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Sample records for constitutively active gpcr

A knockout mutation of a constitutive GPCR in Tetrahymena decreases both G-protein activity and chemoattraction.

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Thomas J Lampert

Full Text Available Although G-protein coupled receptors (GPCRs are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490. Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba²⁺ and K⁺, suggesting a decrease in basal excitability (decrease in Ca²⁺ channel activity. The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA and proteose peptone (PP, two known chemoattractants in Tetrahymena. Using microsomal [³⁵S]GTPγS binding assays, we found that wild-type (CU427 have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor, addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein.
Multiplexed profiling of GPCR activities by combining split TEV assays and EXT-based barcoded readouts.

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Galinski, Sabrina; Wichert, Sven P; Rossner, Moritz J; Wehr, Michael C

2018-05-25

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and are implicated in the physiological regulation of many biological processes. The high diversity of GPCRs and their physiological functions make them primary targets for therapeutic drugs. For the generation of novel compounds, however, selectivity towards a given target is a critical issue in drug development as structural similarities between members of GPCR subfamilies exist. Therefore, the activities of multiple GPCRs that are both closely and distantly related to assess compound selectivity need to be tested simultaneously. Here, we present a cell-based multiplexed GPCR activity assay, termed GPCRprofiler, which uses a β-arrestin recruitment strategy and combines split TEV protein-protein interaction and EXT-based barcode technologies. This approach enables simultaneous measurements of receptor activities of multiple GPCR-ligand combinations by applying massively parallelized reporter assays. In proof-of-principle experiments covering 19 different GPCRs, both the specificity of endogenous agonists and the polypharmacological effects of two known antipsychotics on GPCR activities were demonstrated. Technically, normalization of barcode reporters across individual assays allows quantitative pharmacological assays in a parallelized manner. In summary, the GPCRprofiler technique constitutes a flexible and scalable approach, which enables simultaneous profiling of compound actions on multiple receptor activities in living cells.
Advances in GPCR modeling evaluated by the GPCR Dock 2013 assessment

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Kufareva, Irina; Katritch, Vsevolod; Biggin, Phil

2014-01-01

Despite tremendous successes of GPCR crystallography, the receptors with available structures represent only a small fraction of human GPCRs. An important role of the modeling community is to maximize structural insights for the remaining receptors and complexes. The community-wide GPCR Dock asse...
Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

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Yin, Yanting; de Waal, Parker W.; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H. Eric

2017-01-01

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. PMID:28356352
Conformational entropic maps of functional coupling domains in GPCR activation: A case study with beta2 adrenergic receptor

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Liu, Fan; Abrol, Ravinder; Goddard, William, III; Dougherty, Dennis

2014-03-01

Entropic effect in GPCR activation is poorly understood. Based on the recent solved structures, researchers in the GPCR structural biology field have proposed several ``local activating switches'' that consisted of a few number of conserved residues, but have long ignored the collective dynamical effect (conformational entropy) of a domain comprised of an ensemble of residues. A new paradigm has been proposed recently that a GPCR can be viewed as a composition of several functional coupling domains, each of which undergoes order-to-disorder or disorder-to-order transitions upon activation. Here we identified and studied these functional coupling domains by comparing the local entropy changes of each residue between the inactive and active states of the β2 adrenergic receptor from computational simulation. We found that agonist and G-protein binding increases the heterogeneity of the entropy distribution in the receptor. This new activation paradigm and computational entropy analysis scheme provides novel ways to design functionally modified mutant and identify new allosteric sites for GPCRs. The authors thank NIH and Sanofi for funding this project.
A constitutively activating mutation alters the dynamics and energetics of a key conformational change in a ligand-free G protein-coupled receptor.

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Tsukamoto, Hisao; Farrens, David L

2013-09-27

G protein-coupled receptors (GPCRs) undergo dynamic transitions between active and inactive conformations. Usually, these conversions are triggered when the receptor detects an external signal, but some so-called constitutively activating mutations, or CAMs, induce a GPCR to bind and activate G proteins in the absence of external stimulation, in ways still not fully understood. Here, we investigated how a CAM alters the structure of a GPCR and the dynamics involved as the receptor transitions between different conformations. Our approach used site-directed fluorescence labeling (SDFL) spectroscopy to compare opsin, the ligand-free form of the GPCR rhodopsin, with opsin containing the CAM M257Y, focusing specifically on key movements that occur in the sixth transmembrane helix (TM6) during GPCR activation. The site-directed fluorescence labeling data indicate opsin is constrained to an inactive conformation both in detergent micelles and lipid membranes, but when it contains the M257Y CAM, opsin is more dynamic and can interact with a G protein mimetic. Further study of these receptors using tryptophan-induced quenching (TrIQ) methods indicates that in detergent, the CAM significantly increases the population of receptors in the active state, but not in lipids. Subsequent Arrhenius analysis of the TrIQ data suggests that, both in detergent and lipids, the CAM lowers the energy barrier for TM6 movement, a key transition required for conversion between the inactive and active conformations. Together, these data suggest that the lowered energy barrier is a primary effect of the CAM on the receptor dynamics and energetics.
Glycosylation of KSHV Encoded vGPCR Functions in Its Signaling and Tumorigenicity

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Hui Wu

2015-03-01

Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV is a tumor virus and the etiologic agent of Kaposi’s Sarcoma (KS. KSHV G protein-coupled receptor (vGPCR is an oncogene that is implicated in malignancies associated with KHSV infection. In this study, we show that vGPCR undergoes extensive N-linked glycosylation within the extracellular domains, specifically asparagines 18, 22, 31 and 202. An immunofluorescence assay demonstrates that N-linked glycosylation are necessary for vGPCR trafficking to the cellular membrane. Employing vGPCR mutants whose glycosylation sites were ablated, we show that these vGPCR mutants failed to activate downstream signaling in cultured cells and were severely impaired to induce tumor formation in the xenograph nude mouse model. These findings support the conclusion that glycosylation is critical for vGPCR tumorigenesis and imply that chemokine regulation at the plasma membrane is crucial for vGPCR mediated signaling.
GPCR homomers and heteromers: a better choice as targets for drug development than GPCR monomers?

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Casadó, Vicent; Cortés, Antoni; Mallol, Josefa; Pérez-Capote, Kamil; Ferré, Sergi; Lluis, Carmen; Franco, Rafael; Canela, Enric I

2009-11-01

G protein-coupled receptors (GPCR) are targeted by many therapeutic drugs marketed to fight against a variety of diseases. Selection of novel lead compounds are based on pharmacological parameters obtained assuming that GPCR are monomers. However, many GPCR are expressed as dimers/oligomers. Therefore, drug development may consider GPCR as homo- and hetero-oligomers. A two-state dimer receptor model is now available to understand GPCR operation and to interpret data obtained from drugs interacting with dimers, and even from mixtures of monomers and dimers. Heteromers are distinct entities and therefore a given drug is expected to have different affinities and different efficacies depending on the heteromer. All these concepts would lead to broaden the therapeutic potential of drugs targeting GPCRs, including receptor heteromer-selective drugs with a lower incidence of side effects, or to identify novel pharmacological profiles using cell models expressing receptor heteromers.
Conformational biosensors reveal GPCR signalling from endosomes

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Irannejad, R; Tomshine, Jin C; Tomshine, Jon R

2013-01-01

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited...... or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute...... in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane...
GPCR crystal structures: Medicinal chemistry in the pocket.

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Shonberg, Jeremy; Kling, Ralf C; Gmeiner, Peter; Löber, Stefan

2015-07-15

Recent breakthroughs in GPCR structural biology have significantly increased our understanding of drug action at these therapeutically relevant receptors, and this will undoubtedly lead to the design of better therapeutics. In recent years, crystal structures of GPCRs from classes A, B, C and F have been solved, unveiling a precise snapshot of ligand-receptor interactions. Furthermore, some receptors have been crystallized in different functional states in complex with antagonists, partial agonists, full agonists, biased agonists and allosteric modulators, providing further insight into the mechanisms of ligand-induced GPCR activation. It is now obvious that there is enormous diversity in the size, shape and position of the ligand binding pockets in GPCRs. In this review, we summarise the current state of solved GPCR structures, with a particular focus on ligand-receptor interactions in the binding pocket, and how this can contribute to the design of GPCR ligands with better affinity, subtype selectivity or efficacy. Copyright © 2015 Elsevier Ltd. All rights reserved.
Amplification of the angiogenic signal through the activation of the TSC/mTOR/HIF axis by the KSHV vGPCR in Kaposi's sarcoma.

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Bruno C Jham

2011-04-01

Full Text Available Kaposi's sarcoma (KS is a vascular neoplasm characterized by the dysregulated expression of angiogenic and inflammatory cytokines. The driving force of the KS lesion, the KSHV-infected spindle cell, secretes elevated levels of vascular endothelial growth factor (VEGF, essential for KS development. However, the origin of VEGF in this tumor remains unclear.Here we report that the KSHV G protein-coupled receptor (vGPCR upregulates VEGF in KS through an intricate paracrine mechanism. The cytokines secreted by the few vGPCR-expressing tumor cells activate in neighboring cells multiple pathways (including AKT, ERK, p38 and IKKβ that, in turn, converge on TSC1/2, promoting mTOR activation, HIF upregulation, and VEGF secretion. Conditioned media from vGPCR-expressing cells lead to an mTOR-dependent increase in HIF-1α and HIF-2α protein levels and VEGF upregulation. In a mouse allograft model for KS, specific inhibition of the paracrine activation of mTOR in non-vGPCR-expressing cells was sufficient to inhibit HIF upregulation in these cells, and abolished the ability of the vGPCR-expressing cells to promote tumor formation in vivo. Similarly, pharmacologic inhibition of HIF in this model blocked VEGF secretion and also lead to tumor regression.Our findings provide a compelling explanation for how the few tumor cells expressing vGPCR can contribute to the dramatic amplification of VEGF secretion in KS, and further provide a molecular mechanism for how cytokine dysregulation in KS fuels angiogenesis and tumor development. These data further suggest that activation of HIF by vGPCR may be a vulnerable target for the treatment of patients with KS.
Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

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Yin, Yanting; de Waal, Parker W; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H Eric

2017-06-16

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β 2 -adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
CADM1 is essential for KSHV-encoded vGPCR-and vFLIP-mediated chronic NF-κB activation.

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Richard Hunte

2018-04-01

Full Text Available Approximately 12% of all human cancers worldwide are caused by infections with oncogenic viruses. Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV/HHV8 is one of the oncogenic viruses responsible for human cancers, including Kaposi's sarcoma (KS, Primary Effusion Lymphoma (PEL, and the lymphoproliferative disorder multicentric Castleman's disease (MCD. Chronic inflammation mediated by KSHV infection plays a decisive role in the development and survival of these cancers. NF-κB, a family of transcription factors regulating inflammation, cell survival, and proliferation, is persistently activated in KSHV-infected cells. The KSHV latent and lytic expressing oncogenes involved in NF-κB activation are vFLIP/K13 and vGPCR, respectively. However, the mechanisms by which NF-κB is activated by vFLIP and vGPCR are poorly understood. In this study, we have found that a host molecule, Cell Adhesion Molecule 1 (CADM1, is robustly upregulated in KSHV-infected PBMCs and KSHV-associated PEL cells. Further investigation determined that both vFLIP and vGPCR interacted with CADM1. The PDZ binding motif localized at the carboxyl terminus of CADM1 is essential for both vGPCR and vFLIP to maintain chronic NF-κB activation. Membrane lipid raft associated CADM1 interaction with vFLIP is critical for the initiation of IKK kinase complex and NF-κB activation in the PEL cells. In addition, CADM1 played essential roles in the survival of KSHV-associated PEL cells. These data indicate that CADM1 plays key roles in the activation of NF-κB pathways during latent and lytic phases of the KSHV life cycle and the survival of KSHV-infected cells.
Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling.

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Hidetoshi Kumagai

Full Text Available G protein-coupled receptors (GPCRs play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase to the N-terminal end of the receptor (HT-GPCR. HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays.
Analgesic tone conferred by constitutively active mu opioid receptors in mice lacking β-arrestin 2

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Hales Tim G

2011-04-01

Full Text Available Abstract Hedonic reward, dependence and addiction are unwanted effects of opioid analgesics, linked to the phasic cycle of μ opioid receptor activation, tolerance and withdrawal. In vitro studies of recombinant G protein coupled receptors (GPCRs over expressed in cell lines reveal an alternative tonic signaling mechanism that is independent of agonist. Such studies demonstrate that constitutive GPCR signaling can be inhibited by inverse agonists but not by neutral antagonists. However, ligand-independent activity has been difficult to examine in vivo, at the systems level, due to relatively low levels of constitutive activity of most GPCRs including μ receptors, often necessitating mutagenesis or pharmacological manipulation to enhance basal signaling. We previously demonstrated that the absence of β-arrestin 2 (β-arr2 augments the constitutive coupling of μ receptors to voltage-activated Ca2+ channels in primary afferent dorsal root ganglion neurons from β-arr2-/- mice. We used this in vitro approach to characterize neutral competitive antagonists and inverse agonists of the constitutively active wild type μ receptors in neurons. We administered these agents to β-arr2-/- mice to explore the role of constitutive μ receptor activity in nociception and hedonic tone. This study demonstrates that the induction of constitutive μ receptor activity in vivo in β-arr2-/- mice prolongs tail withdrawal from noxious heat, a phenomenon that was reversed by inverse agonists, but not by antagonists that lack negative efficacy. By contrast, the aversive effects of inverse agonists were similar in β-arr2-/- and β-arr2+/+ mice, suggesting that hedonic tone was unaffected.
Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks.

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Bahouth, Suleiman W; Nooh, Mohammed M

2017-08-01

Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted "barcodes" that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or "barcodes" within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes. Given the vast number of diverse sequences in GPCR, several trafficking mechanisms for endosomal GPCR have been described. The majority of recycling GPCR, are sorted out of endosomes in a "sequence-dependent pathway" anchored around a type-1 PDZ-binding module found in their C-tails. For a subset of these GPCR, a second "barcode" imprinted onto specific GPCR serine/threonine residues by compartmentalized kinase networks was required for their efficient recycling through the "sequence-dependent pathway". Mutating the serine/threonine residues involved, produced dramatic effects on GPCR trafficking, indicating that they played a major role in setting the trafficking itinerary of these GPCR. While endosomal SNX27, retromer/WASH complexes and actin were required for efficient sorting and budding of all these GPCR, additional proteins were required for GPCR sorting via the second "barcode". Here we will review recent developments in GPCR trafficking in general and the human β 1 -adrenergic receptor in particular across the various trafficking roadmaps. In addition, we will discuss the role of GPCR trafficking in regulating endosomal GPCR signaling, which promote biochemical and physiological effects that are distinct from those generated by the GPCR signal transduction
High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

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Bing Xu

Full Text Available G protein-coupled receptors (GPCRs exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(--TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/10⁶ cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.
The essential role of G protein-coupled receptor (GPCR) signaling in regulating T cell immunity.

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Wang, Dashan

2018-06-01

The aim of this paper is to clarify the critical role of GPCR signaling in T cell immunity. The G protein-coupled receptors (GPCRs) are the most common targets in current pharmaceutical industry, and represent the largest and most versatile family of cell surface communicating molecules. GPCRs can be activated by a diverse array of ligands including neurotransmitters, chemokines as well as sensory stimuli. Therefore, GPCRs are involved in many key cellular and physiological processes, such as sense of light, taste and smell, neurotransmission, metabolism, endocrine and exocrine secretion. In recent years, GPCRs have been found to play an important role in immune system. T cell is an important type of immune cell, which plays a central role in cell-mediated immunity. A variety of GPCRs and their signaling mediators (RGS proteins, GRKs and β-arrestin) have been found to express in T cells and involved T cell-mediated immunity. We will summarize the role of GPCR signaling and their regulatory molecules in T cell activation, homeostasis and function in this article. GPCR signaling plays an important role in T cell activation, homeostasis and function. GPCR signaling is critical in regulating T cell immunity.
Platforms for the identification of GPCR targets, and of orthosteric and allosteric modulators.

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Franco, Rafael; Canela, Enric I; Casado, Vicent; Ferre, Sergi

2010-04-01

The review provides a summary of old and new approaches for GPCR target identification and for the screening of molecules acting on GPCR targets. The new findings in the field are presented as well as an opinion about how these developments may help GPCR drug discovery. Importance in the field: GPCRs have been the most useful family of proteins in terms of targets for drug discovery. The expectations for GPCR target identification and discovery of new drugs acting on 'old' or 'new' GPCR targets are very high. Given the fact that the pace at which new 'GPCR drugs' appear in the market is decreasing and since the new developments in the field are not being translated into drug discovery there is a need to review the field from a critical perspective. To overcome the limitation of the old approaches used in GPCR target identification and drugs discovery new approaches are required. In particular successful approaches in GPCR drug discovery should take into account that the real GPCR targets for a given disease are not GPCR monomers but GPCR heteromers. The reader will gain an overview of the strategies currently used and their pros and cons. The reader will also understand that new strategies may help in accelerating the access of GPCR into the market, and also notice that successful strategies should take advantage of the new findings in the field of GPCRs.
Onco-GPCR signaling and dysregulated expression of microRNAs in human cancer.

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Nohata, Nijiro; Goto, Yusuke; Gutkind, J Silvio

2017-01-01

The G-protein-coupled receptor (GPCR) family is the largest family of cell-surface receptors involved in signal transduction. Aberrant expression of GPCRs and G proteins are frequently associated with prevalent human diseases, including cancer. In fact, GPCRs represent the therapeutic targets of more than a quarter of the clinical drugs currently on the market. MiRNAs (miRNAs) are also aberrantly expressed in many human cancers, and they have significant roles in the initiation, development and metastasis of human malignancies. Recent studies have revealed that dysregulation of miRNAs and their target genes expression are associated with cancer progression. The emerging information suggests that miRNAs play an important role in the fine tuning of many signaling pathways, including GPCR signaling. We summarize our current knowledge of the individual functions of miRNAs regulated by GPCRs and GPCR signaling-associated molecules, and miRNAs that regulate the expression and activity of GPCRs, their endogenous ligands and their coupled heterotrimeric G proteins in human cancer.

GPCR & company: databases and servers for GPCRs and interacting partners.

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Kowalsman, Noga; Niv, Masha Y

2014-01-01

G-protein-coupled receptors (GPCRs) are a large superfamily of membrane receptors that are involved in a wide range of signaling pathways. To fulfill their tasks, GPCRs interact with a variety of partners, including small molecules, lipids and proteins. They are accompanied by different proteins during all phases of their life cycle. Therefore, GPCR interactions with their partners are of great interest in basic cell-signaling research and in drug discovery.Due to the rapid development of computers and internet communication, knowledge and data can be easily shared within the worldwide research community via freely available databases and servers. These provide an abundance of biological, chemical and pharmacological information.This chapter describes the available web resources for investigating GPCR interactions. We review about 40 freely available databases and servers, and provide a few sentences about the essence and the data they supply. For simplification, the databases and servers were grouped under the following topics: general GPCR-ligand interactions; particular families of GPCRs and their ligands; GPCR oligomerization; GPCR interactions with intracellular partners; and structural information on GPCRs. In conclusion, a multitude of useful tools are currently available. Summary tables are provided to ease navigation between the numerous and partially overlapping resources. Suggestions for future enhancements of the online tools include the addition of links from general to specialized databases and enabling usage of user-supplied template for GPCR structural modeling.
Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization.

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Fisher, Gregory W; Fuhrman, Margaret H; Adler, Sally A; Szent-Gyorgyi, Christopher; Waggoner, Alan S; Jarvik, Jonathan W

2014-09-01

G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications. © 2014 Society for Laboratory Automation and Screening.
GPCR-autoantibodies in chronic heart failure.

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Boivin-Jahns, Valerie; Jahns, Roland

2018-06-01

Chronic heart failure (CHF) is a syndrome characterized by shortness of breath, fluid retention, and a progressive reduction in cardiac function. More than 60% of the cases are ischemic in origin (i.e., due to myo-cardial infarction) and about 30% are caused by non-ischemic myocardial damage (i.e., due to genetic or non-genetic causes like myocardial inflammation). Because of alterations in both cellular and humoral immunity patients with non-ischemic CHF often develop abnormal or misled immune responses, including cross-reacting antibodies and/or autoantibodies to various cardiac anti-gens. Non-ischemic myo-cardial damage was found to progress to CHF particularly, when associated (a) with the generation of autoantibodies directed against distinct myocyte membrane proteins critically involved in cardiac function - like G-protein coup-led membrane receptors (GPCRs), or (b) with virus persistence in the myocardium. This article will review current knowledge on the pathophysiological relevance of GPCR-autoreactivity in CHF by giving an overview on the so far available evidence from pre-clinical, clinical and epidemiological studies on the CHF-inducing potential of GPCR-autoantibodies and thereon based novel therapeutic approaches in GPCR autoantibody-associated CHF.
GPCR-SSFE: A comprehensive database of G-protein-coupled receptor template predictions and homology models

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Kreuchwig Annika

2011-05-01

Full Text Available Abstract Background G protein-coupled receptors (GPCRs transduce a wide variety of extracellular signals to within the cell and therefore have a key role in regulating cell activity and physiological function. GPCR malfunction is responsible for a wide range of diseases including cancer, diabetes and hyperthyroidism and a large proportion of drugs on the market target these receptors. The three dimensional structure of GPCRs is important for elucidating the molecular mechanisms underlying these diseases and for performing structure-based drug design. Although structural data are restricted to only a handful of GPCRs, homology models can be used as a proxy for those receptors not having crystal structures. However, many researchers working on GPCRs are not experienced homology modellers and are therefore unable to benefit from the information that can be gleaned from such three-dimensional models. Here, we present a comprehensive database called the GPCR-SSFE, which provides initial homology models of the transmembrane helices for a large variety of family A GPCRs. Description Extending on our previous theoretical work, we have developed an automated pipeline for GPCR homology modelling and applied it to a large set of family A GPCR sequences. Our pipeline is a fragment-based approach that exploits available family A crystal structures. The GPCR-SSFE database stores the template predictions, sequence alignments, identified sequence and structure motifs and homology models for 5025 family A GPCRs. Users are able to browse the GPCR dataset according to their pharmacological classification or search for results using a UniProt entry name. It is also possible for a user to submit a GPCR sequence that is not contained in the database for analysis and homology model building. The models can be viewed using a Jmol applet and are also available for download along with the alignments. Conclusions The data provided by GPCR-SSFE are useful for investigating
GPCR-SSFE: a comprehensive database of G-protein-coupled receptor template predictions and homology models.

Science.gov (United States)

Worth, Catherine L; Kreuchwig, Annika; Kleinau, Gunnar; Krause, Gerd

2011-05-23

G protein-coupled receptors (GPCRs) transduce a wide variety of extracellular signals to within the cell and therefore have a key role in regulating cell activity and physiological function. GPCR malfunction is responsible for a wide range of diseases including cancer, diabetes and hyperthyroidism and a large proportion of drugs on the market target these receptors. The three dimensional structure of GPCRs is important for elucidating the molecular mechanisms underlying these diseases and for performing structure-based drug design. Although structural data are restricted to only a handful of GPCRs, homology models can be used as a proxy for those receptors not having crystal structures. However, many researchers working on GPCRs are not experienced homology modellers and are therefore unable to benefit from the information that can be gleaned from such three-dimensional models. Here, we present a comprehensive database called the GPCR-SSFE, which provides initial homology models of the transmembrane helices for a large variety of family A GPCRs. Extending on our previous theoretical work, we have developed an automated pipeline for GPCR homology modelling and applied it to a large set of family A GPCR sequences. Our pipeline is a fragment-based approach that exploits available family A crystal structures. The GPCR-SSFE database stores the template predictions, sequence alignments, identified sequence and structure motifs and homology models for 5025 family A GPCRs. Users are able to browse the GPCR dataset according to their pharmacological classification or search for results using a UniProt entry name. It is also possible for a user to submit a GPCR sequence that is not contained in the database for analysis and homology model building. The models can be viewed using a Jmol applet and are also available for download along with the alignments. The data provided by GPCR-SSFE are useful for investigating general and detailed sequence-structure-function relationships
Trends in GPCR drug discovery: new agents, targets and indications.

