•)) in a magnetic interaction with the CaMn4 cluster in a particular S state. The intensity of the split EPR signals are affected by the addition of the water substrate analogue methanol. This was previously shown by the induction of split EPR signals from the S1, S3, and S0 states [Su, J.-H. et al. (2006) Biochemistry 45, 7617-7627.]. Here, we use two split EPR signals induced from photosystem II trapped in the S2 state to further probe the binding of methanol in an S state dependent manner. The signals are induced with either visible or near-infrared light illumination provided at 5-10 K where methanol cannot bind or unbind from its site. The results imply that the binding of methanol not only changes the magnetic properties of the CaMn4 cluster but also the hydrogen bond network in the oxygen evolving complex (OEC), thereby affecting the relative charge of the S2 state. The induction mechanisms for the two split EPR signals are different resulting in two different redox states, S2YZ(•) and S1YZ(•) respectively. The two states show different methanol dependence for their induction. This indicates the existence of two binding sites for methanol in the CaMn4 cluster. It is proposed that methanol binds to MnA with high affinity and to MnD with lower affinity. The molecular nature and S-state dependence of the methanol binding to each respective site are discussed.

Quantitative correlation between intracellular calcium signals and hapten density or molecular size of antigens was studied for two cell lines; hapten-specific murine B cells (TP67.21) and rat basophilic leukemia cells (RBL-2H3) with hapten-specific IgE. Magnitude of the induced calcium signal in both cells exhibited the same dependence on hapten density of antigen molecules and there existed an optimal hapten density which induced the maximum amount of calcium signal for both cells. However, they responded differently to antigens of various molecular size. In contrast to TP67.21 cells which showed larger response to larger antigen molecules, RBL-2H3 cells showed the largest response to the smallest antigen. This may possibly suggest that there exists an optimal structure of receptor aggregates for each cell. Calcium signal induced in each cell by multivalent antigen was rapidly abrogated by addition of excess hapten and this abrogation occurred both in transmembrane influx and the release from intracellular stores. We directly observed the mobilization of receptor molecules during this calcium signal abrogation at single cell level by using two fluorescent calcium probes, whose fluorescence wavelength ranges have least overlap, and confocal microscopy. During this abrogation, large clusters of receptor molecules were not affected by hapten molecules. We, therefore, conclude that these large clusters are inactive in the induction of calcium signal and smaller clusters of receptor molecules are necessary for calcium signal induction.

In unstimulated cells, the transcription factor NF-kappaB is held in the cytoplasm in an inactive state by the inhibitor protein IkappaBalpha. Stimulation of cells results in rapid phosphorylation and degradation of IkappaBalpha, thus releasing NF-kappaB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor alpha results in the rapid accumulation of higher molecular weight forms of IkappaBalpha that dissociate from NF-kappaB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IkappaBalpha by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IkappaBalpha was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IkappaBalpha from cells expressing epitope-tagged versions of IkappaBalpha and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IkappaBalpha that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-kappaB involves phosphorylation-dependent ubiquitination of IkappaBalpha, which targets the protein for rapid degradation by the proteasome and releases NF-kappaB for translocation to the nucleus.

Communication between cells and extracellular environments is of interest because of its critical roles in cell development and differentiation. Particularly, this signal transduction is commonly believed to rely on the contact and binding of the participating molecules/proteins, suggesting that the binding distance needed is less than a few nanometers. However, it is difficult to precisely match the rapidly binding interaction which depends on the probability of molecular collision in living systems, raising a hypothesis that another mechanism exists, could promote this signal communication, and remains unknown. Here we report that a long-range signal delivery over 10-μm and 20-μm polydimethylsiloxane(PDMS) barriers can be observed in microfluidically tetracycline (Tet) inducible expression systems. Results show that a significant increment of the long-range induced green fluorescent protein in human embryonic kidney 293T (HEK 293T) cells by the stimulation of Tet is demonstrated, and that such a signal induction is not dominated by Tet diffusion and displays a specific bindingless property. In addition, our experimental results, combined with theoretical modeling, suggest that this communication exhibits a bump-shaped characteristic depending on barrier thickness, materially structural property, surface roughness, and agonist concentration. It strongly relies on the PDMS barrier to delivery signal; therefore, we call such a mechanism as “wired” cell communication instead of wireless. These results could ignite interests in the novel and “wired” cell communication, which we call it X-signal, and in the use of such systems for the study of cellular biology and development of new drug.