Science.gov (United States)

Hauser, Alexander S; Attwood, Misty M; Rask-Andersen, Mathias; Schiöth, Helgi B; Gloriam, David E

2017-12-01

G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, mostly due to their substantial involvement in human pathophysiology and their pharmacological tractability. Here, we report an up-to-date analysis of all GPCR drugs and agents in clinical trials, which reveals current trends across molecule types, drug targets and therapeutic indications, including showing that 475 drugs (~34% of all drugs approved by the US Food and Drug Administration (FDA)) act at 108 unique GPCRs. Approximately 321 agents are currently in clinical trials, of which ~20% target 66 potentially novel GPCR targets without an approved drug, and the number of biological drugs, allosteric modulators and biased agonists has increased. The major disease indications for GPCR modulators show a shift towards diabetes, obesity and Alzheimer disease, although several central nervous system disorders are also highly represented. The 224 (56%) non-olfactory GPCRs that have not yet been explored in clinical trials have broad untapped therapeutic potential, particularly in genetic and immune system disorders. Finally, we provide an interactive online resource to analyse and infer trends in GPCR drug discovery.
Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

International Nuclear Information System (INIS)

Kajikawa, Mizuho; Sasaki, Kaori; Wakimoto, Yoshitaro; Toyooka, Masaru; Motohashi, Tomoko; Shimojima, Tsukasa; Takeda, Shigeki; Park, Enoch Y.; Maenaka, Katsumi

2009-01-01

Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G i α) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [ 35 S]GTPγS-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.
Membrane-mediated oligomerization of G protein coupled receptors and its implications for GPCR function

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Stefan Gahbauer

2016-10-01

Full Text Available The dimerization or even oligomerization of G protein coupled receptors (GPCRs causes ongoing, controversial debates about its functional role and the coupled biophysical, biochemical or biomedical implications. A continously growing number of studies hints to a relation between oligomerization and function of GPCRs and strengthens the assumption that receptor assembly plays a key role in the regulation of protein function. Additionally, progress in the structural analysis of GPCR-G protein and GPCR-ligand interactions allows to distinguish between actively functional and non-signalling complexes. Recent findings further suggest that the surrounding membrane, i.e. its lipid composition may modulate the preferred dimerization interface and as a result the abundance of distinct dimeric conformations. In this review, the association of GPCRs and the role of the membrane in oligomerization will be discussed. An overview of the different reported oligomeric interfaces is provided and their capability for signaling discussed. The currently available data is summarized with regard to the formation of GPCR oligomers, their structures and dependency on the membrane microenvironment as well as the coupling of oligomerization to receptor function.
The current state of GPCR-based drug discovery to treat metabolic disease.

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Sloop, Kyle W; Emmerson, Paul J; Statnick, Michael A; Willard, Francis S

2018-02-02

One approach of modern drug discovery is to identify agents that enhance or diminish signal transduction cascades in various cell types and tissues by modulating the activity of GPCRs. This strategy has resulted in the development of new medicines to treat many conditions, including cardiovascular disease, psychiatric disorders, HIV/AIDS, certain forms of cancer and Type 2 diabetes mellitus (T2DM). These successes justify further pursuit of GPCRs as disease targets and provide key learning that should help guide identifying future therapeutic agents. This report reviews the current landscape of GPCR drug discovery with emphasis on efforts aimed at developing new molecules for treating T2DM and obesity. We analyse historical efforts to generate GPCR-based drugs to treat metabolic disease in terms of causal factors leading to success and failure in this endeavour. © 2018 The British Pharmacological Society.
TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation.

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Nadine Kirschmer

Full Text Available The Transient Receptor Potential Channel Subunit 4 (TRPC4 has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II stimulation. NRCs infected with TRPC4β (Ad-C4β also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not
Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

Energy Technology Data Exchange (ETDEWEB)

Kajikawa, Mizuho; Sasaki, Kaori [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Wakimoto, Yoshitaro; Toyooka, Masaru [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Motohashi, Tomoko; Shimojima, Tsukasa [National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 (Japan); Takeda, Shigeki [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Park, Enoch Y. [Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka, Shizuoka 422-8529 (Japan); Maenaka, Katsumi, E-mail: kmaenaka-umin@umin.net [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

2009-07-31

Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.
Functional understanding of the diverse exon-intron structures of human GPCR genes.

Science.gov (United States)

Hammond, Dorothy A; Olman, Victor; Xu, Ying

2014-02-01

The GPCR genes have a variety of exon-intron structures even though their proteins are all structurally homologous. We have examined all human GPCR genes with at least two functional protein isoforms, totaling 199, aiming to gain an understanding of what may have contributed to the large diversity of the exon-intron structures of the GPCR genes. The 199 genes have a total of 808 known protein splicing isoforms with experimentally verified functions. Our analysis reveals that 1301 (80.6%) adjacent exon-exon pairs out of the total of 1,613 in the 199 genes have either exactly one exon skipped or the intron in-between retained in at least one of the 808 protein splicing isoforms. This observation has a statistical significance p-value of 2.051762 * e(-09), assuming that the observed splicing isoforms are independent of the exon-intron structures. Our interpretation of this observation is that the exon boundaries of the GPCR genes are not randomly determined; instead they may be selected to facilitate specific alternative splicing for functional purposes.
Pharmacological profiling an abundantly expressed schistosome serotonergic GPCR identifies nuciferine as a potent antagonist

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John D. Chan

2016-12-01

Full Text Available 5-hydroxytryptamine (5-HT is a key regulator of muscle contraction in parasitic flatworms. In Schistosoma mansoni, the myoexcitatory action of 5-HT is effected through activation of a serotonergic GPCR (Sm.5HTRL, prioritizing pharmacological characterization of this target for anthelmintic drug discovery. Here, we have examined the effects of several aporphine alkaloids on the signaling activity of a heterologously expressed Sm.5HTRL construct using a cAMP biosensor assay. Four structurally related natural products – nuciferine, D-glaucine, boldine and bulbocapnine – were demonstrated to block Sm.5HTRL evoked cAMP generation with the potency of GPCR blockade correlating well with the ability of each drug to inhibit contractility of schistosomule larvae. Nuciferine was also effective at inhibiting both basal and 5-HT evoked motility of adult schistosomes. These data advance our understanding of structure-affinity relationships at Sm.5HTRL, and demonstrate the effectiveness of Sm.5HTRL antagonists as hypomotility-evoking drugs across different parasite life cycle stages.
Prediction of GPCR-Ligand Binding Using Machine Learning Algorithms

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Sangmin Seo

2018-01-01

Full Text Available We propose a novel method that predicts binding of G-protein coupled receptors (GPCRs and ligands. The proposed method uses hub and cycle structures of ligands and amino acid motif sequences of GPCRs, rather than the 3D structure of a receptor or similarity of receptors or ligands. The experimental results show that these new features can be effective in predicting GPCR-ligand binding (average area under the curve [AUC] of 0.944, because they are thought to include hidden properties of good ligand-receptor binding. Using the proposed method, we were able to identify novel ligand-GPCR bindings, some of which are supported by several studies.
Role of spatial inhomogenity in GPCR dimerisation predicted by receptor association-diffusion models

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Deshpande, Sneha A.; Pawar, Aiswarya B.; Dighe, Anish; Athale, Chaitanya A.; Sengupta, Durba

2017-06-01

G protein-coupled receptor (GPCR) association is an emerging paradigm with far reaching implications in the regulation of signalling pathways and therapeutic interventions. Recent super resolution microscopy studies have revealed that receptor dimer steady state exhibits sub-second dynamics. In particular the GPCRs, muscarinic acetylcholine receptor M1 (M1MR) and formyl peptide receptor (FPR), have been demonstrated to exhibit a fast association/dissociation kinetics, independent of ligand binding. In this work, we have developed a spatial kinetic Monte Carlo model to investigate receptor homo-dimerisation at a single receptor resolution. Experimentally measured association/dissociation kinetic parameters and diffusion coefficients were used as inputs to the model. To test the effect of membrane spatial heterogeneity on the simulated steady state, simulations were compared to experimental statistics of dimerisation. In the simplest case the receptors are assumed to be diffusing in a spatially homogeneous environment, while spatial heterogeneity is modelled to result from crowding, membrane micro-domains and cytoskeletal compartmentalisation or ‘corrals’. We show that a simple association-diffusion model is sufficient to reproduce M1MR association statistics, but fails to reproduce FPR statistics despite comparable kinetic constants. A parameter sensitivity analysis is required to reproduce the association statistics of FPR. The model reveals the complex interplay between cytoskeletal components and their influence on receptor association kinetics within the features of the membrane landscape. These results constitute an important step towards understanding the factors modulating GPCR organisation.
G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

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Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

2015-03-13

Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Targeting GPCR-Gβγ-GRK2 signaling as a novel strategy for treating cardiorenal pathologies.

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Rudomanova, Valeria; Blaxall, Burns C

2017-08-01

The pathologic crosstalk between the heart and kidney is known as cardiorenal syndrome (CRS). While the specific mechanisms underlying this crosstalk remain poorly understood, CRS is associated with exacerbated dysfunction of either or both organs and reduced survival. Maladaptive fibrotic remodeling is a key component of both heart and kidney failure pathogenesis and progression. G-protein coupled receptor (GPCR) signaling is a crucial regulator of cardiovascular and renal function. Chronic/pathologic GPCR signaling elicits the interaction of the G-protein Gβγ subunit with GPCR kinase 2 (GRK2), targeting the receptor for internalization, scaffolding to pathologic signals, and receptor degradation. Targeting this pathologic Gβγ-GRK2 interaction has been suggested as a possible strategy for the treatment of HF. In the current review, we discuss recent updates in understanding the role of GPCR-Gβγ-GRK2 signaling as a crucial mediator of maladaptive organ remodeling detected in HF and kidney dysfunction, with specific attention to small molecule-mediated inhibition of pathologic Gβγ-GRK2 interactions. Further, we explore the potential of GPCR-Gβγ-GRK2 signaling as a possible therapeutic target for cardiorenal pathologies. Copyright © 2017 Elsevier B.V. All rights reserved.
Interrogating the Spatiotemporal Landscape of Neuromodulatory GPCR Signaling by Real-Time Imaging of cAMP in Intact Neurons and Circuits

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Brian S. Muntean

2018-01-01

Full Text Available Summary: Modulation of neuronal circuits is key to information processing in the brain. The majority of neuromodulators exert their effects by activating G-protein-coupled receptors (GPCRs that control the production of second messengers directly impacting cellular physiology. How numerous GPCRs integrate neuromodulatory inputs while accommodating diversity of incoming signals is poorly understood. In this study, we develop an in vivo tool and analytical suite for analyzing GPCR responses by monitoring the dynamics of a key second messenger, cyclic AMP (cAMP, with excellent quantitative and spatiotemporal resolution in various neurons. Using this imaging approach in combination with CRISPR/Cas9 editing and optogenetics, we interrogate neuromodulatory mechanisms of defined populations of neurons in an intact mesolimbic reward circuit and describe how individual inputs generate discrete second-messenger signatures in a cell- and receptor-specific fashion. This offers a resource for studying native neuronal GPCR signaling in real time. : Muntean et al. develop an in vivo reagent to study processing of neurotransmitter GPCR signals by monitoring real-time dynamics of cAMP responses. They demonstrate application of this approach, in combination with CRISPR/Cas9 gene editing and optogenetics, to interrogate the functional organization of a striatal circuit. Keywords: cAMP, GPCR, neuromodulation, dopamine, striatum, imaging, optogenetics
Seeking Ligand Bias: Assessing GPCR Coupling to Beta-Arrestins for Drug Discovery

OpenAIRE

Bohn, Laura M.; McDonald, Patricia H.

2010-01-01

G protein-coupled receptors (GPCR) are the major site of action for endogenous hormones and neurotransmitters. Early drug discovery efforts focused on determining whether ligands could engage G protein coupling and subsequently activate or inhibit cognate “second messengers.” Gone are those simple days as we now realize that receptors can also couple βarrestins. As we delve into the complexity of ligand-directed signaling and receptosome scaffolds, we are faced with what may seem like endless...
G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey.

Science.gov (United States)

Kratochwil, Nicole A; Gatti-McArthur, Silvia; Hoener, Marius C; Lindemann, Lothar; Christ, Andreas D; Green, Luke G; Guba, Wolfgang; Martin, Rainer E; Malherbe, Pari; Porter, Richard H P; Slack, Jay P; Winnig, Marcel; Dehmlow, Henrietta; Grether, Uwe; Hertel, Cornelia; Narquizian, Robert; Panousis, Constantinos G; Kolczewski, Sabine; Steward, Lucinda

2011-01-01

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor. © 2011 Bentham Science Publishers

Global medicinal chemistry and GPCR conference: interview with Stevan Djuric.

Science.gov (United States)

Djuric, Stevan

2018-04-01

Stevan Djuric speaks to Benjamin Walden, Commissioning Editor. Stevan Djuric is head of the global Medicinal Chemistry Leadership Team at AbbVie and is also Vice President of the Discovery Chemistry and Technology organization within their Discovery organization and chemistry outsourcing activities. He spoke at the Global-Medicinal-Chemistry and GPCR summit on the imperative to develop chemistry related technology that can reduce cycle time, cost of goods and improve probability of success. To this end, he discussed his efforts in the chemistry technology area with a focus on integrated synthesis-purification bioassay, and flow photochemistry and high temperature chemistry platforms.
iGPCR-drug: a web server for predicting interaction between GPCRs and drugs in cellular networking.

Directory of Open Access Journals (Sweden)

Xuan Xiao

Full Text Available Involved in many diseases such as cancer, diabetes, neurodegenerative, inflammatory and respiratory disorders, G-protein-coupled receptors (GPCRs are among the most frequent targets of therapeutic drugs. It is time-consuming and expensive to determine whether a drug and a GPCR are to interact with each other in a cellular network purely by means of experimental techniques. Although some computational methods were developed in this regard based on the knowledge of the 3D (dimensional structure of protein, unfortunately their usage is quite limited because the 3D structures for most GPCRs are still unknown. To overcome the situation, a sequence-based classifier, called "iGPCR-drug", was developed to predict the interactions between GPCRs and drugs in cellular networking. In the predictor, the drug compound is formulated by a 2D (dimensional fingerprint via a 256D vector, GPCR by the PseAAC (pseudo amino acid composition generated with the grey model theory, and the prediction engine is operated by the fuzzy K-nearest neighbour algorithm. Moreover, a user-friendly web-server for iGPCR-drug was established at http://www.jci-bioinfo.cn/iGPCR-Drug/. For the convenience of most experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results without the need to follow the complicated math equations presented in this paper just for its integrity. The overall success rate achieved by iGPCR-drug via the jackknife test was 85.5%, which is remarkably higher than the rate by the existing peer method developed in 2010 although no web server was ever established for it. It is anticipated that iGPCR-Drug may become a useful high throughput tool for both basic research and drug development, and that the approach presented here can also be extended to study other drug - target interaction networks.
MicroRNA-34a promotes genomic instability by a broad suppression of genome maintenance mechanisms downstream of the oncogene KSHV-vGPCR.

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Krause, Claudia J; Popp, Oliver; Thirunarayanan, Nanthakumar; Dittmar, Gunnar; Lipp, Martin; Müller, Gerd

2016-03-01

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded chemokine receptor vGPCR acts as an oncogene in Kaposi's sarcomagenesis. Until now, the molecular mechanisms by which the vGPCR contributes to tumor development remain incompletely understood. Here, we show that the KSHV-vGPCR contributes to tumor progression through microRNA (miR)-34a-mediated induction of genomic instability. Large-scale analyses on the DNA, gene and protein level of cell lines derived from a mouse model of vGPCR-driven tumorigenesis revealed that a vGPCR-induced upregulation of miR-34a resulted in a broad suppression of genome maintenance genes. A knockdown of either the vGPCR or miR-34a largely restored the expression of these genes and confirmed miR-34a as a downstream effector of the KSHV-vGPCR that compromises genome maintenance mechanisms. This novel, protumorigenic role of miR-34a questions the use of miR-34a mimetics in cancer therapy as they could impair genome stability.
FLIPR assays of intracellular calcium in GPCR drug discovery

DEFF Research Database (Denmark)

Hansen, Kasper Bø; Bräuner-Osborne, Hans

2009-01-01

Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non...
GPCR-SSFE 2.0-a fragment-based molecular modeling web tool for Class A G-protein coupled receptors.

Science.gov (United States)

Worth, Catherine L; Kreuchwig, Franziska; Tiemann, Johanna K S; Kreuchwig, Annika; Ritschel, Michele; Kleinau, Gunnar; Hildebrand, Peter W; Krause, Gerd

2017-07-03

G-protein coupled receptors (GPCRs) are key players in signal transduction and therefore a large proportion of pharmaceutical drugs target these receptors. Structural data of GPCRs are sparse yet important for elucidating the molecular basis of GPCR-related diseases and for performing structure-based drug design. To ameliorate this problem, GPCR-SSFE 2.0 (http://www.ssfa-7tmr.de/ssfe2/), an intuitive web server dedicated to providing three-dimensional Class A GPCR homology models has been developed. The updated web server includes 27 inactive template structures and incorporates various new functionalities. Uniquely, it uses a fingerprint correlation scoring strategy for identifying the optimal templates, which we demonstrate captures structural features that sequence similarity alone is unable to do. Template selection is carried out separately for each helix, allowing both single-template models and fragment-based models to be built. Additionally, GPCR-SSFE 2.0 stores a comprehensive set of pre-calculated and downloadable homology models and also incorporates interactive loop modeling using the tool SL2, allowing knowledge-based input by the user to guide the selection process. For visual analysis, the NGL viewer is embedded into the result pages. Finally, blind-testing using two recently published structures shows that GPCR-SSFE 2.0 performs comparably or better than other state-of-the art GPCR modeling web servers. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Trends in GPCR drug discovery: new agents, targets and indications

DEFF Research Database (Denmark)

Hauser, Alexander Sebastian; Gloriam, David E.; Attwood, Misty M.

2017-01-01

current trends across molecule types, drug targets and therapeutic indications, including showing that 475 drugs (~34% of all drugs approved by the US Food and Drug Administration (FDA)) act at 108 unique GPCRs. Approximately 321 agents are currently in clinical trials, of which ~20% target 66 potentially...... are also highly represented. The 224 (56%) non-olfactory GPCRs that have not yet been explored in clinical trials have broad untapped therapeutic potential, particularly in genetic and immune system disorders. Finally, we provide an interactive online resource to analyse and infer trends in GPCR drug......G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, mostly due to their substantial involvement in human pathophysiology and their pharmacological tractability. Here, we report an up-to-date analysis of all GPCR drugs and agents in clinical trials, which reveals...
Promoter switching allows simultaneous transcription of LANA and K14/vGPCR of Kaposi's sarcoma-associated herpesvirus

International Nuclear Information System (INIS)

Staudt, Michelle R.; Dittmer, Dirk P.

2006-01-01

Latent transcription of the latency-associated nuclear antigen (LANA/ORF73) of Kaposi's sarcoma-associated herpesvirus is driven by the LANAp-c. Complexity arises during lytic reactivation, however, as the bicistronic K14/vGPCR transcript initiates 32 bp downstream of LANAp-c in the opposite orientation. We identify an Rta/ORF50-inducible LANA promoter (LANAp-i) that is distinct from the LANAp-c. LANAp-c is unaffected by Rta/ORF50. Utilization of the second, downstream LANAp-i explains how LANA and K14/vGPCR are simultaneously transcribed during de novo infection or lytic reactivation. Transactivation of LANAp-i and K14/vGPCRp requires the C-terminal activation domain of Rta/ORF50 and is mediated by DNA-binding-dependent and -independent Rta/ORF50 mechanisms. Transcriptional profiling following viral reactivation support promoter reporter phenotypes. In sum, cis-elements within the LANAp were selected to ensure faithful expression of LANA and other genes regulated by LANAp during all stages of the KSHV lifecycle despite potential interference from K14/vGPCRp activity
Controversial constitutive TSHR activity: patients, physiology, and in vitro characterization.

Science.gov (United States)

Huth, S; Jaeschke, H; Schaarschmidt, J; Paschke, R

2014-06-01

G protein-coupled receptors constitute a large family of transmembrane receptors, which activate cellular responses by signal transmission and regulation of second messenger metabolism after ligand binding. For several of these receptors it is known that they also signal ligand-independently. The G protein-coupled thyroid stimulating hormone receptor (TSHR) is characterized by a high level of constitutive activity in the wild type state. However, little is known yet concerning the physiological relevance of the constitutive wild type TSHR activity. Certainly, knowledge of the physiological relevance of constitutive wild type receptor activity is necessary to better understand thyroid physiology and it is a prerequisite for the development of better therapies for nonautoimmune hyperthyroidism and thyroid cancer. Based on a literature search regarding all published TSHR mutations, this review covers several mutations which are clearly associated with a hyperthyroidism-phenotype, but interestingly show a lack of constitutive activity determined by in vitro characterization. Possible reasons for the observed discrepancies between clinical phenotypes and in vitro characterization results for constitutive TSHR activity are reviewed. All current in vitro characterization methods for constitutive TSHR mutations are "preliminary attempts" and may well be revised by more comprehensive and even better approaches. However, a standardized approach for the determination of constitutive activity can help to identify TSHR mutations for which the investigation of additional signaling mechanisms would be most interesting to find explanations for the current clinical phenotype/in vitro discrepancies and thereby also define suitable methods to explore the physiological relevance of constitutive wild type TSHR activity. © Georg Thieme Verlag KG Stuttgart · New York.
Balancing focused combinatorial libraries based on multiple GPCR ligands

Science.gov (United States)

Soltanshahi, Farhad; Mansley, Tamsin E.; Choi, Sun; Clark, Robert D.

2006-08-01

G-Protein coupled receptors (GPCRs) are important targets for drug discovery, and combinatorial chemistry is an important tool for pharmaceutical development. The absence of detailed structural information, however, limits the kinds of combinatorial design techniques that can be applied to GPCR targets. This is particularly problematic given the current emphasis on focused combinatorial libraries. By linking an incremental construction method (OptDesign) to the very fast shape-matching capability of ChemSpace, we have created an efficient method for designing targeted sublibraries that are topomerically similar to known actives. Multi-objective scoring allows consideration of multiple queries (actives) simultaneously. This can lead to a distribution of products skewed towards one particular query structure, however, particularly when the ligands of interest are quite dissimilar to one another. A novel pivoting technique is described which makes it possible to generate promising designs even under those circumstances. The approach is illustrated by application to some serotonergic agonists and chemokine antagonists.
High Content Analysis of Compositional Heterogeneities to Study GPCR Oligomerization

DEFF Research Database (Denmark)

Walsh, Samuel McEwen

In this thesis I demonstrate how the natural compositional heterogeneities of synthetic and living cell model systems can be used to quantitate the mechanics of G-protein coupled receptor (GPCR) oligomerization with Förster resonance energy transfer (FRET). The thesis is structured around three a...
Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

Science.gov (United States)

Harding, Peter J; Attrill, Helen; Boehringer, Jonas; Ross, Simon; Wadhams, George H; Smith, Eleanor; Armitage, Judith P; Watts, Anthony

2009-02-01

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.
Quantification of GPCR internalization by single-molecule microscopy in living cells.