Antigenic modulation is one of many factors determining the effectiveness of monoclonal antibody (MoAb)-mediated therapy. To select the isotype of a CD19 MoAb most suitable for radioimmunotherapy of patients with B-cell malignancies, we studied the influence of MoAb isotype on modulation, after binding of the MoAb to different cell-line cells. The CD19-IgG1 MoAb was found to induce modulation of CD19 antigens on Daudi cell line cells more rapidly than did its IgG2a switch variant. We provide evidence that this difference in modulation rate is caused by the expression of Fc gamma receptor II (Fc gamma RII) on these cells. Experiments aimed at elucidating the mechanism of Fc gamma RII involvement in modulation induction by CD19-IgG1 showed that Fc gamma RII did not comodulate with CD19 MoAbs. However, cocrosslinking of CD19 and Fc gamma RII with CD19-IgG1 MoAb resulted in enhanced calcium mobilization in Daudi cells. This increased signal induction accompanies the enhanced capping and subsequent modulation of CD19 antigens. Because Fc gamma RII is expressed in varying densities on malignant B cells in all differentiation stages, our results have implications for the MoAb isotype most suitable for use in MoAb-based therapy of patients with B-cell malignancies.

The electrons extracted from the CaMn(4) cluster during water oxidation in photosystem II are transferred to P(680)(+) via the redox-active tyrosine D1-Tyr161 (Y(Z)). Upon Y(Z) oxidation a proton moves in a hydrogen bond toward D1-His190 (His(Z)). The deprotonation and reprotonation mechanism of Y(Z)-OH/Y(Z)-O is of key importance for the catalytic turnover of photosystem II. By light illumination at liquid helium temperatures (∼5 K) Y(Z) can be oxidized to its neutral radical, Y(Z)(•). This can be followed by the induction of a split EPR signal from Y(Z)(•) in a magnetic interaction with the CaMn(4) cluster, offering a way to probe for Y(Z) oxidation in active photosystem II. In the S(3) state, light in the near-infrared region induces the split S(3) EPR signal, S(2)'Y(Z)(•). Here we report on the pH dependence for the induction of S(2)'Y(Z)(•) between pH 4.0 and pH 8.7. At acidic pH the split S(3) EPR signal decreases with the apparent pK(a) (pK(app)) ∼ 4.1. This can be correlated to a titration event that disrupts the essential H-bond in the Y(Z)-His(Z) motif. At alkaline pH, the split S(3) EPR signal decreases with the pK(app) ∼ 7.5. The analysis of this pH dependence is complicated by the presence of an alkaline-induced split EPR signal (pK(app) ∼ 8.3) promoted by a change in the redox potential of Y(Z). Our results allow dissection of the proton-coupled electron transfer reactions in the S(3) state and provide further evidence that the radical involved in the split EPR signals is indeed Y(Z)(•).

Notch signaling mediated by Delta-like (Dll) 4 is essential and sufficient for T-cell development in vivo. Stromal cells expressing Dll4 or Dll1, but not Jagged1, support T lymphopoiesis in vitro, but the molecular basis of this functional divergence among Notch ligands remains to be clarified. To examine this, we constructed chimeric variants composed of Dll4 and Jagged1. The intracellular regions were necessary, but interchangeable, for signal induction, and the extracellular regions determined the unique characteristics of the ligands. While Jagged1 induced minimal Notch signaling, Jagged2 elicited substantial levels of Hes1 transcripts and promoted T lymphopoiesis in vitro. Dll4 and Jagged2 showed a quantitative advantage when bound to fringe-modified Notch; this was not due to the Delta-Serrate-Lag2 domain, an extracellular region essential for interaction with Notch. These results suggest that different Notch ligands possess distinct potentials for the induction of Notch signaling through unique interactions of their extracellular regions with fringe-modified Notch. Furthermore, the magnitude of Notch signaling induced is critical for the determination of T-cell fate.

Signal transduction along the Ras/MAPK pathway has been generally thought to take place at the plasma membrane. It is now evident that the plasma membrane is not the only platform capable of Ras/MAPK signal induction. Fusion of Ras with green fluorescent protein and the development of genetically encoded fluorescent probes for Ras activation have revealed signaling events on a variety of intracellular membranes including endosomes, the Golgi apparatus and the endoplasmic reticulum. Thus, the Ras/MAPK pathway is spatially compartmentalized within cells and this may afford greater complexity of signal output.

A split EPR signal can be induced at 5 K in Photosystem II in the S3-state by light in the range of 400–900 nm. To investigate if the same mechanism is involved in the signal induction in the full spectral range we compared the properties of the S3 signal induced by 830 nm light or white light. Our results indicate that the same mechanism is responsible for the formation of the “Split S3” signal in the whole spectral range. The mechanism of the “Split S3” signal is not P680 driven but is instead driven by manganese excitation.