NARCIS (Netherlands)

Serge, A.; Keijzer, S. de; Hemert, F. Van; Hickman, M.R.; Hereld, D.; Spaink, H.P.; Schmidt, T.; Snaar-Jagalska, B.E.

2011-01-01

Receptor internalization upon ligand stimulation is a key component of a cell's response and allows a cell to correctly sense its environment. Novel fluorescent methods have enabled the direct visualization of the agonist-stimulated G-protein-coupled receptors (GPCR) trafficking in living cells.
GPCR-Bench: A Benchmarking Set and Practitioners' Guide for G Protein-Coupled Receptor Docking.

Science.gov (United States)

Weiss, Dahlia R; Bortolato, Andrea; Tehan, Benjamin; Mason, Jonathan S

2016-04-25

Virtual screening is routinely used to discover new ligands and in particular new ligand chemotypes for G protein-coupled receptors (GPCRs). To prepare for a virtual screen, we often tailor a docking protocol that will enable us to select the best candidates for further screening. To aid this, we created GPCR-Bench, a publically available docking benchmarking set in the spirit of the DUD and DUD-E reference data sets for validation studies, containing 25 nonredundant high-resolution GPCR costructures with an accompanying set of diverse ligands and computational decoy molecules for each target. Benchmarking sets are often used to compare docking protocols; however, it is important to evaluate docking methods not by "retrospective" hit rates but by the actual likelihood that they will produce novel prospective hits. Therefore, docking protocols must not only rank active molecules highly but also produce good poses that a chemist will select for purchase and screening. Currently, no simple objective machine-scriptable function exists that can do this; instead, docking hit lists must be subjectively examined in a consistent way to compare between docking methods. We present here a case study highlighting considerations we feel are of importance when evaluating a method, intended to be useful as a practitioners' guide.
Pharmacogenomics of GPCR Drug Targets

DEFF Research Database (Denmark)

Hauser, Alexander Sebastian; Chavali, Sreenivas; Masuho, Ikuo

2018-01-01

Natural genetic variation in the human genome is a cause of individual differences in responses to medications and is an underappreciated burden on public health. Although 108 G-protein-coupled receptors (GPCRs) are the targets of 475 (∼34%) Food and Drug Administration (FDA)-approved drugs...... and account for a global sales volume of over 180 billion US dollars annually, the prevalence of genetic variation among GPCRs targeted by drugs is unknown. By analyzing data from 68,496 individuals, we find that GPCRs targeted by drugs show genetic variation within functional regions such as drug......- and effector-binding sites in the human population. We experimentally show that certain variants of μ-opioid and Cholecystokinin-A receptors could lead to altered or adverse drug response. By analyzing UK National Health Service drug prescription and sales data, we suggest that characterizing GPCR variants...
Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling.

Science.gov (United States)

Semack, Ansley; Sandhu, Manbir; Malik, Rabia U; Vaidehi, Nagarajan; Sivaramakrishnan, Sivaraj

2016-08-19

Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the β2-adrenergic receptor (β2-AR)-Gαs and V1A receptor (V1AR)-Gαq The Gαs and Gαq peptides adopt different orientations in β2-AR and V1AR, respectively. The β2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with β2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of GPCR-mediated smooth muscle contraction : implications for asthma and pulmonary hypertension

NARCIS (Netherlands)

Wright, D B; Tripathi, S; Sikarwar, A; Santosh, K T; Perez-Zoghbi, J; Ojo, O O; Irechukwu, N; Ward, J P T; Schaafsma, D

Contractile G-protein-coupled receptors (GPCRs) have emerged as key regulators of smooth muscle contraction, both under healthy and diseased conditions. This brief review will discuss some key topics and novel insights regarding GPCR-mediated airway and vascular smooth muscle contraction as
Common structural basis for constitutive activity of the ghrelin receptor family

DEFF Research Database (Denmark)

Holst, Birgitte; Holliday, Nicholas D; Bach, Anders

2004-01-01

Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand......-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G...... demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically...
Constitutive NADPH-Dependent Electron Transferase Activity of the Nox4 Dehydrogenase Domain?

OpenAIRE

Nisimoto, Yukio; Jackson, Heather M.; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J. David

2010-01-01

NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47 phox and p67 phox and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K m for NADPH of 55 ? 10 ?M. The concentration of Nox4 in cell lysates was esti...
Interaction of Protease-Activated Receptor 2 with G Proteins and Beta-Arrestin 1 Studied by Bioluminescence Resonance Energy Transfer

Directory of Open Access Journals (Sweden)

Mohammed Akli eAyoub

2013-12-01

Full Text Available G protein-coupled receptors (GPCRs are well recognized as being able to activate several signaling pathways through the activation of different G proteins as well as other signaling proteins such as beta-arrestins. Therefore, understanding how such multiple GPCR-mediated signaling can be integrated constitute an important aspect. Here, we applied bioluminescence resonance energy transfer (BRET to shed more light on the G protein coupling profile of trypsin receptor, or protease-activated receptor 2 (PAR2, and its interaction with beta-arrestin1. Using YFP and Rluc fusion constructs expressed in COS-7 cells, BRET data revealed a pre-assembly of PAR2 with both Galphai1 and Galphao and a rapid and transient activation of these G proteins upon receptor activation. In contrast, no preassembly of PAR2 with Galpha12 could be detected and their physical association can be measured with a very slow and sustained kinetics similar to that of beta-arrestin1 recruitment. These data demonstrate the coupling of PAR2 with Galphai1, Galphao and Galpha12 in COS-7 cells with differences in the kinetics of GPCR-G protein coupling, a parameter that very likely influences the cellular response. Moreover, this further illustrates that preassembly or agonist-induced G protein interaction depends on receptor-G protein pairs indicating another level of complexity and regulation of the signaling of GPCR-G protein complexes and its multiplicity.
Criteria for confirming sequence periodicity identified by Fourier transform analysis: application to GCR2, a candidate plant GPCR?

Science.gov (United States)

Illingworth, Christopher J R; Parkes, Kevin E; Snell, Christopher R; Mullineaux, Philip M; Reynolds, Christopher A

2008-03-01

Methods to determine periodicity in protein sequences are useful for inferring function. Fourier transformation is one approach but care is required to ensure the periodicity is genuine. Here we have shown that empirically-derived statistical tables can be used as a measure of significance. Genuine protein sequences data rather than randomly generated sequences were used as the statistical backdrop. The method has been applied to G-protein coupled receptor (GPCR) sequences, by Fourier transformation of hydrophobicity values, codon frequencies and the extent of over-representation of codon pairs; the latter being related to translational step times. Genuine periodicity was observed in the hydrophobicity whereas the apparent periodicity (as inferred from previously reported measures) in the translation step times was not validated statistically. GCR2 has recently been proposed as the plant GPCR receptor for the hormone abscisic acid. It has homology to the Lanthionine synthetase C-like family of proteins, an observation confirmed by fold recognition. Application of the Fourier transform algorithm to the GCR2 family revealed strongly predicted seven fold periodicity in hydrophobicity, suggesting why GCR2 has been reported to be a GPCR, despite negative indications in most transmembrane prediction algorithms. The underlying multiple sequence alignment, also required for the Fourier transform analysis of periodicity, indicated that the hydrophobic regions around the 7 GXXG motifs commence near the C-terminal end of each of the 7 inner helices of the alpha-toroid and continue to the N-terminal region of the helix. The results clearly explain why GCR2 has been understandably but erroneously predicted to be a GPCR.

Transgenic Expression of Constitutively Active RAC1 Disrupts Mouse Rod Morphogenesis

Science.gov (United States)

Song, Hongman; Bush, Ronald A.; Vijayasarathy, Camasamudram; Fariss, Robert N.; Kjellstrom, Sten; Sieving, Paul A.

2014-01-01

Purpose. Dominant-active RAC1 rescues photoreceptor structure in Drosophila rhodopsin-null mutants, indicating an important role in morphogenesis. This report assesses the morphogenetic effect of activated RAC1 during mammalian rod photoreceptor development using transgenic mice that express constitutively active (CA) RAC1. Methods. Transgenic mice were generated by expressing CA RAC1 under control of the Rhodopsin promoter, and morphological features of the photoreceptors were evaluated by histology, immunohistochemistry, and transmission electron microscopy. Function was evaluated by electroretinography. Potential protein partners of CA RAC1 were identified by co-immunoprecipitation of retinal extracts. Results. Constitutively active RAC1 expression in differentiating rods disrupted outer retinal lamination as early as postnatal day (P)6, and many photoreceptor cell nuclei were displaced apically into the presumptive subretinal space. These photoreceptors did not develop normal inner and outer segments and had abnormal placement of synaptic elements. Some photoreceptor nuclei were also mislocalized into the inner nuclear layer. Extensive photoreceptor degeneration was subsequently observed in the adult animal. Constitutively active RAC1 formed a complex with the polarity protein PAR6 and with microtubule motor dynein in mouse retina. The normal localization of the PAR6 complex was disrupted in CA RAC1-expressing rod photoreceptors. Conclusions. Constitutively active RAC1 had a profound negative effect on mouse rod cell viability and development. Rod photoreceptors in the CA RAC1 retina exhibited a defect in polarity and migration. Constitutively active RAC1 disrupted rod morphogenesis and gave a phenotype resembling that found in the Crumbs mutant. PAR6 and dynein are two potential downstream effectors that may be involved in CA RAC1-mediated defective mouse photoreceptor morphogenesis. PMID:24651551
Morphine withdrawal enhances constitutive μ-opioid receptor activity in the ventral tegmental area.

Science.gov (United States)

Meye, Frank J; van Zessen, Ruud; Smidt, Marten P; Adan, Roger A H; Ramakers, Geert M J

2012-11-14

μ-Opioid receptors (MORs) in the ventral tegmental area (VTA) are pivotally involved in addictive behavior. While MORs are typically activated by opioids, they can also become constitutively active in the absence of any agonist. In the current study, we present evidence that MOR constitutive activity is highly relevant in the mouse VTA, as it regulates GABAergic input to dopamine neurons. Specifically, suppression of MOR constitutive activity with the inverse agonist KC-2-009 enhanced GABAergic neurotransmission onto VTA dopamine neurons. This inverse agonistic effect was fully blocked by the specific MOR neutral antagonist CTOP, which had no effect on GABAergic transmission itself. We next show that withdrawal from chronic morphine further increases the magnitude of inverse agonistic effects at the MOR, suggesting enhanced MOR constitutive activity. We demonstrate that this increase can be an adaptive response to the detrimental elevation in cAMP levels known to occur during morphine withdrawal. These findings offer important insights in the physiological occurrence and function of MOR constitutive activity, and have important implications for therapeutic strategies aimed at normalizing MOR signaling during addiction and opioid overdose.
The systematic annotation of the three main GPCR families in Reactome.

Science.gov (United States)

Jassal, Bijay; Jupe, Steven; Caudy, Michael; Birney, Ewan; Stein, Lincoln; Hermjakob, Henning; D'Eustachio, Peter

2010-07-29

Reactome is an open-source, freely available database of human biological pathways and processes. A major goal of our work is to provide an integrated view of cellular signalling processes that spans from ligand-receptor interactions to molecular readouts at the level of metabolic and transcriptional events. To this end, we have built the first catalogue of all human G protein-coupled receptors (GPCRs) known to bind endogenous or natural ligands. The UniProt database has records for 797 proteins classified as GPCRs and sorted into families A/1, B/2 and C/3 on the basis of amino acid sequence. To these records we have added details from the IUPHAR database and our own manual curation of relevant literature to create reactions in which 563 GPCRs bind ligands and also interact with specific G-proteins to initiate signalling cascades. We believe the remaining 234 GPCRs are true orphans. The Reactome GPCR pathway can be viewed as a detailed interactive diagram and can be exported in many forms. It provides a template for the orthology-based inference of GPCR reactions for diverse model organism species, and can be overlaid with protein-protein interaction and gene expression datasets to facilitate overrepresentation studies and other forms of pathway analysis. Database URL: http://www.reactome.org.
Mutations in the C-terminal region affect subcellular localization of crucian carp herpesvirus (CaHV) GPCR.

Science.gov (United States)

Wang, Jun; Gui, Lang; Chen, Zong-Yan; Zhang, Qi-Ya

2016-08-01

G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture.
F4/80: the macrophage-specific adhesion-GPCR and its role in immunoregulation.

Science.gov (United States)

Lin, Hsi-Hsien; Stacey, Martin; Stein-Streilein, Joan; Gordon, Siamon

2010-01-01

As a macrophage-restricted reagent, the generation and application of the F4/80 mAb has greatly benefited the phenotypic characterization of mouse tissue macrophages for three decades. Following the molecular identification of the F4/80 antigen as an EGF-TM7 member of the adhesion-GPCR family, great interest was ignited to understand its cell type-specific expression pattern as well as its functional role in macrophage biology. Recent studies have shown that the F4/80 gene is regulated by a novel set of transcription factors that recognized a unique promoter sequence. Gene targeting experiments have produced two F4/80 knock out animal models and showed that F4/80 is not required for normal macrophage development. Nevertheless, the F4/80 receptor was found to be necessary for the induction of efferent CD8+ regulatory T cells responsible for peripheral immune tolerance. The identification of cellular ligands for F4/80 and delineation of its signaling pathway remain elusive but are critical to understand the in vivo role of this macrophage-specific adhesion-GPCR.
GPCR Mutation information - GRIPDB | LSDB Archive [Life Science Database Archive metadata

Lifescience Database Archive (English)

Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data List Contact us GRI...s and exposed in molecular surface. Data file File name: gripdb_mutation_info.zip File URL: ftp://ftp.biosciencedbc.jp/archive/gri...pdb/LATEST/gripdb_mutation_info.zip File size: 766 bytes Si...mple search URL http://togodb.biosciencedbc.jp/togodb/view/gripdb_mutation_info#en Data acquisition method P... Site Policy | Contact Us GPCR Mutation information - GRIPDB | LSDB Archive ...
GPCR Interaction - GRIPDB | LSDB Archive [Life Science Database Archive metadata

Lifescience Database Archive (English)

Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data List Contact us GRI...ed information (disease etc.). Data file File name: gripdb_main.zip File URL: ftp://ftp.biosciencedbc.jp/archive/gripdb/LATEST/gri...godb.biosciencedbc.jp/togodb/view/gripdb_main#en Data acquisition method PDB, Refseq Data analysis method - ...Number of data entries 409 entries Data item Description GRIP ID Interaction ID Main Title Interaction title...tabase Database Description Download License Update History of This Database Site Policy | Contact Us GPCR Interaction - GRIPDB | LSDB Archive ...
Stable GPR101 over-Expressing Cell Lines As an Invaluable Tool for Functional Studies, Ligand Screening, and the Identification of Deregulated Genes/Pathways in Patients with X-Linked Acrogigantism

OpenAIRE

Trivellin, Giampaolo; Janjic, Maria; Larco, Darwin; Tomic, Melanija; Daly, Adrian; Palmeira, Leonor; Faucz; BECKERS, Albert; Wu, T John; Calebiro, Davide; Stojilkovic, Stanko; Stratakis, Constantine

2017-01-01

Background: GPR101 is an orphan G protein-coupled receptor (GPCR) that is duplicated in patients with X-linked acrogigantism (X-LAG) and over-expressed in their GH- and PRL-secreting tumors. GPR101 is a constitutively active GPCR that strongly activates the cAMP pathway. To elucidate the mechanisms through which GPR101 causes GH over-secretion we generated HEK293 and GH/PRL-secreting (GH3) cells with stable GPR101 expression. Methods: Both cell lines were created via direct integration of ...
Morphine withdrawal enhances constitutive μ-opioid receptor activity in the ventral tegmental area

NARCIS (Netherlands)

Meye, F.J.; van Zessen, R.; Smidt, M.P.; Adan, R.A.H.; Ramakers, G.M.J.

2012-01-01

μ-opioid receptors (MORs) in the ventral tegmental area (VTA) are pivotally involved in addictive behavior. While MORs are typically activated by opioids, they can also become constitutively active in the absence of any agonist. In the current study, we present evidence that MOR constitutive
Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

Directory of Open Access Journals (Sweden)

Adam L Martin

Full Text Available The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1 200% elevation over baseline reporter gene expression; 2 40% inhibition of baseline expression; and 3 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1 inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176; 2 no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119; 3 elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12; 4 elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65; and 5 no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87. Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40. This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65% than stimulation of expression (15%. Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.
Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

Science.gov (United States)

Martin, Adam L; Steurer, Michael A; Aronstam, Robert S

2015-01-01

The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.
Rac1-stimulated macropinocytosis enhances Gβγ activation of PI3Kβ.

Science.gov (United States)

Erami, Zahra; Khalil, Bassem D; Salloum, Gilbert; Yao, Yanhua; LoPiccolo, Jaclyn; Shymanets, Aliaksei; Nürnberg, Bernd; Bresnick, Anne R; Backer, Jonathan M

2017-11-16

Phosphoinositide 3-kinases (PI 3-kinases) are regulated by a diverse range of upstream activators, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and small GTPases from the Ras, Rho and Rab families. For the Class IA PI 3-kinase PI3Kβ, two mechanisms for GPCR-mediated regulation have been described: direct binding of Gβγ subunits to the C2-helical domain linker of p110β, and Dock180/Elmo1-mediated activation of Rac1, which binds to the Ras-Binding Domain of p110β. We now show that the integration of these dual pathways is unexpectedly complex. In breast cancer cells, expression of constitutively activated Rac1 (CA-Rac1) along with either GPCR stimulation or expression of Gβγ led to an additive PI3Kβ-dependent activation of Akt. Whereas CA-Rac1-mediated activation of Akt was blocked in cells expressing a mutated PI3Kβ that cannot bind Gβγ, Gβγ and GPCR-mediated activation of Akt was preserved when Rac1 binding to PI3Kβ was blocked. Surprisingly, PI3Kβ-dependent CA-Rac1 signaling to Akt was still seen in cells expressing a mutant p110β that cannot bind Rac1. Instead of directly binding to PI3Kβ, CA-Rac1 acts by enhancing Gβγ coupling to PI3Kβ, as CA-Rac1-mediated Akt activation was blocked by inhibitors of Gβγ. Cells expressing CA-Rac1 exhibited a robust induction of macropinocytosis, and inhibitors of macropinocytosis blocked the activation of Akt by CA-Rac1 or lysophosphatidic acid. Our data suggest that Rac1 can potentiate the activation of PI3Kβ by GPCRs through an indirect mechanism, by driving the formation of macropinosomes that serve as signaling platforms for Gβγ coupling to PI3Kβ. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
Gs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake.

Science.gov (United States)

Nakajima, Ken-ichiro; Cui, Zhenzhong; Li, Chia; Meister, Jaroslawna; Cui, Yinghong; Fu, Ou; Smith, Adam S; Jain, Shalini; Lowell, Bradford B; Krashes, Michael J; Wess, Jürgen

2016-01-08

Agouti-related peptide (AgRP) neurons of the hypothalamus play a key role in regulating food intake and body weight, by releasing three different orexigenic molecules: AgRP; GABA; and neuropeptide Y. AgRP neurons express various G protein-coupled receptors (GPCRs) with different coupling properties, including Gs-linked GPCRs. At present, the potential role of Gs-coupled GPCRs in regulating the activity of AgRP neurons remains unknown. Here we show that the activation of Gs-coupled receptors expressed by AgRP neurons leads to a robust and sustained increase in food intake. We also provide detailed mechanistic data linking the stimulation of this class of receptors to the observed feeding phenotype. Moreover, we show that this pathway is clearly distinct from other GPCR signalling cascades that are operative in AgRP neurons. Our data suggest that drugs able to inhibit this signalling pathway may become useful for the treatment of obesity.
Transgenic mice expressing constitutive active MAPKAPK5 display gender-dependent differences in exploration and activity

Directory of Open Access Journals (Sweden)

Moens Ugo

2007-11-01

Full Text Available Abstract Background The mitogen-activated protein kinases, MAPKs for short, constitute cascades of signalling pathways involved in the regulation of several cellular processes that include cell proliferation, differentiation and motility. They also intervene in neurological processes like fear conditioning and memory. Since little remains known about the MAPK-Activated Protein Kinase, MAPKAPK5, we constructed the first MAPKAPK knockin mouse model, using a constitutive active variant of MAPKAPK5 and analyzed the resulting mice for changes in anxiety-related behaviour. Methods We performed primary SHIRPA observations during background breeding into the C57BL/6 background and assessed the behaviour of the background-bred animals on the elevated plus maze and in the light-dark test. Our results were analyzed using Chi-square tests and homo- and heteroscedatic T-tests. Results Female transgenic mice displayed increased amounts of head dips and open arm time on the maze, compared to littermate controls. In addition, they also explored further into the open arm on the elevated plus maze and were less active in the closed arm compared to littermate controls. Male transgenic mice displayed no differences in anxiety, but their locomotor activity increased compared to non-transgenic littermates. Conclusion Our results revealed anxiety-related traits and locomotor differences between transgenic mice expressing constitutive active MAPKAPK5 and control littermates.
Constitutional changes and the dilemmas of constitutionalism

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Arsen Bačić

2009-01-01

Full Text Available The need to develop constitutional mechanisms whose aim is to resolve fundamental relations in society demands the widest possible inclusion of all of society’s active participants in the discussion on the need to adopt or revise the Constitution. The opening of every new round of constitutional changes is of great importance because it always unlocks certain new and important questions. The answers to those questions should be offered by state authority (policy and civil society including science and its disciplines. In this paper, the author mentions several topics which are of interest in the current discussion on the significance of current constitutional changes for the future of the development of constitutionalism and democracy in the Republic of Croatia. These are above all topics of political and legal constitutionalism and suggestions linked to strengthening the independence of judicial powers. The author advocates consistent application of constitutional control and check mechanisms which exclude all insularity of judicial powers in relation to democratic control.
A method for the quantification of biased signalling at constitutively active receptors.

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Hall, David A; Giraldo, Jesús

2018-06-01

Biased agonism, the ability of an agonist to differentially activate one of several signal transduction pathways when acting at a given receptor, is an increasingly recognized phenomenon at many receptors. The Black and Leff operational model lacks a way to describe constitutive receptor activity and hence inverse agonism. Thus, it is impossible to analyse the biased signalling of inverse agonists using this model. In this theoretical work, we develop and illustrate methods for the analysis of biased inverse agonism. Methods were derived for quantifying biased signalling in systems that demonstrate constitutive activity using the modified operational model proposed by Slack and Hall. The methods were illustrated using Monte Carlo simulations. The Monte Carlo simulations demonstrated that, with an appropriate experimental design, the model parameters are 'identifiable'. The method is consistent with methods based on the measurement of intrinsic relative activity (RA i ) (ΔΔlogR or ΔΔlog(τ/K a )) proposed by Ehlert and Kenakin and their co-workers but has some advantages. In particular, it allows the quantification of ligand bias independently of 'system bias' removing the requirement to normalize to a standard ligand. In systems with constitutive activity, the Slack and Hall model provides methods for quantifying the absolute bias of agonists and inverse agonists. This provides an alternative to methods based on RA i and is complementary to the ΔΔlog(τ/K a ) method of Kenakin et al. in systems where use of that method is inappropriate due to the presence of constitutive activity. © 2018 The British Pharmacological Society.
Fighting obesity with a sugar-based library: discovery of novel MCH-1R antagonists by a new computational-VAST approach for exploration of GPCR binding sites.

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Heifetz, Alexander; Barker, Oliver; Verquin, Geraldine; Wimmer, Norbert; Meutermans, Wim; Pal, Sandeep; Law, Richard J; Whittaker, Mark

2013-05-24

Obesity is an increasingly common disease. While antagonism of the melanin-concentrating hormone-1 receptor (MCH-1R) has been widely reported as a promising therapeutic avenue for obesity treatment, no MCH-1R antagonists have reached the market. Discovery and optimization of new chemical matter targeting MCH-1R is hindered by reduced HTS success rates and a lack of structural information about the MCH-1R binding site. X-ray crystallography and NMR, the major experimental sources of structural information, are very slow processes for membrane proteins and are not currently feasible for every GPCR or GPCR-ligand complex. This situation significantly limits the ability of these methods to impact the drug discovery process for GPCR targets in "real-time", and hence, there is an urgent need for other practical and cost-efficient alternatives. We present here a conceptually pioneering approach that integrates GPCR modeling with design, synthesis, and screening of a diverse library of sugar-based compounds from the VAST technology (versatile assembly on stable templates) to provide structural insights on the MCH-1R binding site. This approach creates a cost-efficient new avenue for structure-based drug discovery (SBDD) against GPCR targets. In our work, a primary VAST hit was used to construct a high-quality MCH-1R model. Following model validation, a structure-based virtual screen yielded a 14% hit rate and 10 novel chemotypes of potent MCH-1R antagonists, including EOAI3367472 (IC50 = 131 nM) and EOAI3367474 (IC50 = 213 nM).
Constitutive stimulatory G protein activity in limb mesenchyme impairs bone growth.

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Karaca, Anara; Malladi, Vijayram Reddy; Zhu, Yan; Tafaj, Olta; Paltrinieri, Elena; Wu, Joy Y; He, Qing; Bastepe, Murat

2018-05-01

GNAS mutations leading to constitutively active stimulatory G protein alpha-subunit (Gsα) cause different tumors, fibrous dysplasia of bone, and McCune-Albright syndrome, which are typically not associated with short stature. Enhanced signaling of the parathyroid hormone/parathyroid hormone-related peptide receptor, which couples to multiple G proteins including Gsα, leads to short bones with delayed endochondral ossification. It has remained unknown whether constitutive Gsα activity also impairs bone growth. Here we generated mice expressing a constitutively active Gsα mutant (Gsα-R201H) conditionally upon Cre recombinase (cGsα R201H mice). Gsα-R201H was expressed in cultured bone marrow stromal cells from cGsα R201H mice upon adenoviral-Cre transduction. When crossed with mice in which Cre is expressed in a tamoxifen-regulatable fashion (CAGGCre-ER™), tamoxifen injection resulted in mosaic expression of the transgene in double mutant offspring. We then crossed the cGsα R201H mice with Prx1-Cre mice, in which Cre is expressed in early limb-bud mesenchyme. The double mutant offspring displayed short limbs at birth, with narrow hypertrophic chondrocyte zones in growth plates and delayed formation of secondary ossification center. Consistent with enhanced Gsα signaling, bone marrow stromal cells from these mice demonstrated increased levels of c-fos mRNA. Our findings indicate that constitutive Gsα activity during limb development disrupts endochondral ossification and bone growth. Given that Gsα haploinsufficiency also leads to short bones, as in patients with Albright's hereditary osteodystrophy, these results suggest that a tight control of Gsα activity is essential for normal growth plate physiology. Copyright © 2018 Elsevier Inc. All rights reserved.
Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

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Bachmann, Verena A; Mayrhofer, Johanna E; Ilouz, Ronit; Tschaikner, Philipp; Raffeiner, Philipp; Röck, Ruth; Courcelles, Mathieu; Apelt, Federico; Lu, Tsan-Wen; Baillie, George S; Thibault, Pierre; Aanstad, Pia; Stelzl, Ulrich; Taylor, Susan S; Stefan, Eduard

2016-07-12

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.
Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity

DEFF Research Database (Denmark)

Holst, P J; Rosenkilde, M M; Manfra, D

2001-01-01

sarcoma (KS). Here we demonstrate that several lines of mice carrying mutated receptors deficient in either constitutive activity or chemokine regulation fail to develop KS-like disease. In addition, animals expressing a receptor that preserves chemokine binding and constitutive activity but that does...... not respond to agonist stimulation have a much lower incidence of angiogenic lesions and tumors. These results indicate that induction of the KS-like disease in transgenic mice by ORF74 requires not only high constitutive signaling activity but also modulation of this activity by endogenous chemokines....

Gq activity- and β-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility

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Lin, Hui; Li, Rui-Rui; Liang, Zong-Lai; Gao, Yuan; Yang, Zhao; He, Dong-Fang; Lin, Amy; Mo, Hui; Lu, Yu-Jing; Li, Meng-Jing; Kong, Wei; Chung, Ka Young; Yi, Fan; Li, Jian-Yuan; Qin, Ying-Ying; Li, Jingxin; Thomsen, Alex R B; Kahsai, Alem W; Chen, Zi-Jiang; Xu, Zhi-Gang; Liu, Mingyao

2018-01-01

Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and β-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or β-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that β-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/β-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility. PMID:29393851
Rare platelet GPCR variants: what can we learn?

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Nisar, S P; Jones, M L; Cunningham, M R; Mumford, A D; Mundell, S J

2015-07-01

Platelet-expressed GPCRs are critical regulators of platelet function. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis associated with coronary atherosclerosis and ischaemic stroke. However, anti-thrombotic drug therapy is associated with high inter-patient variability in therapeutic response and adverse bleeding side effects. In order to optimize the use of existing anti-platelet drugs and to develop new therapies, more detailed knowledge is required relating to the molecular mechanisms that regulate GPCR and therefore platelet function. One approach has been to identify rare, function-disrupting mutations within key platelet proteins in patients with bleeding disorders. In this review, we describe how an integrated functional genomics strategy has contributed important structure-function information about platelet GPCRs with specific emphasis upon purinergic and thromboxane A2 receptors. We also discuss the potential implications these findings have for pharmacotherapy and for understanding the molecular basis of mild bleeding disorders. © 2014 The British Pharmacological Society.
Helix 11 Dynamics is Critical for Constitutive Androstane Receptor Activity

OpenAIRE

Wright, Edward; Busby, Scott A.; Wisecarver, Sarah; Vincent, Jeremy; Griffin, Patrick R.; Fernandez, Elias J.

2011-01-01

The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizi...
Synthesis and structure-activity relationship of the first nonpeptidergic inverse agonists for the human cytomegalovirus encoded chemokine receptor US28.

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Hulshof, Janneke W; Casarosa, Paola; Menge, Wiro M P B; Kuusisto, Leena M S; van der Goot, Henk; Smit, Martine J; de Esch, Iwan J P; Leurs, Rob

2005-10-06

US28 is a human cytomegalovirus (HCMV) encoded G-protein-coupled receptor that signals in a constitutively active manner. Recently, we identified 1 [5-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylpentanenitrile] as the first reported nonpeptidergic inverse agonist for a viral-encoded chemokine receptor. Interestingly, this compound is able to partially inhibit the viral entry of HIV-1. In this study we describe the synthesis of 1 and several of its analogues and unique structure-activity relationships for this first class of small-molecule ligands for the chemokine receptor US28. Moreover, the compounds have been pharmacologically characterized as inverse agonists on US28. By modification of lead structure 1, it is shown that a 4-phenylpiperidine moiety is essential for affinity and activity. Other structural features of 1 are shown to be of less importance. These compounds define the first SAR of ligands on a viral GPCR (US28) and may therefore serve as important tools to investigate the significance of US28-mediated constitutive activity during viral infection.
Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

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Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

2010-03-23

NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.
Constitutive activation of Gli2 impairs bone formation in postnatal growing mice.

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Kyu Sang Joeng

Full Text Available Indian hedgehog (Ihh signaling is indispensable for osteoblast differentiation during endochondral bone development in the mouse embryo. We have previously shown that the Gli2 transcription activator critically mediates Ihh function in osteoblastogenesis. To explore the possibility that activation of Hedgehog (Hh signaling may enhance bone formation, we generated mice that expressed a constitutively active form of Gli2 in the Osx-lineage cells. Unexpectedly, these mice exhibited severe osteopenia due to a marked decrease in osteoblast number and function, although bone resorption was not affected. Quantitative analyses of the molecular markers indicated that osteoblast differentiation was impaired in the mutant mouse. However, the osteoblast-lineage cells isolated from these mice exhibited more robust osteoblast differentiation than normal in vitro. Similarly, pharmacological stimulation of Hh signaling enhanced osteoblast differentiation from Osx-expressing cells isolated from the wild-type mouse. Thus, even though Hh signaling directly promotes osteoblast differentiation in vitro, constitutive activation of this pathway impairs bone formation in vivo, perhaps through an indirect mechanism.
A new inhibitor of the ?-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

OpenAIRE

Beautrait, Alexandre; Paradis, Justine S.; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M.; Khoury, Etienne; Audet, Martin; Roux, Philippe P.; Veprintsev, Dmitry B.; Laporte, St?phane A.

2017-01-01

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, ?-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of ?-arrestin recruitment to the receptor and ?-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecul...
NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

International Nuclear Information System (INIS)

Nishina, Takashi; Yamaguchi, Noritaka; Gohda, Jin; Semba, Kentaro; Inoue, Jun-ichiro

2009-01-01

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-κB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-κB activation. Here, we show that the alternative pathway is constitutively activated and NF-κB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.
NIK is involved in constitutive activation of the alternative NF-{kappa}B pathway and proliferation of pancreatic cancer cells

Energy Technology Data Exchange (ETDEWEB)

Nishina, Takashi [Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Yamaguchi, Noritaka [Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041 (Japan); Gohda, Jin [Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Semba, Kentaro [Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Wasedatsurumaki-cho, Shinjuku-ku, Tokyo 162-0041 (Japan); Department of Life Science and Medical Bio-science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan); Inoue, Jun-ichiro, E-mail: jun-i@ims.u-tokyo.ac.jp [Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan)

2009-10-09

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-{kappa}B is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-{kappa}B activation. Here, we show that the alternative pathway is constitutively activated and NF-{kappa}B-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.
Quantitative GPCR and ion channel transcriptomics in primary alveolar macrophages and macrophage surrogates

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Groot-Kormelink Paul J

2012-10-01

Full Text Available Abstract Background Alveolar macrophages are one of the first lines of defence against invading pathogens and play a central role in modulating both the innate and acquired immune systems. By responding to endogenous stimuli within the lung, alveolar macrophages contribute towards the regulation of the local inflammatory microenvironment, the initiation of wound healing and the pathogenesis of viral and bacterial infections. Despite the availability of protocols for isolating primary alveolar macrophages from the lung these cells remain recalcitrant to expansion in-vitro and therefore surrogate cell types, such as monocyte derived macrophages and phorbol ester-differentiated cell lines (e.g. U937, THP-1, HL60 are frequently used to model macrophage function. Methods The availability of high throughput gene expression technologies for accurate quantification of transcript levels enables the re-evaluation of these surrogate cell types for use as cellular models of the alveolar macrophage. Utilising high-throughput TaqMan arrays and focussing on dynamically regulated families of integral membrane proteins, we explore the similarities and differences in G-protein coupled receptor (GPCR and ion channel expression in alveolar macrophages and their widely used surrogates. Results The complete non-sensory GPCR and ion channel transcriptome is described for primary alveolar macrophages and macrophage surrogates. The expression of numerous GPCRs and ion channels whose expression were hitherto not described in human alveolar macrophages are compared across primary macrophages and commonly used macrophage cell models. Several membrane proteins known to have critical roles in regulating macrophage function, including CXCR6, CCR8 and TRPV4, were found to be highly expressed in macrophages but not expressed in PMA-differentiated surrogates. Conclusions The data described in this report provides insight into the appropriate choice of cell models for
Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

Science.gov (United States)

Li, Ting; Liu, Nannan

2017-12-01

This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.
Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells.

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Katie M Lowther

Full Text Available G protein-coupled receptor 3 (GPR3 is a constitutively active receptor that maintains high 3'-5'-cyclic adenosine monophosphate (cAMP levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2 and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third
Constitutively active transforming growth factor β receptor 1 in the mouse ovary promotes tumorigenesis

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Gao, Yang; Vincent, David F.; Davis, Anna Jane; Sansom, Owen J.; Bartholin, Laurent; Li, Qinglei

2016-01-01

Despite the well-established tumor suppressive role of TGFβ proteins, depletion of key TGFβ signaling components in the mouse ovary does not induce a growth advantage. To define the role of TGFβ signaling in ovarian tumorigenesis, we created a mouse model expressing a constitutively active TGFβ receptor 1 (TGFBR1) in ovarian somatic cells using conditional gain-of-function approach. Remarkably, these mice developed ovarian sex cord-stromal tumors with complete penetrance, leading to reproductive failure and mortality. The tumors expressed multiple granulosa cell markers and caused elevated serum inhibin and estradiol levels, reminiscent of granulosa cell tumors. Consistent with the tumorigenic effect, overactivation of TGFBR1 altered tumor microenvironment by promoting angiogenesis and enhanced ovarian cell proliferation, accompanied by impaired cell differentiation and dysregulated expression of critical genes in ovarian function. By further exploiting complementary genetic models, we substantiated our finding that constitutively active TGFBR1 is a potent oncogenic switch in mouse granulosa cells. In summary, overactivation of TGFBR1 drives gonadal tumor development. The TGFBR1 constitutively active mouse model phenocopies a number of morphological, hormonal, and molecular features of human granulosa cell tumors and are potentially valuable for preclinical testing of targeted therapies to treat granulosa cell tumors, a class of poorly defined ovarian malignancies. PMID:27344183
Growth hormone (GH)-independent dimerization of GH receptor by a leucine zipper results in constitutive activation

DEFF Research Database (Denmark)

Behncken, S N; Billestrup, Nils; Brown, R

2000-01-01

Growth hormone initiates signaling by inducing homodimerization of two GH receptors. Here, we have sought to determine whether constitutively active receptor can be created in the absence of the extracellular domain by substituting it with high affinity leucine zippers to create dimers of the gro......Growth hormone initiates signaling by inducing homodimerization of two GH receptors. Here, we have sought to determine whether constitutively active receptor can be created in the absence of the extracellular domain by substituting it with high affinity leucine zippers to create dimers...
Constitutive activation of a slowly migrating isoform of Stat3 in mycosis fungoides: tyrphostin AG490 inhibits Stat3 activation and growth of mycosis fungoides tumor cell lines

DEFF Research Database (Denmark)

Nielsen, M; Kaltoft, K; Nordahl, M

1997-01-01

. Jaks link cytokine receptors to Stats, and abnormal Jak/Stat signaling has been observed in some hemopoietic cancers. In MF tumor cells, a slowly migrating isoform of Stat3, Stat3(sm), was found to be constitutively activated, i.e., (i) Stat3(sm) was constitutively phosphorylated on tyrosine residues...... specific. Thus, neither the fast migrating isoform of Stat3 (Stat3(fm)) nor other Stats (Stat1, Stat2, and Stat4 through Stat6) were constitutively activated. The Jak kinase inhibitor, tyrphostin AG490, blocked the constitutive activation of Stat3(sm) and inhibited spontaneous as well as interleukin 2...
Constitutive nitric oxide synthase (cNOS activity in Langerhans islets from streptozotocin diabetic rats

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Fonovich de Schroeder T.M.

1998-01-01

Full Text Available Nitric oxide synthase activity was measured in Langerhans islets isolated from control and streptozotocin diabetic rats. The activity of the enzyme was linear up to 150 µg of protein from control rats and was optimal at 0.1 µM calcium, when it was measured after 45 min of incubation at 37oC in the presence of 200 µM arginine. Specific activity of the enzyme was 25 x 10-4 nmol [3H]citrulline 45 min-1 mg protein-1. Streptozotocin diabetic rats exhibited less enzyme activity both in total pancreas homogenate and in isolated Langerhans islets when compared to control animals. Nitric oxide synthase activity measured in control and diabetic rats 15 days after the last streptozotocin injection in the second group of animals corresponded only to a constitutive enzyme since it was not inhibited by aminoguanidine in any of the mentioned groups. Hyperglycemia in diabetic rats may be the consequence of impaired insulin release caused at least in part by reduced positive modulation mediated by constitutive nitric oxide synthase activity, which was dramatically reduced in islets severely damaged after streptozotocin treatment.
Constitutively active signaling by the G protein βγ-subunit mediates intrinsically increased phosphodiesterase-4 activity in human asthmatic airway smooth muscle cells.

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Aihua Hu

Full Text Available Signaling by the Gβγ subunit of Gi protein, leading to downstream c-Src-induced activation of the Ras/c-Raf1/MEK-ERK1/2 signaling pathway and its upregulation of phosphodiesterase-4 (PDE4 activity, was recently shown to mediate the heightened contractility in proasthmatic sensitized isolated airway smooth muscle (ASM, as well as allergen-induced airway hyperresponsiveness and inflammation in an in vivo animal model of allergic asthma. This study investigated whether cultured human ASM (HASM cells derived from asthmatic donor lungs exhibit constitutively increased PDE activity that is attributed to intrinsically upregulated Gβγ signaling coupled to c-Src activation of the Ras/MEK/ERK1/2 cascade. We show that, relative to normal cells, asthmatic HASM cells constitutively exhibit markedly increased intrinsic PDE4 activity coupled to heightened Gβγ-regulated phosphorylation of c-Src and ERK1/2, and direct co-localization of the latter with the PDE4D isoform. These signaling events and their induction of heightened PDE activity are acutely suppressed by treating asthmatic HASM cells with a Gβγ inhibitor. Importantly, along with increased Gβγ activation, asthmatic HASM cells also exhibit constitutively increased direct binding of the small Rap1 GTPase-activating protein, Rap1GAP, to the α-subunit of Gi protein, which serves to cooperatively facilitate Ras activation and, thereby, enable enhanced Gβγ-regulated ERK1/2-stimulated PDE activity. Collectively, these data are the first to identify that intrinsically increased signaling via the Gβγ subunit, facilitated by Rap1GAP recruitment to the α-subunit, mediates the constitutively increased PDE4 activity detected in asthmatic HASM cells. These new findings support the notion that interventions targeted at suppressing Gβγ signaling may lead to novel approaches to treat asthma.
The GTPase Rab43 Controls the Anterograde ER-Golgi Trafficking and Sorting of GPCRs

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Chunman Li

2017-10-01

Full Text Available G-protein-coupled receptors (GPCRs constitute the largest superfamily of cell-surface signaling proteins. However, mechanisms underlying their surface targeting and sorting are poorly understood. Here, we screen the Rab family of small GTPases in the surface transport of multiple GPCRs. We find that manipulation of Rab43 function significantly alters the surface presentation and signaling of all GPCRs studied without affecting non-GPCR membrane proteins. Rab43 specifically regulates the transport of nascent GPCRs from the endoplasmic reticulum (ER to the Golgi. More interestingly, Rab43 directly interacts with GPCRs in an activation-dependent fashion. The Rab43-binding domain identified in the receptors effectively converts non-GPCR membrane protein transport into a Rab43-dependent pathway. These data reveal a crucial role for Rab43 in anterograde ER-Golgi transport of nascent GPCRs, as well as the ER sorting of GPCR members by virtue of its ability to interact directly.
Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

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Navarro, Gemma; Cordomí, Arnau; Casadó-Anguera, Verónica; Moreno, Estefanía; Cai, Ning-Sheng; Cortés, Antoni; Canela, Enric I; Dessauer, Carmen W; Casadó, Vicent; Pardo, Leonardo; Lluís, Carme; Ferré, Sergi

2018-03-28

G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A 2A receptor and dopamine D 2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.
Legitimacy of Constitutional Justice: Democracy, Constitutional Court and Theory Against Majority Interest

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Thaminne Nathalia Cabral Moraes e Silva

2016-12-01

Full Text Available This article has as its theme the analysis of the separation of powers and the rule of democracy, in addition to the possibility of the Constitutional Court be composed of people appointed by the President of the Republic, not fulfilling the democratic rule, and make the control of constitutionality of laws, created through democratic process. Will be answered: the separation of powers obey the democratic rule? When the Legislature fails to fulfill its function of legislating, opens the opportunity for the Supreme Court, as the Constitutional Court that is, create, through judicial activism, silent rules? That injured the democratic rule?

Constitutively active Notch1 converts cranial neural crest-derived frontonasal mesenchyme to perivascular cells in vivo

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Sophie R. Miller

2017-03-01

Full Text Available Perivascular/mural cells originate from either the mesoderm or the cranial neural crest. Regardless of their origin, Notch signalling is necessary for their formation. Furthermore, in both chicken and mouse, constitutive Notch1 activation (via expression of the Notch1 intracellular domain is sufficient in vivo to convert trunk mesoderm-derived somite cells to perivascular cells, at the expense of skeletal muscle. In experiments originally designed to investigate the effect of premature Notch1 activation on the development of neural crest-derived olfactory ensheathing glial cells (OECs, we used in ovo electroporation to insert a tetracycline-inducible NotchΔE construct (encoding a constitutively active mutant of mouse Notch1 into the genome of chicken cranial neural crest cell precursors, and activated NotchΔE expression by doxycycline injection at embryonic day 4. NotchΔE-targeted cells formed perivascular cells within the frontonasal mesenchyme, and expressed a perivascular marker on the olfactory nerve. Hence, constitutively activating Notch1 is sufficient in vivo to drive not only somite cells, but also neural crest-derived frontonasal mesenchyme and perhaps developing OECs, to a perivascular cell fate. These results also highlight the plasticity of neural crest-derived mesenchyme and glia.
Constitutive activation of the thyroid-stimulating hormone receptor (TSHR by mutating Ile691 in the cytoplasmic tail segment.

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Zheng Liu

Full Text Available BACKGROUND: Autosomal dominant non-autoimmune hyperthyroidism (ADNAH is a rare genetic disorder of the endocrine system. Molecular genetic studies in ADNAH have revealed heterozygous germline mutations in the TSHR. To data, mutations leading to an increase in the constitutive activation of the TSHR have been described in the transmembrane segments, exoloops and cytoplasmic loop of TSHR. These mutations result in constitutive activation of the G(αs/cAMP or G(αq/11/inositol phosphate (IP pathways, which stimulate thyroid hormone production and thyroid proliferation. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study, we reported a new TSHR mutation located in the C-terminal domain of TSHR, which results in a substitution of the conserved Ile(691 for Phe. In this study, to address the question of whether the I691F mutated receptor could be responsible for G(αs/cAMP or G(αq/11/IP constitutive activity, wild-type and TSHR mutants were expressed in COS-7 cells to determine cAMP constitutive activity and IP formation. Compared to the cell surface with expression of the A623V mutated receptor as positive control, the I691F mutated receptor showed a slight increase of cAMP accumulation. Furthermore, I691F resulted in constitutive activation of the G(αq/11/IP signaling pathway. CONCLUSIONS/SIGNIFICANCE: Our results indicate that Ile(691 not only contributes to keeping TSHR inactive in the G(αs/cAMP pathways but also in the G(αq/11/IP cascade.
Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive G{sub s}–G protein signaling in osteoblasts

Energy Technology Data Exchange (ETDEWEB)

Wattanachanya, Lalita, E-mail: lalita_md@yahoo.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Division of Endocrinology and Metabolism, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok (Thailand); Wang, Liping, E-mail: lipingwang05@yahoo.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Millard, Susan M., E-mail: susan.millard@mater.uq.edu.au [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Lu, Wei-Dar, E-mail: weidar_lu@yahoo.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); O’Carroll, Dylan, E-mail: dylancocarroll@gmail.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Hsiao, Edward C., E-mail: Edward.Hsiao@ucsf.edu [Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Francisco, CA (United States); Conklin, Bruce R., E-mail: bconklin@gladstone.ucsf.edu [Gladstone Institute of Cardiovascular Disease, San Francisco, CA (United States); Department of Medicine, University of California, San Francisco, CA (United States); Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA (United States); Nissenson, Robert A., E-mail: Robert.Nissenson@ucsf.edu [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States)

2015-05-01

G protein-coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active G{sub s}-coupled GPCR, under the control of the 2.3 kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced G{sub s} signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated G{sub s} G protein-coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3 kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of G{sub s}-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of G{sub s} signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to G{sub s} signaling in mature OBs. - Highlights: • OB expression of an engineered G{sub s}-coupled receptor dramatically increases bone mass. • We investigated the changes in gene expression in vivo in enhanced OB G{sub s} signaling. • Genes in cell cycle and transcription were increased in
Discovery and development of inhibitors selective for human constitutive proteasome and immunoproteasome active sites

NARCIS (Netherlands)

Xin, B.