The mechanisms governing transition of the thyroid stimulating hormone (TSH) receptor (TSHR) from basal to active conformations are poorly understood. Considering that constitutively activating mutations (CAMs) and inactivating mutations in each of the extracellular loops (ECLs) trigger only partial TSHR activation or inactivation, respectively, we hypothesized that full signaling occurs via multiple extracellular signal propagation events. Therefore, individual CAMs in the extracellular region were combined to create double and triple mutants. In support of our hypothesis, combinations of mutants in the ECLs are in some cases additive, while in others they are even synergistic, with triple mutant I486A/I568V/V656F exhibiting a 70-fold increase in TSH-independent signaling. The proximity but likely different spatial orientation of the residues of activating and inactivating mutations in each ECL supports a dual functionality to facilitate signal induction and conduction, respectively. This is the first report for G-protein coupled receptors, suggesting that multiple and cooperative signal propagating events at all three ECLs are required for full receptor activation. Our findings provide new insights concerning molecular signal transmission from extracellular domains toward the transmembrane helix bundle of the glycoprotein hormone receptors.—Kleinau, G., Jaeschke, H., Mueller, S., Raaka, B. M., Neumann, S., Paschke, R., Krause, G. Evidence for cooperative signal triggering at the extracellular loops of the TSH receptor.

SUMMARY For proper understanding of image formation using charge carriers, it is shown that signal detection by means of induction must be considered. This explains the possibility of imaging insulators as well as other phenomena especially in the conditions of the environmental scanning electron microscope. In addition, a basic principle and method to separate the secondary and backscattered electrons is demonstrated.

Signal transduction pathways regulating NF-κB activation essential for microenvironment formation in secondary lymphoid organs remain to be determined. We investigated the effect of a deficiency of TNFR-associated factor 6 (TRAF6), which activates the classical NF-κB pathway, in splenic microenvironment formation. Two-week-old TRAF6-deficient mice showed severe defects in B cell follicle and marginal zone formation, similar to mutant mice defective in lymphotoxin (Lt) β receptor (LtβR) signal induction of nonclassical NF-κB activation. However, analysis revealed a TRAF6 role in architecture formation distinct from its role in the early neonatal Lt signaling pathway. LtβR signal was essential for primary B cell cluster formation with initial differentiation of follicular dendritic cells (FDCs) in neonatal mice. In contrast, TRAF6 was dispensable for progression to this stage but was required for converting B cell clusters to B cell follicles and maintaining FDCs through to later stages. Fetal liver transfer experiments suggested that TRAF6 in radiation-resistant cells is responsible for follicle formation. Despite FDC-specific surface marker expression, FDCs in neonatal TRAF6-deficient mice had lost the capability to express CXCL13. These data suggest that developmentally regulated activation of TRAF6 in FDCs is required for inducing CXCL13 expression to maintain B cell follicles.

We have discovered the signal initiation structure of the relaxin-like factor and shown its function to be independent of the amino acid side chains in the contact region. Evidence presented in this article suggests that signal induction is a function of the peptide bond and that completion of the signaling contact is initiated by ligand binding to the leucine-rich repeat G-protein coupled receptor 8 (LGR8). The specific mode of binding forces certain peptide bonds into a signaling position. This observation implies that the receiving structures are equally nonspecific so that signaling should occur at any peptide bond of the receptor or the trans-membrane loop that is within reach of the signaling wires of the receptor-bound ligand. Our observations offer an explanation for ligand cross-talk as well as for the ability of some antibodies to elicit the biological response normally associated with a specific ligand.

The first report of the crystal structure of the whole insulin receptor ectodomain dimer reveals how its multiple domains are organized. The structure also identifies the elusive second binding site on the receptor, and suggests how the high-affinity insulin–receptor complex, which leads to signal induction, is generated. The structure as now published differs dramatically from previous versions based on reconstructions from cryoelectron microscopy data. The crystal structure of the entire ectodomain dimer of the insulin receptor A isoform details the domain arrangement and defines the two sites for insulin binding. The insulin receptor is a phylogenetically ancient tyrosine kinase receptor found in organisms as primitive as cnidarians and insects. In higher organisms it is essential for glucose homeostasis1, whereas the closely related insulin-like growth factor receptor (IGF-1R) is involved in normal growth and development2. The insulin receptor is expressed in two isoforms, IR-A and IR-B; the former also functions as a high-affinity receptor for IGF-II and is implicated, along with IGF-1R, in malignant transformation3. Here we present the crystal structure at 3.8 A resolution of the IR-A ectodomain dimer, complexed with four Fabs from the monoclonal antibodies 83-7 and 83-14 (ref. 4), grown in the presence of a fragment of an insulin mimetic peptide5. The structure reveals the domain arrangement in the disulphide-linked ectodomain dimer, showing that the insulin receptor adopts a folded-over conformation that places the ligand-binding regions in juxtaposition. This arrangement is very different from previous models6. It shows that the two L1 domains are on opposite sides of the dimer, too far apart to allow insulin to bind both L1 domains simultaneously as previously proposed7. Instead, the structure implicates the carboxy-terminal surface of the first fibronectin type III domain as the second binding site involved in high-affinity binding.