2017-01-01

This thesis describes the design and development of subunit‐selective inhibitors of particular catalytically active subunits of human constitutive proteasomes and immunoproteasomes. Most existing proteasome inhibitors are oligopeptides composed of 2‐4 amino acid residues, N‐terminally
Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop

DEFF Research Database (Denmark)

Mokrosinski, Jacek; Frimurer, Thomas M; Sivertsen, Bjoern

2012-01-01

Based on a rare, natural Glu for Ala204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2 (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional....... Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal-ion site stabilizing an a-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment...
Prevalence of bortezomib-resistant constitutive NF-kappaB activity in mantle cell lymphoma

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Kahl Brad S

2008-05-01

Full Text Available Abstract Background The proteasome inhibitor bortezomib can inhibit activation of the transcription factor NF-κB, a mechanism implicated in its anti-neoplastic effects observed in mantle cell lymphoma (MCL. However, NF-κB can be activated through many distinct mechanisms, including proteasome independent pathways. While MCL cells have been shown to harbor constitutive NF-κB activity, what fraction of this activity in primary MCL samples is sensitive or resistant to inhibition by bortezomib remains unclear. Results Proteasome activity in the EBV-negative MCL cell lines Jeko-1 and Rec-1 is inhibited by greater than 80% after exposure to 20 nM bortezomib for 4 hours. This treatment decreased NF-κB activity in Jeko-1 cells, but failed to do so in Rec-1 cells when assessed by electrophoretic mobility shift assay (EMSA. Concurrently, Rec-1 cells were more resistant to the cytotoxic effects of bortezomib than Jeko-1 cells. Consistent with a proteasome inhibitor resistant pathway of activation described in mouse B-lymphoma cells (WEHI231 and a breast carcinoma cell line (MDA-MB-468, the bortezomib-resistant NF-κB activity in Rec-1 cells is inhibited by calcium chelators, calmodulin inhibitors, and perillyl alcohol, a monoterpene capable of blocking L-type calcium channels. Importantly, the combination of perillyl alcohol and bortezomib is synergistic in eliciting Rec-1 cell cytotoxicity. The relevance of these results is illuminated by the additional finding that a considerable fraction of primary MCL samples (8 out of 10 displayed bortezomib-resistant constitutive NF-κB activity. Conclusion Our findings show that bortezomib-resistant NF-κB activity is frequently observed in MCL samples and suggest that this activity may be relevant to MCL biology as well as serve as a potential therapeutic target.
Constitutive activation of the ERK pathway in melanoma and skin melanocytes in Grey horses.

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Jiang, Lin; Campagne, Cécile; Sundström, Elisabeth; Sousa, Pedro; Imran, Saima; Seltenhammer, Monika; Pielberg, Gerli; Olsson, Mats J; Egidy, Giorgia; Andersson, Leif; Golovko, Anna

2014-11-21

Constitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses. Grey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting. We found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway. The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases. Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses. These findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this
Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

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Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

2013-05-07

Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.
The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

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Pack, Thomas F; Orlen, Margo I; Ray, Caroline; Peterson, Sean M; Caron, Marc G

2018-04-20

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and β-arrestins (βarrs). Selectively engaging either the G protein or βarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to βarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates βarr recruitment, bridge these pathways. Therefore, βarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on βarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or βarrs as well as a βarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the βarr-preferring D2R achieves βarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the βarr-preferring D2R and also contributes to βarr recruitment at the WT D2R. Additionally, we found an additive interaction between the βarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving βarr-biased signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Single amino acid change in STING leads to constitutive active signaling.

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Eric D Tang

Full Text Available The production of cytokines by the immune system in response to cytosolic DNA plays an important role in host defense, autoimmune disease, and cancer immunogenicity. Recently a cytosolic DNA signaling pathway that is dependent on the endoplasmic reticulum adaptor and cyclic dinucleotide sensor protein STING has been identified. Association of cytosolic DNA with cyclic-GMP-AMP synthase (cGAS activates its enzymatic activity to synthesize the cyclic dinucleotide second messenger cGAMP from GTP and ATP. Direct detection of cGAMP by STING triggers the activation of IRF3 and NF-kB, and the production of type I interferons and proinflammatory cytokines. The mechanism of how STING is able to mediate downstream signaling remains incompletely understood although it has been shown that dimerization is a prerequisite. Here, we identify a single amino acid change in STING that confers constitutive active signaling. This mutation appears to both enhance ability of STING to both dimerize and associate with its downstream target TBK1.
Single amino acid change in STING leads to constitutive active signaling.

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Tang, Eric D; Wang, Cun-Yu

2015-01-01

The production of cytokines by the immune system in response to cytosolic DNA plays an important role in host defense, autoimmune disease, and cancer immunogenicity. Recently a cytosolic DNA signaling pathway that is dependent on the endoplasmic reticulum adaptor and cyclic dinucleotide sensor protein STING has been identified. Association of cytosolic DNA with cyclic-GMP-AMP synthase (cGAS) activates its enzymatic activity to synthesize the cyclic dinucleotide second messenger cGAMP from GTP and ATP. Direct detection of cGAMP by STING triggers the activation of IRF3 and NF-kB, and the production of type I interferons and proinflammatory cytokines. The mechanism of how STING is able to mediate downstream signaling remains incompletely understood although it has been shown that dimerization is a prerequisite. Here, we identify a single amino acid change in STING that confers constitutive active signaling. This mutation appears to both enhance ability of STING to both dimerize and associate with its downstream target TBK1.
Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.

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Stolwijk, Judith A; Matrougui, Khalid; Renken, Christian W; Trebak, Mohamed

2015-10-01

The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate.
Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor

OpenAIRE

Cherian, Milu T.; Yang, Lei; Chai, Sergio C.; Lin, Wenwei; Chen, Taosheng

2016-01-01

The constitutive androstane receptor (CAR) regulates the expression of genes involved in drug metabolism and other processes. A specific inhibitor of CAR is critical for modulating constitutive CAR activity. We recently described a specific small-molecule inhibitor of CAR, CINPA1 (ethyl (5-(diethylglycyl)-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)carbamate), which is capable of reducing CAR-mediated transcription by changing the coregulator recruitment pattern and reducing CAR occupancy at the...
Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

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Saadat U Aleem

Full Text Available The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.
The Constitutional Court and the Imperative of its Reform

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Claudia Gilia

2012-12-01

Full Text Available Recent debates on the upcoming review of the Constitution have determined us to pay close attention to the basic institution in a democratic state, that is the Constitutional Court. Being caught in the crossfire between power and opposition, the Constitutional Court had a hard time lately, facing severe attacks. The aim of our study is to analyze the evolution of the Constitutional Court within the inland constitutional system, particularly bringing up the flaws describing the Court’s activity. We have also analyzed the proposals put forth by several bodies or experts regarding the constitutional contentious court. At the end of our study, following an analysis of different constitutional types of constitutional review, used by a number of states in Europe, we introduced several resolutions that may improve the role, the course and, last but not least, the activity of the Romanian Constitutional Court.
Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity

DEFF Research Database (Denmark)

Rosenkilde, Mette M; Benned-Jensen, Tau; Holst, Peter J

2006-01-01

Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Galpha(i) as determined by a receptor...
Pinning down the Constitution: Interactively Teaching Congress's Power, Federalism, and Constitutional Interpretation

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Emenaker, Ryan

2014-01-01

"Pin-the-Tail-on-the-Constitution" is an engaged-learning activity that has been conducted in 26 classes over the past four years. The activity teaches multiple themes commonly included in a variety of courses on American politics such as federalism, congressional powers, the role of the federal courts, and the relevance of the commerce…
Integrin and GPCR Crosstalk in the Regulation of ASM Contraction Signaling in Asthma.

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Teoh, Chun Ming; Tam, John Kit Chung; Tran, Thai

2012-01-01

Airway hyperresponsiveness (AHR) is one of the cardinal features of asthma. Contraction of airway smooth muscle (ASM) cells that line the airway wall is thought to influence aspects of AHR, resulting in excessive narrowing or occlusion of the airway. ASM contraction is primarily controlled by agonists that bind G protein-coupled receptor (GPCR), which are expressed on ASM. Integrins also play a role in regulating ASM contraction signaling. As therapies for asthma are based on symptom relief, better understanding of the crosstalk between GPCRs and integrins holds good promise for the design of more effective therapies that target the underlying cellular and molecular mechanism that governs AHR. In this paper, we will review current knowledge about integrins and GPCRs in their regulation of ASM contraction signaling and discuss the emerging concept of crosstalk between the two and the implication of this crosstalk on the development of agents that target AHR.
A Prospect and Challenges for Adopting Constitutional Complaint and Constitutional Question in the Indonesian Constitutional Court

OpenAIRE

Faiz, Pan Mohamad

2016-01-01

A jurisdiction of the Indonesian Constitutional Court concerning constitutional adjudication is only limited to review the constitutionality of national law. There is no mechanism for challenging any decision or action made by public authorities that violate fundamental rights enshrined in the Indonesian Constitution. This article argues that constitutional complaint and constitutional question might be adopted as new jurisdictions of the Indonesian Constitutional Court in order to strengthen...
NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice

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Song, Hongman; Vijayasarathy, Camasamudram; Zeng, Yong; Marangoni, Dario; Bush, Ronald A.; Wu, Zhijian; Sieving, Paul A.

2016-01-01

Purpose The active form of small GTPase RAC1 is required for activation of NADPH oxidase (NOX), which in turn generates reactive oxygen species (ROS) in nonphagocytic cells. We explored whether NOX-induced oxidative stress contributes to rod degeneration in retinas expressing constitutively active (CA) RAC1. Methods Transgenic (Tg)–CA-RAC1 mice were given apocynin (10 mg/kg, intraperitoneal), a NOX inhibitor, or vehicle daily for up to 13 weeks. Superoxide production and oxidative damage were assessed by dihydroethidium staining and by protein carbonyls and malondialdehyde levels, respectively. Outer nuclear layer (ONL) cells were counted and electroretinogram (ERG) amplitudes measured in Tg-CA-RAC1 mice. Outer nuclear layer cells were counted in wild-type (WT) mice after transfer of CA-Rac1 gene by subretinal injection of AAV8-pOpsin-CA Rac1-GFP. Results Transgenic-CA-RAC1 retinas had significantly fewer photoreceptor cells and more apoptotic ONL cells than WT controls from postnatal week (Pw) 3 to Pw13. Superoxide accumulation and protein and lipid oxidation were increased in Tg-CA-RAC1 retinas and were reduced in mice treated with apocynin. Apocynin reduced the loss of photoreceptors and increased the rod ERG a- and b-wave amplitudes when compared with vehicle-injected transgenic controls. Photoreceptor loss was also observed in regions of adult WT retina transduced with AAV8-pOpsin-CA Rac1-GFP but not in neighboring regions that were not transduced or in AAV8-pOpsin-GFP–transduced retinas. Conclusions Constitutively active RAC1 promotes photoreceptor cell death by oxidative damage that occurs, at least partially, through NOX-induced ROS. Reactive oxygen species are likely involved in multiple forms of retinal degenerations, and our results support investigating RAC1 inhibition as a therapeutic approach that targets this disease pathway. PMID:27233035

Structural basis for constitutive activity and agonist-induced activation of the enteroendocrine fat sensor GPR119

DEFF Research Database (Denmark)

Engelstoft, Maja Storm; Norn, C; Pedersen, Maria Hauge

2014-01-01

BACKGROUND AND PURPOSE: GPR119 is a Gαs-coupled 7TM receptor activated by endogenous lipids such as oleoylethanolamide (OEA) and by the dietary triglyceride metabolite 2-monoacylglycerol. GPR119 stimulates enteroendocrine hormone and insulin secretion. But despite massive drug discovery efforts...... activation (AR231453 and OEA). Novel Rosetta-based receptor modelling was applied, using a composite template approach with segments from different X-ray structures and fully flexible ligand docking. KEY RESULTS: The increased signalling induced by increasing the cell surface expression of GPR119...... in the absence of agonist and the inhibitory effect of two synthetic inverse agonists demonstrated that GRP119 signals with a high degree of constitutive activity through the Gαs pathway. The mutational maps for AR231453 and OEA were very similar and, surprisingly, also similar to the mutational map for residues...
Navigating the conformational landscape of G protein-coupled receptor kinases during allosteric activation.

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Yao, Xin-Qiu; Cato, M Claire; Labudde, Emily; Beyett, Tyler S; Tesmer, John J G; Grant, Barry J

2017-09-29

G protein-coupled receptors (GPCRs) are essential for transferring extracellular signals into carefully choreographed intracellular responses controlling diverse aspects of cell physiology. The duration of GPCR-mediated signaling is primarily regulated via GPCR kinase (GRK)-mediated phosphorylation of activated receptors. Although many GRK structures have been reported, the mechanisms underlying GRK activation are not well-understood, in part because it is unknown how these structures map to the conformational landscape available to this enzyme family. Unlike most other AGC kinases, GRKs rely on their interaction with GPCRs for activation and not phosphorylation. Here, we used principal component analysis of available GRK and protein kinase A crystal structures to identify their dominant domain motions and to provide a framework that helps evaluate how close each GRK structure is to being a catalytically competent state. Our results indicated that disruption of an interface formed between the large lobe of the kinase domain and the regulator of G protein signaling homology domain (RHD) is highly correlated with establishment of the active conformation. By introducing point mutations in the GRK5 RHD-kinase domain interface, we show with both in silico and in vitro experiments that perturbation of this interface leads to higher phosphorylation activity. Navigation of the conformational landscape defined by this bioinformatics-based study is likely common to all GPCR-activated GRKs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Combining on-chip synthesis of a focused combinatorial library with computational target prediction reveals imidazopyridine GPCR ligands.

Science.gov (United States)

Reutlinger, Michael; Rodrigues, Tiago; Schneider, Petra; Schneider, Gisbert

2014-01-07

Using the example of the Ugi three-component reaction we report a fast and efficient microfluidic-assisted entry into the imidazopyridine scaffold, where building block prioritization was coupled to a new computational method for predicting ligand-target associations. We identified an innovative GPCR-modulating combinatorial chemotype featuring ligand-efficient adenosine A1/2B and adrenergic α1A/B receptor antagonists. Our results suggest the tight integration of microfluidics-assisted synthesis with computer-based target prediction as a viable approach to rapidly generate bioactivity-focused combinatorial compound libraries with high success rates. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse.

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Helen Waller-Evans

2010-11-01

Full Text Available Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.
Constitutive STAT5 Activation Correlates With Better Survival in Cervical Cancer Patients Treated With Radiation Therapy

Energy Technology Data Exchange (ETDEWEB)

Chen, Helen H.W. [Department of Radiation Oncology, National Cheng Kung University, Medical College and Hospital, Tainan, Taiwan (China); Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Chou, Cheng-Yang [Department of Obstetrics and Gynecology, National Cheng Kung University, Medical College and Hospital, Tainan, Taiwan (China); Wu, Yuan-Hua; Hsueh, Wei-Ting; Hsu, Chiung-Hui [Department of Radiation Oncology, National Cheng Kung University, Medical College and Hospital, Tainan, Taiwan (China); Guo, How-Ran [Department of Environmental and Occupational Health, National Cheng Kung University, Medical College and Hospital, Tainan, Taiwan (China); Lee, Wen-Ying, E-mail: 7707@so-net.net.tw [Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan (China) and Department of Pathology, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Su, Wu-Chou, E-mail: sunnysu@mail.ncku.edu.tw [Department of Internal Medicine, National Cheng Kung University, Medical College and Hospital, Tainan, Taiwan (China)

2012-02-01

Purpose: Constitutively activated signal transducers and activators of transcription (STAT) factors, in particular STAT1, STAT3, and STAT5, have been detected in a wide variety of human primary tumors and have been demonstrated to directly contribute to oncogenesis. However, the expression pattern of these STATs in cervical carcinoma is still unknown, as is whether or not they have prognostic significance. This study investigated the expression patterns of STAT1, STAT3, and STAT5 in cervical cancer and their associations with clinical outcomes in patients treated with radical radiation therapy. Methods and Materials: A total of 165 consecutive patients with International Federation of Gynecology and Obstetrics (FIGO) Stages IB to IVA cervical cancer underwent radical radiation therapy, including external beam and/or high-dose-rate brachytherapy between 1989 and 2002. Immunohistochemical studies of their formalin-fixed, paraffin-embedded tissues were performed. Univariate and multivariate analyses were performed to identify and to evaluate the effects of these factors affecting patient survival. Results: Constitutive activations of STAT1, STAT3, and STAT5 were observed in 11%, 22%, and 61% of the participants, respectively. While STAT5 activation was associated with significantly better metastasis-free survival (p < 0.01) and overall survival (p = 0.04), STAT1 and STAT3 activation were not. Multivariate analyses showed that STAT5 activation, bulky tumor ({>=}4 cm), advanced stage (FIGO Stages III and IV), and brachytherapy (yes vs. no) were independent prognostic factors for cause-specific overall survival. None of the STATs was associated with local relapse. STAT5 activation (odds ratio = 0.29, 95% confidence interval = 0.13-0.63) and advanced stage (odds ratio = 2.54; 95% confidence interval = 1.03-6.26) were independent predictors of distant metastasis. Conclusions: This is the first report to provide the overall expression patterns and prognostic significance of
Curcumin induces growth-arrest and apoptosis in association with the inhibition of constitutively active JAK-STAT pathway in T cell leukemia

International Nuclear Information System (INIS)

Rajasingh, Johnson; Raikwar, Himanshu P.; Muthian, Gladson; Johnson, Caroline; Bright, John J.

2006-01-01

Adult T cell leukemia is an aggressive and frequently fatal malignancy that expressess constitutively activated growth-signaling pathways in association with deregulated growth and resistance to apoptosis. Curcumin (diferuloylmethane) is a naturally occurring yellow pigment, isolated from the rhizomes of the plant Curcuma longa that has traditionally been used in the treatment of injury and inflammation. But the effect and mechanism of action of curcumin on T cell leukemia is not known. To investigate the antitumor activity of curcumin in T cell leukemia, we examined its effect on constitutive phosphorylation of JAK and STAT proteins, proliferation, and apoptosis in HTLV-I-transformed T cell lines. HTLV-I-transformed T cell leukemia lines, MT-2, HuT-102, and SLB-1, express constitutively phosphorylated JAK3, TYK2, STAT3, and STAT5 signaling proteins. In vitro treatment with curcumin induced a dose-dependent decrease in JAK and STAT phosphorylation resulting in the induction of growth-arrest and apoptosis in T cell leukemia. The induction of growth-arrest and apoptosis in association with the blockade of constitutively active JAK-STAT pathway suggests this be a mechanism by which curcumin induces antitumor activity in T cell leukemia
COUNTERMAJORITARIAN INSTITUTIONS IN THE RUSSIAN CONSTITUTION OF 1993 AS AN INSTRUMENT ENSURING CONSTITUTIONAL AND POLITICAL STABILITY

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S. Shakhray

2018-01-01

Full Text Available The article enriches the discussion on the legal reasons and conditions fostering the viability of democratic constitutions by analyzing the rich experience of the Russian Constitution of 1993. Particular attention is paid to the concept of countermajoritarian institutions. The authors elaborate the idea that countermajoritarian institutions can play an important role in ensuring the viability (put in other terms, the proper balance between stability, adaptability, and dynamic development of modern democratic constitutions as well as political regimes.The article presents evidence-based data showing that the President and the Constitutional Court of the Russian Federation systematically acted as countermajoritarian institutions at the initial stage of the implementation of the “blueprint for the future” set down in the 1993 Russian Constitution. As a result of the activities of these institutions, strong legal frameworks were created that are necessary for the establishment of anew constitutional system of the Russian state and law.Today, the Russian Constitution of 1993 is one of the longest lasting democratic constitutions in the world (the average “life expectancy” of democratic constitutions adopted over the past 250 years is about seventeen years. The study of the countermajoritarian provisions in the 1993 Russian Constitution is of both theoretical and practical importance. In particular, the results of the study can be useful in creating efficient legal instruments for the maintenance of political stability and social development management both within sovereign states and within interstate communities.
Functional characterization of human RSK4, a new 90-kDa ribosomal S6 kinase, reveals constitutive activation in most cell types

DEFF Research Database (Denmark)

Dümmler, Bettina A; Hauge, Camilla; Silber, Joachim

2005-01-01

characterization of a predicted new human RSK homologue, RSK4. We showed that RSK4 is a predominantly cytosolic protein with very low expression and several characteristics of the RSK family kinases, including the presence of two functional kinase domains and a C-terminal docking site for ERK. Surprisingly......, however, in all cell types analyzed, endogenous RSK4 was maximally (constitutively) activated under serum-starved conditions where other RSKs are inactive due to their requirement for growth factor stimulation. Constitutive activation appeared to result from constitutive phosphorylation of Ser232, Ser372...
Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

Energy Technology Data Exchange (ETDEWEB)

Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher (Stanford); (Stanford-MED); (Whitehead); (MIT)

2015-03-05

Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.
Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

Science.gov (United States)

Luchetti, Giovanni; Sircar, Ria; Kong, Jennifer H; Nachtergaele, Sigrid; Sagner, Andreas; Byrne, Eamon FX; Covey, Douglas F; Siebold, Christian; Rohatgi, Rajat

2016-01-01

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility. DOI: http://dx.doi.org/10.7554/eLife.20304.001 PMID:27705744
A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

Science.gov (United States)

Beautrait, Alexandre; Paradis, Justine S.; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M.; Khoury, Etienne; Audet, Martin; Roux, Philippe P.; Veprintsev, Dmitry B.; Laporte, Stéphane A.; Bouvier, Michel

2017-04-01

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.
Constitutional judges (guarantee of the Constitution and responsibility

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Francisco Javier Ansuátegui Roig

2012-06-01

Full Text Available My aim in this paper is to propose a reflection on the position and the importance that the constitutional judge has in the legal systems of contemporary constitutionalism. The figure of the judge responsible of protecting the Constitution is a key institution, without which we cannot understand the laws of constitutional democracies, their current lines of development, and the guarantee of rights and freedoms that constitute the normative core of these systems. Moreover, the reflection on the exercise of the powers of the judge, its scope and its justification is an important part of contemporary legal discussion, still relevant, albeit not exclusively - in the field of legal philosophy. The object of attention of my reflection is the judge who has the power of judicial review, in a scheme of defense of the Constitution, regardless the specific ways of this defense.
The Australian Constitution and the Aid/Watch Case

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George Williams

2011-11-01

Full Text Available The Australian Constitution played a significant role in underpinning the result in the Aid/Watch Case. It was invoked by the majority to support their conclusion that a body can be a ‘charitable institution’ despite engaging in political activities. The use of the Constitution in this way came as a surprise. The case extended an existing constitutional principle relating to freedom of political communication from its electoral base into the protection of the political activities of non-government organisations. This may have future ramifications for those organisations in other areas, as well as further implications for the development of what it means to be a charity in Australia. This article examines the use of the Australian Constitution in the Aid/Watch Case. It explains how the High Court was able to invoke the Constitution in defining what it means to be a ‘charitable institution’. It also examines the implications of that reasoning for the development of charitable law in Australia.
A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

DEFF Research Database (Denmark)

Whorton, Matthew R; Bokoch, Michael P; Rasmussen, Søren Gøgsig Faarup

2007-01-01

G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet...... the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However......, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein...
Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

Science.gov (United States)

Alcázar, Isabela; Marqués, Miriam; Kumar, Amit; Hirsch, Emilio; Wymann, Matthias; Carrera, Ana C.; Barber, Domingo F.

2007-01-01

Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation. PMID:17998387
Control of ADAM17 activity by regulation of its cellular localisation

Science.gov (United States)

Lorenzen, Inken; Lokau, Juliane; Korpys, Yvonne; Oldefest, Mirja; Flynn, Charlotte M.; Künzel, Ulrike; Garbers, Christoph; Freeman, Matthew; Grötzinger, Joachim; Düsterhöft, Stefan

2016-01-01

An important, irreversible step in many signalling pathways is the shedding of membrane-anchored proteins. A Disintegrin And Metalloproteinase (ADAM) 17 is one of the major sheddases involved in a variety of physiological and pathophysiological processes including regeneration, differentiation, and cancer progression. This central role in signalling implies that ADAM17 activity has to be tightly regulated, including at the level of localisation. Most mature ADAM17 is localised intracellularly, with only a small amount at the cell surface. We found that ADAM17 is constitutively internalised by clathrin-coated pits and that physiological stimulators such as GPCR ligands induce ADAM17-mediated shedding, but do not alter the cell-surface abundance of the protease. In contrast, the PKC-activating phorbol ester PMA, often used as a strong inducer of ADAM17, causes not only proteolysis by ADAM17 but also a rapid increase of the mature protease at the cell surface. This is followed by internalisation and subsequent degradation of the protease. Eventually, this leads to a substantial downregulation of mature ADAM17. Our results therefore imply that physiological activation of ADAM17 does not rely on its relocalisation, but that PMA-induced PKC activity drastically dysregulates the localisation of ADAM17. PMID:27731361
National constitutional courts in the European Constitutional Democracy

DEFF Research Database (Denmark)

Komárek, Jan

2014-01-01

This article critically assesses the transformation of national constitutional courts’ place in the law and politics of the EU and its member states. This process eliminates the difference between constitutional and ordinary national courts, which is crucial for the institutional implementation...... of the discourse theory of law and democracy. It also disrupts the symbiotic relationship between national constitutional democracies established after World War II and European integration. The article argues that maintaining the special place of national constitutional courts is in the vital interest of both...... the EU and its member states, understood together as the European Constitutional Democracy—the central notion developed in this article in order to support an argument that should speak to both EU lawyers and national constitutionalists....
The extracellular loop 2 (ECL2 of the human histamine H4 receptor substantially contributes to ligand binding and constitutive activity.

Directory of Open Access Journals (Sweden)

David Wifling

Full Text Available In contrast to the corresponding mouse and rat orthologs, the human histamine H4 receptor (hH4R shows extraordinarily high constitutive activity. In the extracellular loop (ECL, replacement of F169 by V as in the mouse H4R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH4R-F168A mutant. The receptor was co-expressed in Sf9 insect cells with the G-protein subunits Gαi2 and Gβ1γ2, and the membranes were studied in [3H]histamine binding and functional [35S]GTPγS assays. The potency of various ligands at the hH4R-F168A mutant decreased compared to the wild-type hH4R, for example by 30- and more than 100-fold in case of the H4R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH4R was completely lost in the hH4R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH4R. By analogy, JNJ7777120 was a partial inverse agonist at the hH4R, but a partial agonist at the hH4R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH4R.
Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma

Science.gov (United States)

Yang, Jing; He, Jin; Wang, Ji; Cao, Yabing; Ling, Jianhua; Qian, Jianfei; Lu, Yong; Li, Haiyan; Zheng, Yuhuan; Lan, Yongsheng; Hong, Sungyoul; Matthews, Jairo; Starbuck, Michael W; Navone, Nora M; Orlowski, Robert Z.; Lin, Pei; Kwak, Larry W.; Yi, Qing

2012-01-01

Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into SCID or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism—sactivation of p38 signaling in myeloma cells—by which myeloma cells induce osteolytic bone lesions and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease. PMID:22425892
Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

Energy Technology Data Exchange (ETDEWEB)

Nguyen, Minh Vu Chuong, E-mail: mvchuong@yahoo.fr [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Zhang, Leilei [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Lhomme, Stanislas; Mouz, Nicolas [PX' Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble (France); Lenormand, Jean-Luc [HumProTher Laboratory, TheReX/TIMC-IMAG UMR 5525 CNRS UJF, Universite Joseph Fourier, UFR de Medecine, Domaine de la Merci, 38706 La Tronche (France); Lardy, Bernard; Morel, Francoise [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France)

2012-03-16

Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

Pivotal role of extended linker 2 in the activation of Gα by G protein-coupled receptor.

Science.gov (United States)

Huang, Jianyun; Sun, Yutong; Zhang, J Jillian; Huang, Xin-Yun

2015-01-02

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gαs mutant proteins (from a screen of 66 Gαs mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gαs. This extended Linker 2 is the target site of a natural product inhibitor of Gq. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β2/β3 loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
The Obligations on Government and Society in our Constitutional State to Respect and Support Independent Constitutional Structures

Directory of Open Access Journals (Sweden)

LWH Ackermann

2000-05-01

these institutions is vitally important for creating and sustaining an ethos of constitutionalism among the inhabitants of the Republic.The Constitution makes explicit provision for the protection of the judiciary and the other independent state institutions and thereby indirectly for the development of habits of constitutionalism. The constitutional protection and support given to the independent state institutions are very similar to that given to the courts. One important distinction is to be noted. In the case of the courts, the Constitution provides that they "are subject only to the Constitution and the law" and no provision is made for them to be accountable to any other organ of state or any other institution or person, for that matter. By contrast, the independent institutions envisaged in section 181 of the Constitution are expressly made accountable to the National Assembly and are obliged to report on their activities and the performance of their functions to the Assembly at least once a year. While the formal independence of state institutions may at all times be scrupulously recognised by the legislature and the executive, their substantive independence can easily be undermined by fiscal starvation and their ability to function properly impeded by bureaucratic administrative obstruction or obfuscation or even, quite innocently, by a lack of appreciation of what the Constitution demands from public administration in support of these institutions. Adequate financial and administrative resources are required to achieve aspects of judicial independence. All South Africans must still be vigilant to ensure, from the outset, that all state organs develop habits and practices of constitutionalism and that they do not, whether by omission, error, or otherwise endanger the independence of our independent state institutions by neglecting their constitutional obligations.For the Constitutional Court to fulfil its role as the ultimate guardian of the Constitution, it
A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor

DEFF Research Database (Denmark)

Grundmann, Manuel; Tikhonova, Irina G; Hudson, Brian D

2016-01-01

Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand...
DEVELOPMENTS IN THE CONSTITUTIONAL REVIEW. CONSTITUTIONAL COURT BETWEEN THE STATUS OF NEGATIVE LEGISLATOR AND THE STATUS OF POSITIVE CO-LEGISLATOR

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Marieta Safta

2012-11-01

Full Text Available The study wants to emphasize that Constitutional Courts belonging to the European model depart from their traditional role as ”negative legislator” – which refers to the effect of their acts consisting in removal from the legal system of those rules contrary to the Basic Law -, becoming, to a certain extent, a ”positive legislator”. Official interpreters of the Constitution, Constitutional Courts assume, sometimes, a role of co-legislators, creating provisions they deduct from the Constitution - when controlling the absence of legislation or legislative omissions -, and revealing the content of constitutional and even infraconstitutional rules accordingly with the Constitution in their case-law, whose effects are nothing but specific forms of „impulse” or „coercion” of the legislator to proceed in a certain sense, and whose continuous development guides the evolution of the entire legal system. Case – law selected presents ways in which the Constitutional Court of Romania is associated to law-making activity. Without minimizing in any way its traditional role as "negative legislator", the study refers mainly to acts and situations that give expression to the creative role of the Constitutional Court of Romania.
Agonists and inverse agonists for the herpesvirus 8-encoded constitutively active seven-transmembrane oncogene product, ORF-74

DEFF Research Database (Denmark)

Rosenkilde, M M; Kledal, T N; Bräuner-Osborne, Hans

1999-01-01

A number of CXC chemokines competed with similar, nanomolar affinity against 125I-interleukin-8 (IL-8) binding to ORF-74, a constitutively active seven-transmembrane receptor encoded by human herpesvirus 8. However, in competition against 125I-labeled growth-related oncogene (GRO)-alpha, the ORF-74...
Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

LENUS (Irish Health Repository)

Toumi, F

2011-02-01

Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.
A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

Science.gov (United States)

Levoye, Angélique; Zwier, Jurriaan M; Jaracz-Ros, Agnieszka; Klipfel, Laurence; Cottet, Martin; Maurel, Damien; Bdioui, Sara; Balabanian, Karl; Prézeau, Laurent; Trinquet, Eric; Durroux, Thierry; Bachelerie, Françoise

2015-01-01

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.
A broad G protein-coupled receptor internalization assay that combines SNAP-tag labeling, diffusion-enhanced resonance energy transfer, and a highly emissive terbium cryptate acceptor

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Angélique eLEVOYE

2015-11-01

Full Text Available Although G protein-coupled receptor (GPCR internalization has long been considered a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z’-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS of compounds that may modulate GPCRs internalization.
Constitutive Activity of the Arabidopsis MAP Kinase 3 Confers Resistance to Pseudomonas syringae and Drives Robust Immune Responses

KAUST Repository

Lang, Julien; Genot, Baptiste; Hirt, Heribert; Colcombet, Jean

2017-01-01

of a constitutively active (CA) form of the protein led to auto-immune phenotypes. CA-MPK3 plants are dwarf and display defense responses that are characterized by the accumulation of salicylic acid and phytoalexins as well as by the upregulation
Enhanced constitutive invasion activity in human nontumorigenic keratinocytes exposed to a low level of barium for a long time.

Science.gov (United States)

Thang, Nguyen D; Yajima, Ichiro; Ohnuma, Shoko; Ohgami, Nobutaka; Kumasaka, Mayuko Y; Ichihara, Gaku; Kato, Masashi

2015-02-01

We have recently demonstrated that exposure to barium for a short time (≤4 days) and at a low level (5 µM = 687 µg/L) promotes invasion of human nontumorigenic HaCaT cells, which have characteristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time enhances malignant characteristics. Here we examined the effect of exposure to low level of barium for a long time, a condition mimicking the exposure to barium through well water, on malignant characteristics of HaCaT keratinocytes. Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma cells were augmented following an increase in malignancy grade of the cells. Constitutive invasion activity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with 5 µM barium for 4 months were significantly higher than those of control untreated HaCaT keratinocytes. Taken together, our results suggest that exposure to a low level of barium for a long time enhances constitutive malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion. © 2013 Wiley Periodicals, Inc.
Constitutive STAT3-activation in Sezary syndrome: tyrphostin AG490 inhibits STAT3-activation, interleukin-2 receptor expression and growth of leukemic Sezary cells

DEFF Research Database (Denmark)

Eriksen, K W; Kaltoft, K; Mikkelsen, G

2001-01-01

are IL-2Ralpha negative. An aberrant expression of IL-2Ralpha has recently been described in cutaneous T-cell lymphoma (CTCL). Here, we study the regulation of IL-2Ralpha expression and STATs in a tumor cell line obtained from peripheral blood from a patient with Sezary syndrome (SS), a leukemic variant...... of CTCL. We show that (1) STAT3 (a transcription factor known to regulate IL-2Ralpha transcription) is constitutively tyrosine-phosphorylated in SS tumor cells, but not in non-malignant T cells; (2) STAT3 binds constitutively to a STAT-binding sequence in the promotor of the IL-2Ralpha gene; (3) the Janus...... kinase inhibitor, tyrphostine AG490, inhibits STAT3 activation, STAT3 DNA binding, and IL-2Ralpha mRNA and protein expression in parallel; and (4) tyrphostine AG490 inhibits IL-2 driven mitogenesis and triggers apoptosis in SS tumor cells. In conclusion, we provide the first example of a constitutive...
Constitutive expression of TNF-related activation-induced cytokine (TRANCE/receptor activating NF-κB ligand (RANK-L by rat plasmacytoid dendritic cells.

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Thomas Anjubault

Full Text Available Plasmacytoid dendritic cells (pDCs are a subset of DCs whose major function relies on their capacity to produce large amount of type I IFN upon stimulation via TLR 7 and 9. This function is evolutionary conserved and place pDC in critical position in the innate immune response to virus. Here we show that rat pDC constitutively express TNF-related activation-induced cytokine (TRANCE also known as Receptor-activating NF-κB ligand (RANKL. TRANCE/RANKL is a member of the TNF superfamily which plays a central role in osteoclastogenesis through its interaction with its receptor RANK. TRANCE/RANK interaction are also involved in lymphoid organogenesis as well as T cell/DC cross talk. Unlike conventional DC, rat CD4(high pDC were shown to constitutively express TRANCE/RANKL both at the mRNA and the surface protein level. TRANCE/RANKL was also induced on the CD4(low subsets of pDC following activation by CpG. The secreted form of TRANCE/RANKL was also produced by rat pDC. Of note, levels of mRNA, surface and secreted TRANCE/RANKL expression were similar to that observed for activated T cells. TRANCE/RANKL expression was found on pDC in all lymphoid organs as well blood and BM with a maximum expression in mesenteric lymph nodes. Despite this TRANCE/RANKL expression, we were unable to demonstrate in vitro osteoclastogenesis activity for rat pDC. Taken together, these data identifies pDC as novel source of TRANCE/RANKL in the immune system.
Constitutively active Arabidopsis MAP Kinase 3 triggers defense responses involving salicylic acid and SUMM2 resistance protein

KAUST Repository

Genot, Baptiste

2017-04-12

Mitogen-activated protein kinases (MAPKs) are important regulators of plant immunity. Most of the knowledge about the function of these pathways is derived from loss-of-function approaches. Using a gain-of-function approach, we investigated the responses controlled by a constitutively active (CA) MPK3 in Arabidopsis thaliana. CA-MPK3 plants are dwarfed and display a massive de-repression of defense genes associated with spontaneous cell death as well as accumulation of reactive oxygen species (ROS), phytoalexins and the stress-related hormones ethylene and salicylic acid (SA). Remarkably CA-MPK3/sid2 and CA-MPK3/ein2-50 lines which are impaired in SA synthesis and ethylene signaling, respectively, retain most of the CA-MPK3-associated phenotypes, indicating that constitutive activity of MPK3 can bypass SA and ethylene signaling to activate defense responses. A comparative analysis of the molecular phenotypes of CA-MPK3 and mpk4 autoimmunity suggested convergence between the MPK3 and MPK4-guarding modules. In support of this model, CA-MPK3 crosses with summ1 and summ2, two known suppressors of mpk4, resulted in a partial reversion of the CA-MPK3 phenotypes. Overall, our data unravel a novel mechanism by which the MAPK signaling network contributes to a robust defense response system.
Ligand binding modes from low resolution GPCR models and mutagenesis: chicken bitter taste receptor as a test-case.

Science.gov (United States)

Di Pizio, Antonella; Kruetzfeldt, Louisa-Marie; Cheled-Shoval, Shira; Meyerhof, Wolfgang; Behrens, Maik; Niv, Masha Y

2017-08-15

Bitter taste is one of the basic taste modalities, warning against consuming potential poisons. Bitter compounds activate members of the bitter taste receptor (Tas2r) subfamily of G protein-coupled receptors (GPCRs). The number of functional Tas2rs is species-dependent. Chickens represent an intriguing minimalistic model, because they detect the bitter taste of structurally different molecules with merely three bitter taste receptor subtypes. We investigated the binding modes of several known agonists of a representative chicken bitter taste receptor, ggTas2r1. Because of low sequence similarity between ggTas2r1 and crystallized GPCRs (~10% identity, ~30% similarity at most), the combination of computational approaches with site-directed mutagenesis was used to characterize the agonist-bound conformation of ggTas2r1 binding site between TMs 3, 5, 6 and 7. We found that the ligand interactions with N93 in TM3 and/or N247 in TM5, combined with hydrophobic contacts, are typically involved in agonist recognition. Next, the ggTas2r1 structural model was successfully used to identify three quinine analogues (epiquinidine, ethylhydrocupreine, quinidine) as new ggTas2r1 agonists. The integrated approach validated here may be applicable to additional cases where the sequence identity of the GPCR of interest and the existing experimental structures is low.
CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor

OpenAIRE

Cherian, Milu T; Lin, Wenwei; Wu, Jing; Chen, Taosheng

2015-01-01

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug ...
cAMP-dependent cell differentiation triggered by activated CRHR1 in hippocampal neuronal cells.

Science.gov (United States)

Inda, Carolina; Bonfiglio, Juan José; Dos Santos Claro, Paula A; Senin, Sergio A; Armando, Natalia G; Deussing, Jan M; Silberstein, Susana

2017-05-16

Corticotropin-releasing hormone receptor 1 (CRHR1) activates the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). Both cAMP sources were shown to be required for the phosphorylation of ERK1/2 triggered by activated G protein coupled receptor (GPCR) CRHR1 in neuronal and neuroendocrine contexts. Here, we show that activated CRHR1 promotes growth arrest and neurite elongation in neuronal hippocampal cells (HT22-CRHR1 cells). By characterising CRHR1 signalling mechanisms involved in the neuritogenic effect, we demonstrate that neurite outgrowth in HT22-CRHR1 cells takes place by a sAC-dependent, ERK1/2-independent signalling cascade. Both tmACs and sAC are involved in corticotropin-releasing hormone (CRH)-mediated CREB phosphorylation and c-fos induction, but only sAC-generated cAMP pools are critical for the neuritogenic effect of CRH, further highlighting the engagement of two sources of cAMP downstream of the activation of a GPCR, and reinforcing the notion that restricted cAMP microdomains may regulate independent cellular processes.
Regulator of G protein signaling 2 (RGS2 and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1 in live cells.

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Sungho Ghil

Full Text Available Protease-activated receptor 1 (PAR1 is a G-protein coupled receptor (GPCR that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET, we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven and either RGS2-Luciferase (RGS2-Luc or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.
G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs).

Science.gov (United States)

Li, Lingyong; Homan, Kristoff T; Vishnivetskiy, Sergey A; Manglik, Aashish; Tesmer, John J G; Gurevich, Vsevolod V; Gurevich, Eugenia V

2015-04-24

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Constitutive activation of a variant of the env-mpl oncogene product by disulfide-linked homodimerization.

OpenAIRE

Courtois, G; Bénit, L; Mikaeloff, Y; Pauchard, M; Charon, M; Varlet, P; Gisselbrecht, S

1995-01-01

The myeloproliferative leukemia retrovirus (MPLV) has the v-mpl cellular sequences transduced in frame with the deleted and rearranged Friend murine leukemia virus env gene. The resulting env-mpl fusion oncogene is responsible for an acute myeloproliferative disorder induced in mice by MPLV. v-mpl is a truncated form of the c-mpl gene which encodes the receptor for thrombopoietin. We investigated the contribution of the Env-Mpl extracellular domain in the constitutive activation of this trunc...
The comparative constitutional law on national constitutional system: with regard to the IX World Congress of Constitutional Law

OpenAIRE

Landa Arroyo, César

2015-01-01

From the process of globalization of law, the comparative constitutional law has gained a leading role for a better understanding and solving old and new constitutional national and international challenges. Therefore, some assumptions and considerations to take into account are presented for the development of the national constitutional order within the framework of the comparative constitutional law, such as universality and relativism of human rights; the concept of power and cons...

Constitutive properties of salt from four sites

International Nuclear Information System (INIS)

Pfeifle, T.W.; Mellegard, K.D.; Senseny, P.E.