The Her2/neu oncogene is overexpressed in various human cancers of epithelial origin and is associated with increased metastatic potential and poor prognosis. Blocking the Her2/neu signalling has been the focus of most therapeutic approaches. In this paper, the Her2/neu extracellular domain expressed in soluble form in yeast Pichia pastoris was used in order to isolate a fully human Fab fragment from a combinatorial Fab phage display library, derived from invaded lymph nodes of a breast cancer patient. The isolated fully human Fab63 binds specifically the native Her2/neu receptor and competes with Herceptin for binding to soluble Her2/neu receptor. In Her2/neu overexpressing cancer cells, Fab63 is rapidly internalized and has significant antiproliferative effects, where ligand-independent mechanisms dominate signal induction. Moreover, in the presence of the ligand heregulin, growth inhibition was also detected by Fab63. The human Fab63 is a non-immunogenic agent with unique properties that can be applied in diagnosis and cancer therapy, with great potential for further manipulation towards the generation of an effective anticancer molecule.

Differential expression of TBP-2 and Trx-1 occurs during osteoclastogenesis. Adenoviral overexpression of TBP-2 in osteoclast precursors inhibits Trx-1 expression, osteoclast formation, and AP-1 binding activity. TBP-2 and Trx-1 are key regulators of osteoclastogenesis. Introduction: Thioredoxin binding protein-2 (TBP-2) negatively regulates thioredoxin-1 (Trx-1), a key endogenous modulator of cellular redox and signaling. In gene array analysis, we found that TBP-2 expression was reduced during human osteoclast differentiation compared with macrophage differentiation. Our aim was to determine the roles of TBP-2 and Trx-1 in human osteoclastogenesis and RANKL signaling. Materials and Methods: Osteoclasts or macrophages were generated from colony-forming unit-granulocyte macrophage (CFU-GM) precursors treated with sRANKL and macrophage-colony-stimulating factor (M-CSF), or M-CSF alone, respectively. Expression of TBP-2 and Trx-1 was quantified by real-time PCR and Western analysis. Adenoviral gene transfer was used to overexpress TBP-2 in precursors. NF-κB and activator protein 1 (AP-1) signaling was assessed with EMSA. Results: In the presence of sRANKL, expression of TBP-2 was decreased, whereas Trx-1 expression was increased. The antioxidant N-acetylcysteine reversed this pattern and markedly inhibited osteoclastogenesis. Adenoviral overexpression of human TBP-2 in precursors inhibited osteoclastogenesis and Trx-1 expression, inhibited sRANKL-induced DNA binding of AP-1, but enhanced sRANKL-induced DNA binding of NF-κB. Conclusions: These data support significant roles for TBP-2 and the Trx system in osteoclast differentiation that are mediated by redox regulation of AP-1 transcription. A likely mechanism of stress signal induction of bone resorption is provided. Modulators of the Trx system such as antioxidants have potential as antiresorptive therapies.

The B cell antigen receptor (BCR) is a multimeric protein complex consisting of the ligand binding immunoglobulin molecule and the Ig-alpha/beta heterodimer that mediates intracellular signalling by coupling the receptor to protein tyrosine kinases (PTKs). Transfection of the Ig-alpha deficient myeloma cell line J558L microns with expression vectors coding for mutated Ig-alpha allowed us to test the function of the tyrosines in the cytoplasmic region of Ig-alpha in the context of the BCR. Furthermore we expressed Ig-alpha mutations as chimeric CD8-Ig-alpha molecules on K46 B lymphoma cells and tested their signalling capacity in terms of PTK activation and release of calcium. We show here that the conserved tyrosine residues in the cytoplasmic portion of Ig-alpha have a dual role. First, they are required for efficient activation of PTKs during signal induction and second, one of them is subject to phosphorylation by activated src-related PTKs. Phosphorylation on tyrosine in the cytoplasmic portion of Ig-alpha is discussed as a possible mechanism to couple the BCR to SH2 domain-carrying molecules.