1983-04-01

Results are presented from laboratory strength and creep tests performed on salt specimens from the Richton dome in Mississippi, the Vacherie dome in Louisiana, the Permian basin in Texas, and the Paradox basin in Utah. The constitutive properties obtained are the elastic moduli and the failure envelope at 24 0 C and parameter values for the baseline creep law. Some additional data are presented to indicate how the elastic moduli and strength change with temperature. The constitutive properties given in this report and subsequent numerical simulations will serve as input to the screening of site locations for a nuclear-waste repository. The matrix of tests performed is the minimum effort required to obtain these constitutive properties. Comparison of results with those obtained for sites that have been characterized in greater detail suggests that the constitutive parameter values obtained are adequate for site-screening activity
Constitutive Modeling of Geomaterials Advances and New Applications

CERN Document Server

Zhang, Jian-Min; Zheng, Hong; Yao, Yangping

2013-01-01

The Second International Symposium on Constitutive Modeling of Geomaterials: Advances and New Applications (IS-Model 2012), is to be held in Beijing, China, during October 15-16, 2012. The symposium is organized by Tsinghua University, the International Association for Computer Methods and Advances in Geomechanics (IACMAG), the Committee of Numerical and Physical Modeling of Rock Mass, Chinese Society for Rock Mechanics and Engineering, and the Committee of Constitutive Relations and Strength Theory, China Institution of Soil Mechanics and Geotechnical Engineering, China Civil Engineering Society. This Symposium follows the first successful International Workshop on Constitutive Modeling held in Hong Kong, which was organized by Prof. JH Yin in 2007. Constitutive modeling of geomaterials has been an active research area for a long period of time. Different approaches have been used in the development of various constitutive models. A number of models have been implemented in the numerical analyses of geote...
Development of a Systems Computational Model to Investigate Early Biological Events in Hepatic Activation of Constitutive Androstane Receptor (CAR) by Phenobarbital

Science.gov (United States)

Activation of the nuclear receptor CAR (constitutive active/androstane receptor) is implicated in the control several key biological events such as metabolic pathways. Here, we combined data from literature with information obtained from in vitro assays in the US EPA ToxCast dat...
Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

Science.gov (United States)

Fischer, Jana; Kleinau, Gunnar; Rutz, Claudia; Zwanziger, Denise; Khajavi, Noushafarin; Müller, Anne; Rehders, Maren; Brix, Klaudia; Worth, Catherine L; Führer, Dagmar; Krude, Heiko; Wiesner, Burkhard; Schülein, Ralf; Biebermann, Heike

2018-06-01

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.
UNDERSTANDING INFORMAL CONSTITUTIONAL CHANGE

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Stephen M. Griffin

2016-01-01

Full Text Available Amid much recent American work on the problem of informal constitutional change, this article stakes out a distinctive position. I argue that theories of constitutional change in the US must address the question of the relationship between the “small c” and “big C” Constitution and treat seriously the possibility of conflict between them. I stress the unavoidable role the text of the Constitution and structural doctrines of federalism and separation of powers play in this relationship and thus in constitutional change, both formal and informal. I therefore counsel against theories that rely solely on a practice-based approach or analogies between “small c” constitutional developments and British or Commonwealth traditions of the “unwritten” constitution and constitutional “conventions.” The alternative I advocate is to approach constitutional change from a historicist perspective that focuses attention on state building and the creation of new institutional capacities. This approach will allow us to make progress by highlighting that there can be multiple constitutional orders in a given historical era, thus accounting for the conflictual nature of contemporary constitutional development in the US.
Transnational Constitutional Law

NARCIS (Netherlands)

Zumbansen, P (Peer); K.I. Bhatt (Kinnari)

2018-01-01

textabstractThis chapter provides an overview of the emerging field of transnational constitutional law (TCL). Whilst questions of constitutional law are typically discussed in the context of a specific domestic legal setting, a salient strategy of TCL is to understand constitutional law and its
Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation

NARCIS (Netherlands)

Rossaint, Jan; Herter, Jan M.; van Aken, Hugo; Napirei, Markus; Döring, Yvonne; Weber, Christian; Soehnlein, Oliver; Zarbock, Alexander

2014-01-01

There is emerging evidence that neutrophil extracellular traps (NETs) play important roles in inflammatory processes. Here we report that neutrophils have to be simultaneously activated by integrin-mediated outside-in- and G-protein-coupled receptor (GPCR) signaling to induce NET formation in acute
Right Product, Wrong Packaging: Not 'Constitution', but 'Constitutional Charter'

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John Law

2007-05-01

Full Text Available The article seeks to locate the principal cause of Europe’s prevailing ratification crisis in the inappropriate title arrived at in the European Convention, Treaty Establishing a Constitution for Europe. This over-ambitious styling led the media to characterise the text as simply an ‘EU Constitution’. Yet, the text was not a Constitution as we traditionally understand the term, i.e. the founding document of a State: scholars are agreed that the EU is not, and will not become upon ratification, a State.In terms of substance, whilst the text certainly strengthened some emerging constitutional aspects, it was not a major departure from the status quo like the Single European Act and Treaty on European Union had been; and it remained technically a treaty like all its predecessors. Arguably, therefore, it did not require referenda to ratify. However, confusion over the scale and importance of what was proposed, stemming from ambiguity in the title, pushed politicians down this unfortunate path.The article identifies a high level of consensus among commentators as to the true nature of the text: most are happy designating it a treaty (noun with constitutional (adjective aspects. The early proposed title Constitutional Treaty for Europe was arguably, therefore, the correct one; but it is now too late to choose this option, as the terms Constitution and Constitutional Treaty have already been muddled in debate. A more distinctive change is required. One idea could be to follow the principle employed elsewhere in the text of codifying the generally accepted but presently unwritten legal concepts of the European Court of Justice, as was done for example for ‘primacy’ and ‘direct effect’. The Court has characterised the EU treaties as a ‘constitutional charter’ for over twenty years now, and on this basis a modified title could read Treaty Establishing a Constitutional Charter for Europe. Importantly, the term ‘charter’ is recognised
Structural basis for activation of G-protein-coupled receptors

DEFF Research Database (Denmark)

Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine

2002-01-01

into conformational changes accompanying GPCR activation and the underlying molecular mechanism governing transition of the receptor between its active and inactive states. Using the beta2-adrenergic receptor as a model system we have obtained evidence for an evolutionary conserved activation mechanism where...... changes and receptor activation. At the current stage we are exploring the possibility of reaching this goal by direct in situ labeling of the beta2-adrenergic receptor in Xenopus laevis oocytes with conformationally sensitive fluorescent probes and parallel detection of receptor activation by co...
Constitutive Activation of Smoothened in the Renal Collecting Ducts Leads to Renal Hypoplasia, Hydronephrosis, and Hydroureter.

Science.gov (United States)

Gupta, Deepak Prasad; Hwang, Jae-Won; Cho, Eui-Sic; Kim, Won; Song, Chang Ho; Chai, Ok Hee

2017-01-01

Sonic Hedgehog (Shh) signaling plays a major role in and is essential for regulation, patterning, and proliferation during renal development. Smoothened (Smo) plays a pivot role in transducing the Shh-glioma-associated oncogene Kruppel family member. However, the cellular and molecular mechanism underlying the role of sustained Smo activation in postnatal kidney development is still not clearly understood. Using a conditional knockin mouse model that expresses a constitutively activated form of Smo (SmoM2) upon Homeobox-B7-mediated recombination (Hoxb7-Cre), the effects of Shh signaling were determined in postnatal kidney development. SmoM2;Hoxb7-Cre mutant mice showed growth retardation with a reduction of body weight. Constitutive activation of Smo in the renal collecting ducts caused renal hypoplasia, hydronephrosis, and hydroureter. The parenchymal area and glomerular numbers were reduced, but the glomerular density was increased in SmoM2;Hoxb7-Cre mutant mice. The expression of Patched 1, the receptor of Shh and a downstream target gene of the Shh signaling pathway, was highly restricted and it was upregulated in the inner medullary collecting ducts of the kidney. The proliferative cells in the mesenchyme and collecting ducts were decreased in SmoM2;Hoxb7-Cre mutant mice. This study showed for the first time that sustained Smo inhibits postnatal kidney development by suppressing the proliferation of the mesenchyme and medullary collecting ducts in mice. © 2017 S. Karger AG, Basel.
The Constitutional Court in light of interpretive decisions in normative control proceedings

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Stojanović Dragan

2016-01-01

Full Text Available In a legal system based on the principle of the separation of powers, constitutional judicature is necessarily (given the nature of its social function at the intersection of law and politics. Thus, constitutional judiciary is required to preserve political neutrality, particularly in relation to political (legislative and executive authorities. The paper analyzes the principal issues pertaining to constitutional court interpretation, particularly in light of observing the principle of the separation of powers that the constitutional judiciary is bound to abide by and considering the role of the constitutional court as an institution standing at the intersection of law and politics. Every constitutional court is required to be politically neutral and independent from daily politics, which is the major factor in delineating not only the overall boundaries of the constitutional control of the normative framework but also in ensuring the independent and unbiased activity of the constitutional court in the process of interpreting the Constitution and the laws. The constitutional control function shall not be politicized, and it must be exercised only through legal reasoning. Consequently, in the process of constitutional interpretation, the Constitutional Court of Serbia has to develop and consistently pursue a doctrine of self-restraint, thus refraining from politically-driven assessment which is the exclusive duty of political authorities. A closer examination of the doctrine of self-restraint in recent constitutional practice shows that the most prominent elements of this doctrine are relatively new interpretative constructions and legal formulations of constitutional court, which reinforce not only the political neutrality of the constitutional judiciary but also its role as 'the negative legislator'. Yet, some of these constructions may be challenged because their excessive and often inadequate application has resulted in a kind of 'self
Conditional expression of constitutively active estrogen receptor α in chondrocytes impairs longitudinal bone growth in mice

International Nuclear Information System (INIS)

Ikeda, Kazuhiro; Tsukui, Tohru; Imazawa, Yukiko; Horie-Inoue, Kuniko; Inoue, Satoshi

2012-01-01

Highlights: ► Conditional transgenic mice expressing constitutively active estrogen receptor α (caERα) in chondrocytes were developed. ► Expression of caERα in chondrocytes impaired longitudinal bone growth in mice. ► caERα affects chondrocyte proliferation and differentiation. ► This mouse model is useful for understanding the physiological role of ERαin vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caERα ColII , expressing constitutively active mutant estrogen receptor (ER) α in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caERα ColII mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caERα ColII mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caERα ColII mice. These results suggest that ERα is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.
Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

DEFF Research Database (Denmark)

Bartkova, J; Hamerlik, P; Stockhausen, Marie

2010-01-01

brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...... and indicate that replication stress, rather than oxidative stress, fuels the DNA damage signalling in early stages of astrocytoma development.......Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA...
Rights of Minors and Constitutional Politics in the German Länder. Legal Framework, Party Strategies, and Constitutional Amendments

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Lorenz Astrid

2015-11-01

Full Text Available The article analyses constitutional politics in the German Länder in the field of minors’ rights. Since this issue seems a purely legal matter dealt with at the federal, European and international level, we should expect similar, almost identically shaped policies at the Länder level. However, the analysis brings considerable variations of constitutional activities in this field to the fore: time, frequency, and contents of respective initiatives vary significantly in the period from 1999 to 2014. These variations were due to different party strategies, diverging party platforms and majority constellations in the Länder. The analysis also shows that the public arguments brought forward in favour of constitutional amendments refer only weakly and randomly to legal provisions and processes at other levels. The political debate supporting extended children's rights rather refers to general observations, to the specific regional context, and constitutional provisions in other Länder. At least with regard to this issue, the multi-level system did not systematically impact on constitutional politics in the Länder. It rather can be understood as an opportunity structure providing parties with multiple realms in which they can pursue their goals. Thus the study shows that federal and regional party strategies are key factors in explaining policy diffusion in multilevel systems.
Active learning of constitutive relation from mesoscopic dynamics for macroscopic modeling of non-Newtonian flows

Science.gov (United States)

Zhao, Lifei; Li, Zhen; Caswell, Bruce; Ouyang, Jie; Karniadakis, George Em

2018-06-01

We simulate complex fluids by means of an on-the-fly coupling of the bulk rheology to the underlying microstructure dynamics. In particular, a continuum model of polymeric fluids is constructed without a pre-specified constitutive relation, but instead it is actively learned from mesoscopic simulations where the dynamics of polymer chains is explicitly computed. To couple the bulk rheology of polymeric fluids and the microscale dynamics of polymer chains, the continuum approach (based on the finite volume method) provides the transient flow field as inputs for the (mesoscopic) dissipative particle dynamics (DPD), and in turn DPD returns an effective constitutive relation to close the continuum equations. In this multiscale modeling procedure, we employ an active learning strategy based on Gaussian process regression (GPR) to minimize the number of expensive DPD simulations, where adaptively selected DPD simulations are performed only as necessary. Numerical experiments are carried out for flow past a circular cylinder of a non-Newtonian fluid, modeled at the mesoscopic level by bead-spring chains. The results show that only five DPD simulations are required to achieve an effective closure of the continuum equations at Reynolds number Re = 10. Furthermore, when Re is increased to 100, only one additional DPD simulation is required for constructing an extended GPR-informed model closure. Compared to traditional message-passing multiscale approaches, applying an active learning scheme to multiscale modeling of non-Newtonian fluids can significantly increase the computational efficiency. Although the method demonstrated here obtains only a local viscosity from the polymer dynamics, it can be extended to other multiscale models of complex fluids whose macro-rheology is unknown.
Constitutional Rights in Indonesia

OpenAIRE

Judhariksawan

2018-01-01

The constitution is fundamental to the life of the modern state as a major foothold in state governance. Includes the guarantee of constitutional rights of citizens. The The constitution is the basis of state organizers to be implemented so that the state is obliged to guarantee the fulfillment of citizens' constitutional rights. Human rights have become an important part of the modern constitution. This study will describe how human rights guarantees become part of consti...
The activation mechanisms of G protein-coupled receptors : the case of the adenosine A2B and HCA2/3 receptors

NARCIS (Netherlands)

Liu, R.

2016-01-01

Identifying and elucidating the functions and activation of GPCRs will provide opportunities for novel drug discovery. We confirmed that a yeast system with an extended library of G proteins is very well suited for the study of GPCR activation, G protein coupling profiles, receptor-G protein binding
Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

Science.gov (United States)

Ngo, Tony; Coleman, James L J; Smith, Nicola J

2015-01-01

Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.
Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator.

Science.gov (United States)

Zhang, Xiaoyue; Li, Yonghao; Zhao, Xinqing; Bai, Fengwu

2017-01-01

The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved. Copyright © 2016. Published by Elsevier Ltd.
The Constitutional Amendment Process

Science.gov (United States)

Chism, Kahlil

2005-01-01

This article discusses the constitutional amendment process. Although the process is not described in great detail, Article V of the United States Constitution allows for and provides instruction on amending the Constitution. While the amendment process currently consists of six steps, the Constitution is nevertheless quite difficult to change.…

Constitutive activation of IKK2/NF-κB impairs osteogenesis and skeletal development.

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Gaurav Swarnkar

Full Text Available Pathologic conditions impair bone homeostasis. The transcription factor NF-κB regulates bone homeostasis and is central to bone pathologies. Whereas contribution of NF-κB to heightened osteoclast activity is well-documented, the mechanisms underlying NF-κB impact on chondrocytes and osteoblasts are scarce. In this study, we examined the effect of constitutively active IKK2 (IKK2ca on chondrogenic and osteogenic differentiation. We show that retroviral IKK2ca but not GFP, IKK2WT, or the inactive IKK2 forms IKK2KM and IKK2SSAA, strongly suppressed osteogenesis and chondrogenesis, in vitro. In order to explore the effect of constitutive NF-κB activation on bone formation in vivo, we activated this pathway in a conditional fashion. Specifically, we crossed the R26StopIKK2ca mice with mice carrying the Col2-cre in order to express IKK2ca in osteoblasts and chondrocytes. Both chondrocytes and osteoblasts derived from Col2Cre/IKK2ca expressed IKK2ca. Mice were born alive yet died shortly thereafter. Histologically, newborn Col2Cre+/RosaIKK2ca heterozygotes (Cre+IKK2ca_w/f (het and homozygotes (Cre+IKK2ca_f/f (KI showed smaller skeleton, deformed vertebrate and reduced or missing digit ossification. The width of neural arches, as well as ossification in vertebral bodies of Cre+IKK2ca_w/f and Cre+IKK2ca_f/f, was reduced or diminished. H&E staining of proximal tibia from new born pups revealed that Cre+IKK2ca_f/f displayed disorganized hypertrophic zones within the smaller epiphysis. Micro-CT analysis indicated that 4-wk old Cre+IKK2ca_w/f has abnormal trabecular bone in proximal tibia compared to WT littermates. Mechanistically, ex-vivo experiments showed that expression of differentiation markers in calvarial osteoblasts derived from newborn IKK2ca knock-in mice was diminished compared to WT-derived cells. In situ hybridization studies demonstrated that the hypertrophic chondrocyte marker type-X collagen, the pre-hypertrophic chondrocyte markers
Constitutional Politics, Constitutional Texts and Democratic Variety in Central and Eastern Europe

OpenAIRE

Blokker, Paul

2008-01-01

In the paper, it is argued that democratization in Central and Eastern Europe involves important forms of differentiation of democracy, rather than merely convergence to a singular – liberal-democratic, constitutional - model. One way of taking up democratic differentiation in post-communist societies is by analysing the constitutional documents of the new democratic orders, and the constitutional politics leading to the foundational documents. In a first step, the paper analyses constitution...
Constitutive Activation of NF-KB in Prostate Carcinoma Cells Through a Positive Feedback Loop: Implication of Inducible IKK-Related Kinase (IKKi)

National Research Council Canada - National Science Library

Budunova, Irina V

2005-01-01

The overall goal of this project is to understand the role of inducible IKK-related kinase IKKi in constitutive activation of anti-apoptotic transcription factor NF-KB prostate carcinoma (PC) cells...
On the constitutionality of dose limiting values

International Nuclear Information System (INIS)

Goetz, V.

1976-01-01

The fundamental right according to Art. 2 par. 2 sentence 1 of the German Constitution is relevant for the set-up and application of radiation protection law. Resulting from Art. 2 par. 2 sentence 1 of the Constitution it is a general obligation of the state to protect life (Federal Constitutional Court, judgment of 25th Feb., 1975, BVerfGE 39.1) and physical soundness. The subjective basic right of everybody to defend against official encroachments his personal integrity corresponds to the right of the individual within the framework of the official obligation for protection from the state (to ward off danger). The term of danger, as to the degree of its determination, corresponds to that of the encroachment. To speak of danger in a legal sense, the causal connection between a certain source of danger and certain damage must be ascertained and proved. Topical controversies as to the admissibility of activity discharges of low doses range in the field of risk reduction and thus in the field of the duty of the state to take precautionary steps against risks (Art. 2 par. 2 sentence 1 of the Constitution). The constitution, however, does not contain any basic right that every risk has to be avoided. On the other hand, the necessity of cautions valuation of radiation risks can be derived from the Constitution. The fixation of dose limits and their application in connection with general radiation protection principles (paragraph 28 E of the Radiation Protection Ordinance) do not contain any 'interference' with the basic right in the sense of Art. 2 par. 2 sentence 3 of the Constitution. Neither from aspects of the principle of the legal state nor from Art. 80 par. 1 of the Constitution can the use of the legal form of the Ordinance be doubted. (orig./HP) [de
PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95

DEFF Research Database (Denmark)

Møller, Thor C; Wirth, Volker F; Roberts, Nina Ingerslev

2013-01-01

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present...
A monoclonal antibody for G protein-coupled receptor crystallography

DEFF Research Database (Denmark)

Day, Peter W; Rasmussen, Søren Gøgsig Faarup; Parnot, Charles

2007-01-01

G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural inf...
Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

Science.gov (United States)

Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

2016-07-01

The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.
Gene network inference and biochemical assessment delineates GPCR pathways and CREB targets in small intestinal neuroendocrine neoplasia.

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Ignat Drozdov

Full Text Available Small intestinal (SI neuroendocrine tumors (NET are increasing in incidence, however little is known about their biology. High throughput techniques such as inference of gene regulatory networks from microarray experiments can objectively define signaling machinery in this disease. Genome-wide co-expression analysis was used to infer gene relevance network in SI-NETs. The network was confirmed to be non-random, scale-free, and highly modular. Functional analysis of gene co-expression modules revealed processes including 'Nervous system development', 'Immune response', and 'Cell-cycle'. Importantly, gene network topology and differential expression analysis identified over-expression of the GPCR signaling regulators, the cAMP synthetase, ADCY2, and the protein kinase A, PRKAR1A. Seven CREB response element (CRE transcripts associated with proliferation and secretion: BEX1, BICD1, CHGB, CPE, GABRB3, SCG2 and SCG3 as well as ADCY2 and PRKAR1A were measured in an independent SI dataset (n = 10 NETs; n = 8 normal preparations. All were up-regulated (p<0.035 with the exception of SCG3 which was not differently expressed. Forskolin (a direct cAMP activator, 10(-5 M significantly stimulated transcription of pCREB and 3/7 CREB targets, isoproterenol (a selective ß-adrenergic receptor agonist and cAMP activator, 10(-5 M stimulated pCREB and 4/7 targets while BIM-53061 (a dopamine D(2 and Serotonin [5-HT(2] receptor agonist, 10(-6 M stimulated 100% of targets as well as pCREB; CRE transcription correlated with the levels of cAMP accumulation and PKA activity; BIM-53061 stimulated the highest levels of cAMP and PKA (2.8-fold and 2.5-fold vs. 1.8-2-fold for isoproterenol and forskolin. Gene network inference and graph topology analysis in SI NETs suggests that SI NETs express neural GPCRs that activate different CRE targets associated with proliferation and secretion. In vitro studies, in a model NET cell system, confirmed that transcriptional
Transnational Constitutional Law

OpenAIRE

Zumbansen, P (Peer); Bhatt, Kinnari

2018-01-01

textabstractThis chapter provides an overview of the emerging field of transnational constitutional law (TCL). Whilst questions of constitutional law are typically discussed in the context of a specific domestic legal setting, a salient strategy of TCL is to understand constitutional law and its values by placing them ‘in context’ with existing and evolving cultural norms and political, social and economic discourses and struggles. Drawing on socio-legal investigations into the relationships ...
Development of operational models of receptor activation including constitutive receptor activity and their use to determine the efficacy of the chemokine CCL17 at the CC chemokine receptor CCR4.

Science.gov (United States)

Slack, R J; Hall, D A

2012-07-01

BACKGROUND AND PURPOSE The operational model provides a key conceptual framework for the analysis of pharmacological data. However, this model does not include constitutive receptor activity, a frequent phenomenon in modern pharmacology, particularly in recombinant systems. Here, we developed extensions of the operational model which include constitutive activity and applied them to effects of agonists at the chemokine receptor CCR4. EXPERIMENTAL APPROACH The effects of agonists of CCR4 on [(35) S]GTPγS binding to recombinant cell membranes and on the filamentous (F-) actin content of human CD4(+) CCR4(+) T cells were determined. The basal [(35) S]GTPγS binding was changed by varying the GDP concentration whilst the basal F-actin contents of the higher expressing T cell populations were elevated, suggesting constitutive activity of CCR4. Both sets of data were analysed using the mathematical models. RESULTS The affinity of CCL17 (also known as TARC) derived from analysis of the T cell data (pK(a) = 9.61 ± 0.17) was consistent with radioligand binding experiments (9.50 ± 0.11) while that from the [(35) S]GTPγS binding experiments was lower (8.27 ± 0.09). Its intrinsic efficacy differed between the two systems (110 in T cells vs. 11). CONCLUSIONS AND IMPLICATIONS The presence of constitutive receptor activity allows the absolute intrinsic efficacy of agonists to be determined without a contribution from the signal transduction system. Intrinsic efficacy estimated in this way is consistent with Furchgott's definition of this property. CCL17 may have a higher intrinsic efficacy at CCR4 in human T cells than that expressed recombinantly in CHO cells. © 2012 GSK Services Unlimited. British Journal of Pharmacology © 2012 The British Pharmacological Society.
The GPCR membrane receptor, DopEcR, mediates the actions of both dopamine and ecdysone to control sex pheromone perception in an insect

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Antoine eAbrieux

2014-09-01

Full Text Available Olfactory information mediating sexual behavior is crucial for reproduction in many animals, including insects. In male moths, the macroglomerular complex of the primary olfactory center, the antennal lobe (AL is specialized in the treatment of information on the female-emitted sex pheromone. Evidence is accumulating that modulation of behavioral pheromone responses occurs through neuronal plasticity via the action of hormones and/or catecholamines. We recently showed that a G-protein-coupled receptor (GPCR, AipsDopEcR, with its homologue known in Drosophila for its double affinity to the main insect steroid hormone 20-hydroxyecdysone (20E, and dopamine (DA, present in the ALs, is involved in the behavioral response to pheromone in the moth, Agrotis ipsilon. Here we tested the role of AipsDopEcR as compared to nuclear 20E receptors in central pheromone processing combining receptor inhibition with intracellular recordings of AL neurons. We show that the sensitivity of AL neurons for the pheromone in males decreases strongly after AipsDopEcR-dsRNA injection but also after inhibition of nuclear 20E receptors. Moreover we tested the involvement of 20E and DA in the receptor-mediated behavioral modulation in wind tunnel experiments, using ligand applications and receptor inhibition treatments. We show that both ligands are necessary and act on AipsDopEcR-mediated behavior. Altogether these results indicate that the GPCR membrane receptor, AipsDopEcR, controls sex pheromone perception through the action of both 20E and DA in the central nervous system, probably in concert with 20E action through nuclear receptors.
CAN THE MUSLIM WORLD BORROW FROM INDONESIAN CONSTITUTIONAL REFORM? A Comparative Constitutional Approach

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Nadirsyah Hosen

2007-06-01

Full Text Available This paper attempts to analytically examine the possibility of constitutional borrowing for the Muslim world regardless the differences in history, system, culture, language, and characteristics. It discusses this issue by looking at the arguments put forth by the opponents of comparative constitutional interpretation and their counter arguments. It will consider materials from Canada, USA, South Africa, Singapore, Malaysia, and Hungary, taking the position that constitutional borrowing can be justified. The paper argues that the 1999-2002 Indonesian constitutional reform should be taken into account by other Muslim countries in undertaking their constitutional reform. The substantive approach of the Shari‘ah that has been used in Indonesia has shown that Muslim world can reform its constitutions without the “assistance” of Western foreign policy. Indonesian constitutional reform has demonstrated that Islamic constitutionalism comes from within Islamic teaching and the Islamic community itself; it is a home grown product.
Constitutional Issues--Watergate and the Constitution. Teaching with Documents.

Science.gov (United States)

National Archives and Records Administration, Washington, DC.

When U.S. President Richard Nixon resigned in 1974 in the wake of the Watergate scandal, it was only the second time that impeachment of a president had been considered. Although the U.S. Constitution has provisions for a person removed from office to be indicted, there are no guidelines in the Constitution about a President who has resigned. The…
Selective elimination of high constitutive activity or chemokine binding in the human herpesvirus 8 encoded seven transmembrane oncogene ORF74

DEFF Research Database (Denmark)

Rosenkilde, M M; Kledal, T N; Holst, Peter Johannes

2000-01-01

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice...
Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications

Science.gov (United States)

Cvicek, Vaclav; Goddard, William A.; Abrol, Ravinder

2016-01-01

The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs
Tracking G-protein-coupled receptor activation using genetically encoded infrared probes.

Science.gov (United States)

Ye, Shixin; Zaitseva, Ekaterina; Caltabiano, Gianluigi; Schertler, Gebhard F X; Sakmar, Thomas P; Deupi, Xavier; Vogel, Reiner

2010-04-29

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.
Essential Medicines in National Constitutions

Science.gov (United States)

Toebes, Brigit; Hogerzeil, Hans

2016-01-01

Abstract A constitutional guarantee of access to essential medicines has been identified as an important indicator of government commitment to the progressive realization of the right to the highest attainable standard of health. The objective of this study was to evaluate provisions on access to essential medicines in national constitutions, to identify comprehensive examples of constitutional text on medicines that can be used as a model for other countries, and to evaluate the evolution of constitutional medicines-related rights since 2008. Relevant articles were selected from an inventory of constitutional texts from WHO member states. References to states’ legal obligations under international human rights law were evaluated. Twenty-two constitutions worldwide now oblige governments to protect and/or to fulfill accessibility of, availability of, and/or quality of medicines. Since 2008, state responsibilities to fulfill access to essential medicines have expanded in five constitutions, been maintained in four constitutions, and have regressed in one constitution. Government commitments to essential medicines are an important foundation of health system equity and are included increasingly in state constitutions. PMID:27781006
Triclocarban mediates induction of xenobiotic metabolism through activation of the constitutive androstane receptor and the estrogen receptor alpha.

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Mei-Fei Yueh

Full Text Available Triclocarban (3,4,4'-trichlorocarbanilide, TCC is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs. To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR and estrogen receptor alpha (ERα activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR because no induction occurred in hUGT1Car(-/- mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for
The Causes of Failure of the European Constitution From the Perspective of the Constitution-Making Process

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Robert Podolnjak

2006-01-01

Full Text Available The basic argument of the article is that the main causes of failure of the European Constitution stem from an inadequate preparation and implementation of a complex procedure of constitution-making for a federation of countries on a continental scale. This process includes the issues of temporal aspects of constitutionmaking, the subject of constitution-making, the text of the constitution, the strategy of constitutional ratifi cation and the constitution-makers themselves. The principal causes of failure of the European Constitution will be presented in the form of certain preliminary assumptions, which will then be examined in the light of certain comparative experiences of constitution-making in two federal systems – the American and the Swiss system. The primary mistakes of the European constitution-making are refl ected in the lack of an appropriate moment for making the constitution, in the vagueness of the document in terms of its constitutional or contractual quality, in the creation of a text of the Constitution which is completely incomprehensible to the average citizen, in the making of the Constitution without a vision or ambition, in the complete lack of any strategy of ratifi cation of the Constitution, in the insistence on the direct participation of the people in the adoption of the Constitution, which is legally and politically considered primarily an international treaty, and in badly managed media presentation and defence of the Constitution before the European public. The most important mistakes, crucial to the failure of the Constitution, are the ambivalent approach of the European constitutionmakers to the mode of ratifi cation of the Constitution, and their disregard of the constitution-making experience of other federal countries.
Constitutive Activation of NF-kappaB in Prostate Carcinoma Cells Through a Positive Feedback Loop: Implication of Inducible IKK-Related Kinase (IKKi)

National Research Council Canada - National Science Library

Budunova, Irina V

2004-01-01

The overall goal of this project is to understand the role of inducible IKK-related kinase IKKi in constitutive activation of anti-apoptotic transcription factor NF-kB prostate carcinoma (PC) cells...

ECHR and national constitutional courts

Directory of Open Access Journals (Sweden)

Nastić Maja

2015-01-01

Full Text Available Comprising fundamental rights and freedoms and establishing the effective control system, the European Convention on Human Rights (ECHR encroaches upon the area that is traditional reserved for constitutional law. Although built on the doctrine reserved for international treaty law, the Convention goes beyond the traditional boundaries that exist between international and constitutional law. It has gradually infiltrated into the national legal systems. Constitutional courts have had the crucial role in this process. This paper will focus on the applicability of the ECHR in proceedings before national constitutional courts. Having in mind the jurisdiction of the national constitutional court, the ECHR may be applied in two ways: first, in the process of constitutional review by national constitutional courts and, second, in the process of deciding on constitutional complaints.
RECEPTING THE PRINCIPLE OF SUPREMACY OF CONSTITUTION ON THE NEW PENAL CODE

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Marius ANDREESCU

2016-07-01

Full Text Available The supremacy of Constitution has as main consequence the compliance of entire law with the constitutional norms. Guaranteeing of the observance of this principle is essential for the rule of law, is primarily an attribute of the Constitutional Court, but also an obligation of the legislator to receive by texts adopted, within its content and form, the constitutional norms. Entering into force of the new criminal codes generated a significant jurisprudence of the Constitutional Court on the verification of constitutionality of some regulations in the Criminal Code and Criminal Procedure Code. Through this study we intend to analyze the following key issues: a how were the constitutional principles and values embodied in some criminal and criminal procedural norms of the new codes; b the effects of Constitutional Court decisions in the process of constitutionalizing of the criminal law; c applying into judicial activities of the Constitutional Court decisions, particularly those through which the new Criminal Code regulations were found unconstitutional.
Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.

Science.gov (United States)

Petersen, Jan; Inoue, Shin-Ichiro; Kelly, Sharon M; Sullivan, Stuart; Kinoshita, Toshinori; Christie, John M

2017-08-18

Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Conditional expression of constitutively active estrogen receptor {alpha} in chondrocytes impairs longitudinal bone growth in mice

Energy Technology Data Exchange (ETDEWEB)

Ikeda, Kazuhiro [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Tsukui, Tohru [Experimental Animal Laboratory, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Imazawa, Yukiko; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Inoue, Satoshi, E-mail: INOUE-GER@h.u-tokyo.ac.jp [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo (Japan)

2012-09-07

Highlights: Black-Right-Pointing-Pointer Conditional transgenic mice expressing constitutively active estrogen receptor {alpha} (caER{alpha}) in chondrocytes were developed. Black-Right-Pointing-Pointer Expression of caER{alpha} in chondrocytes impaired longitudinal bone growth in mice. Black-Right-Pointing-Pointer caER{alpha} affects chondrocyte proliferation and differentiation. Black-Right-Pointing-Pointer This mouse model is useful for understanding the physiological role of ER{alpha}in vivo. -- Abstract: Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caER{alpha}{sup ColII}, expressing constitutively active mutant estrogen receptor (ER) {alpha} in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caER{alpha}{sup ColII} mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caER{alpha}{sup ColII} mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caER{alpha}{sup ColII} mice. These results suggest that ER{alpha} is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.
Institute of constitutional revision in the Constitution of the Republic of Albania, comparative view

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Makbule Çeço

2014-07-01

Full Text Available In its very dynamic essence, a democratic society bears the need for continuous reformation and perfection, and that is why the application of reforms represents an inseparable feature for this type of society. The consolidation of the rule of law, the institutional independence, and the cause of justice itself comprise, inter alia, the need for constitutional revision. This study puts forward a theoretical-historical comparative view of the relevant and dynamic issue of the institute of constitutional revision in the framework of the Constitution of the Republic of Albania, as a complex process accompanied by limitations on constitutional revision. The historical evolution of constitutional drafting, modern constitutions, relevant issues, political and social circumstances as well as drafting and adoption procedures, dynamism of constitutions to cope with the course of time achieved by revisions for the purpose of their stability as well as consolidation of the role of constitutions as a factor that facilitates and precedes social development, comprise the pillar of this study addressed in a comparative point of view.
Constitutive activation of a plasma membrane H+-ATPase prevents abscisic acid-mediated stomatal closure

Science.gov (United States)

Merlot, Sylvain; Leonhardt, Nathalie; Fenzi, Francesca; Valon, Christiane; Costa, Miguel; Piette, Laurie; Vavasseur, Alain; Genty, Bernard; Boivin, Karine; Müller, Axel; Giraudat, Jérôme; Leung, Jeffrey

2007-01-01

Light activates proton (H+)-ATPases in guard cells, to drive hyperpolarization of the plasma membrane to initiate stomatal opening, allowing diffusion of ambient CO2 to photosynthetic tissues. Light to darkness transition, high CO2 levels and the stress hormone abscisic acid (ABA) promote stomatal closing. The overall H+-ATPase activity is diminished by ABA treatments, but the significance of this phenomenon in relationship to stomatal closure is still debated. We report two dominant mutations in the OPEN STOMATA2 (OST2) locus of Arabidopsis that completely abolish stomatal response to ABA, but importantly, to a much lesser extent the responses to CO2 and darkness. The OST2 gene encodes the major plasma membrane H+-ATPase AHA1, and both mutations cause constitutive activity of this pump, leading to necrotic lesions. H+-ATPases have been traditionally assumed to be general endpoints of all signaling pathways affecting membrane polarization and transport. Our results provide evidence that AHA1 is a distinct component of an ABA-directed signaling pathway, and that dynamic downregulation of this pump during drought is an essential step in membrane depolarization to initiate stomatal closure. PMID:17557075
The TLR4 D299G and T399I SNPs are constitutively active to up-regulate expression of Trif-dependent genes.

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Georgina L Hold

Full Text Available Dysregulated Toll-Like Receptor (TLR signalling and genetic polymorphisms in these proteins are linked to many human diseases. We investigated TLR4 functional variants D299G and T399I to assess the impact on LPS-induced responsiveness in comparison to wild-type TLR4. The mechanism by which this occurs in unclear as these SNPs do not lie within the lipid A binding domain or dimerisation sites of the LPS-TLR4/MD2 receptor complexes. Transfection of TLR4D299G, TLR4T399I or TLR4D299G. T399I into HEK cells resulted in constitutive activation of an NF-κB reporter gene and a blunting of the LPS-induced reporter activation compared to WT-TLR4. Unstimulated human monocyte/macrophages, from patients with the D299G and T399I SNPs demonstrated a downregulation of many genes, particularly Tram/Trif signalling pathway constitutents compared to the TLR4 wild-type subjects supporting the concept of basal receptor activity. Monocyte/macrophages from carriers of the TLR4 D299G and T399I polymorphisms stimulated with LPS showed >6 fold lower levels of NF-κB and ∼12 fold higher IFN-β gene expression levels compared to wild-type subjects (P<0.05; MWU test and dramatically altered resultant cytokine profiles. We conclude that these TLR4 SNPs affect constitutive receptor activity which impacts on the hosts ability to respond to LPS challenge leading to a dysregulated sub-optimal immune response to infection.
The role of opioid antagonist efficacy and constitutive opioid receptor activity in the opioid withdrawal syndrome in mice

OpenAIRE

Navani, Dipesh M.; Sirohi, Sunil; Madia, Priyanka A.; Yoburn, Byron C.

2011-01-01

On the basis of efficacy, opioid antagonists are classified as inverse opioid agonists (e.g. naltrexone) or neutral opioid antagonists (e.g. 6β-naltrexol). This study examined the interaction between naltrexone and 6β-naltrexol in the precipitated opioid withdrawal syndrome in morphine dependent mice. Furthermore, the possible contribution of constitutive opioid receptor activity to precipitated withdrawal was evaluated using increasing levels of morphine dependence. In the first experiment, ...
The Possibility of Vice-Presidents’ Authority Arrangement in the 1945 Constitution through Constitutional Amendment

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Fajar L. Suroso

2016-04-01

Full Text Available The debate over the vice-presidents’ authority reappeared in the administration of President Jokowi after the “authority expansion of the Chief of Presidency Staff” and the case of “Rizal Ramli vs Jusuf Kalla”. This article is intended to provide arguments for the idea of some parties to organize more explicit and detailed the authority of vice-president in the 1945 Constitution. The idea arises from the absence of further arrangement on the authority of vice-president in the 1945 Constitution. This article is systematized into 3 (three sub-theme; 1 the arrangement of the vice-presidents’ authority in the constitution for several countries; 2 The authority of the vice-president according to the 1945 Constitution, and 3 New resultant and the possibility of 1945 Constitution amendment. The result revealed a number of interesting things; 1 the constitutions of other countries do not specify the authority of the vice-president and put the vice-president as a “spare tire” when the president is absent; 2 no new resultant about the position and authority of the vice-president so that theoretically is not reason enough to regulate in detail the authority of the vice-president through the 1945 Constitution amendment; 3 arrangement in detail in the authority of vice-president in the 1945 Constitution holds the potential to confuse the presidential system design as the 1945 Constitution. Therefore, the possibility of vice-presidents’ authority arrangement in the 1945 Constitution through amendment is very small, both in terms of momentum and the substance of issues.
Plasticizers May Activate Human Hepatic Peroxisome Proliferator-Activated Receptor α Less Than That of a Mouse but May Activate Constitutive Androstane Receptor in Liver

Science.gov (United States)

Ito, Yuki; Nakamura, Toshiki; Yanagiba, Yukie; Ramdhan, Doni Hikmat; Yamagishi, Nozomi; Naito, Hisao; Kamijima, Michihiro; Gonzalez, Frank J.; Nakajima, Tamie

2012-01-01

Dibutylphthalate (DBP), di(2-ethylhexyl)phthalate (DEHP), and di(2-ethylhexyl)adipate (DEHA) are used as plasticizers. Their metabolites activate peroxisome proliferator-activated receptor (PPAR) α, which may be related to their toxicities. However, species differences in the receptor functions between rodents and human make it difficult to precisely extrapolate their toxicity from animal studies to human. In this paper, we compared the species differences in the activation of mouse and human hepatic PPARα by these plasticizers using wild-type (mPPARα) and humanized PPARα (hPPARα) mice. At 12 weeks old, each genotyped male mouse was classified into three groups, and fed daily for 2 weeks per os with corn oil (vehicle control), 2.5 or 5.0 mmol/kg DBP (696, 1392 mg/kg), DEHP (977, 1953 mg/kg), and DEHA (926, 1853 mg/kg), respectively. Generally, hepatic PPARα of mPPARα mice was more strongly activated than that of hPPARα mice when several target genes involving β-oxidation of fatty acids were evaluated. Interestingly, all plasticizers also activated hepatic constitutive androstane receptor (CAR) more in hPPARα mice than in mPPARα mice. Taken together, these plasticizers activated mouse and human hepatic PPARα as well as CAR. The activation of PPARα was stronger in mPPARα mice than in hPPARα mice, while the opposite was true of CAR. PMID:22792086
Plasticizers May Activate Human Hepatic Peroxisome Proliferator-Activated Receptor α Less Than That of a Mouse but May Activate Constitutive Androstane Receptor in Liver

Directory of Open Access Journals (Sweden)

Yuki Ito

2012-01-01

Full Text Available Dibutylphthalate (DBP, di(2-ethylhexylphthalate (DEHP, and di(2-ethylhexyladipate (DEHA are used as plasticizers. Their metabolites activate peroxisome proliferator-activated receptor (PPAR α, which may be related to their toxicities. However, species differences in the receptor functions between rodents and human make it difficult to precisely extrapolate their toxicity from animal studies to human. In this paper, we compared the species differences in the activation of mouse and human hepatic PPARα by these plasticizers using wild-type (mPPARα and humanized PPARα (hPPARα mice. At 12 weeks old, each genotyped male mouse was classified into three groups, and fed daily for 2 weeks per os with corn oil (vehicle control, 2.5 or 5.0 mmol/kg DBP (696, 1392 mg/kg, DEHP (977, 1953 mg/kg, and DEHA (926, 1853 mg/kg, respectively. Generally, hepatic PPARα of mPPARα mice was more strongly activated than that of hPPARα mice when several target genes involving β-oxidation of fatty acids were evaluated. Interestingly, all plasticizers also activated hepatic constitutive androstane receptor (CAR more in hPPARα mice than in mPPARα mice. Taken together, these plasticizers activated mouse and human hepatic PPARα as well as CAR. The activation of PPARα was stronger in mPPARα mice than in hPPARα mice, while the opposite was true of CAR.
Constitutional Fundamentals of Conscription and Some Aspects of the Ordinary Legal Regulation of Constitutionality

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Kenstavičienė Kristina

2015-12-01

Full Text Available Article 139 of the Constitution of the Republic of Lithuania is one of the constitutional fundamentals of state defense and stipulates the defense of the state as the right of citizens on the one hand and the duty on the other. This article of the Constitution gives the legislative power the right of discretion to detail by law the order of the implementation of citizens’ duty to perform military or alternative country defense service. Due to the reorganization of the armed forces into a professional and volunteer army, the issue of some ordinary regulation rules concerning the constitutionality of nationwide conscription, though at present suspended but not abolished, is becoming urgent. Though the Constitutional Court of the Republic of Lithuania presented their ruling on the constitutionality of the suspension of military conscription, it does not mean that all problems related to conscription have been settled. The aim of this article is to analyze the constitutional basis of nationwide conscription as well as the constitutionality of some ordinary regulation provisions related to nationwide conscription. Therefore, the issue to be analyzed is whether nationwide conscription, if it were to be implemented, complies with the constitutional principles of human equality and military justice1. Consequently, the question is posed how the constitutional objective of ensuring the defense of the state determines conscription. Because of the growing employment of the army abroad, yet the dwindling demand for conscripts, it should be explored whether the suspension of the nationwide conscription as a part of the defense reform is further feasible in order to guarantee the defense of the state. In answering the raised questions, the author will analyze the abundant and long-lasting constitutional doctrine of Germany which provides clarifications of the Basic Law, as the legal act of the establishing power, which can doubtless be of assistance in
Increase of anthraquinone content in Rubia cordifolia cells transformed by native and constitutively active forms of the AtCPK1 gene.

Science.gov (United States)

Shkryl, Yury N; Veremeichik, G N; Makhazen, D S; Silantieva, S A; Mishchenko, N P; Vasileva, E A; Fedoreyev, S A; Bulgakov, V P

2016-09-01

Overexpression of both native and mutant forms of AtCPK1 in Rubia cordifolia cells increased anthraquinone production and transcript abundance of the RcIPPI, RcOSBL, RcOSBS , and RcICS genes to different extents. Calcium-dependent protein kinases (CDPKs) are involved in various cell processes and are regulated by a calcium signal system. CDPKs also function in plant defense against stress factors such as pathogens, temperature, and salinity. In this study, we compared the effect of heterologous expression of two forms of the Arabidopsis AtCPK1 gene, native and constitutively active (Ca(2+)-independent), on anthraquinone production in transgenic Rubia cordifolia cells. Significant qualitative and quantitative differences were found in the content of anthraquinone derivatives in control and AtCPK1-transgenic calli. Expression of the AtCPK1 gene increased anthraquinone production by 3 and 12 times for native and constitutively active forms, respectively, compared with control cells. In addition, we identified and quantified the expression of genes encoding key enzymes of the anthraquinone biosynthesis pathway, including isochorismate synthase (ICS), o-succinylbenzoate synthase (OSBS), o-succinylbenzoate ligase (OSBL), and isopentenyl diphosphate isomerase (IPPi). In all AtCPK1-transgenic cell lines, expression of ICS, OSBS, OSBL, and IPPi increased considerably at 14-15 days of subculture and decreased at the end of cultivation (30 days). The results suggest that both native and constitutively active AtCPK1 forms induced anthraquinone accumulation at the logarithmic growth stage via enhancement of expression of genes involved in the metabolism of anthraquinones or their regulatory mechanisms.
Osteopontin is an endogenous modulator of the constitutively activated phenotype of pulmonary adventitial fibroblasts in hypoxic pulmonary hypertension

Science.gov (United States)

Anwar, Adil; Li, Min; Frid, Maria G.; Kumar, Binod; Gerasimovskaya, Evgenia V.; Riddle, Suzette R.; McKeon, B. Alexandre; Thukaram, Roopa; Meyrick, Barbara O.; Fini, Mehdi A.

2012-01-01

Increased cell proliferation and migration, of several cell types are key components of vascular remodeling observed in pulmonary hypertension (PH). Our previous data demonstrate that adventitial fibroblasts isolated from pulmonary arteries of chronically hypoxic hypertensive calves (termed PH-Fibs) exhibit a “constitutively activated” phenotype characterized by high proliferative and migratory potential. Osteopontin (OPN) has been shown to promote several cellular activities including growth and migration in cancer cells. We thus tested the hypothesis that elevated OPN expression confers the “activated” highly proproliferative and promigratory/invasive phenotype of PH-Fibs. Our results demonstrate that, both in vivo and ex vivo, PH-Fibs exhibited increased expression of OPN, as well as its cognate receptors, αVβ3 and CD44, compared with control fibroblasts (CO-Fibs). Augmented OPN expression in PH-Fibs corresponded to their high proliferative, migratory, and invasive properties and constitutive activation of ERK1/2 and AKT signaling. OPN silencing via small interfering RNA or sequestering OPN production by specific antibodies led to decreased proliferation, migration, invasion, and attenuated ERK1/2, AKT phosphorylation in PH-Fibs. Furthermore, increasing OPN levels in CO-Fibs via recombinant OPN resulted in significant increases in their proliferative, migratory, and invasive capabilities to the levels resembling those of PH-Fibs. Thus our data suggest OPN as an essential contributor to the activated (highly proliferative, migratory, and proinvasive) phenotype of pulmonary adventitial fibroblasts in hypoxic PH. PMID:22582113
High constitutive activity of a virus-encoded seven transmembrane receptor in the absence of the conserved DRY motif (Asp-Arg-Tyr) in transmembrane helix 3

DEFF Research Database (Denmark)

Rosenkilde, Mette M; Kledal, Thomas N; Schwartz, Thue W

2005-01-01

-driven transcriptional activity through a pertussis toxin-sensitive manner. Gs and Gq were not activated constitutively as determined by the lack of inositol phosphate turnover and activities of the three transcription factors: cAMP response element-binding protein (CREB), nuclear factor-kappaB, and nuclear factor...
Chimeric microbial rhodopsins for optical activation of Gs-proteins

Science.gov (United States)

Yoshida, Kazuho; Yamashita, Takahiro; Sasaki, Kengo; Inoue, Keiichi; Shichida, Yoshinori; Kandori, Hideki

2017-01-01

We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins. PMID:29362703
Interpretative decisions in the practice of the Constitutional Court of Serbia

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Rajić Nataša

2014-01-01

Full Text Available The relationship between the Constitutional Court and Parliament is marked by envolving process. The desired balance between these two state bodies was gradually disturbed by the Constitutional Court. Consequently, the substrate of the constitutional function originally designed as a function of 'negative legislator' is also changed in that process. Interpretative decision is one of the model of the activity of the Constitutional Court which makes questionable the position of the Parliament as a state body which regulates social relations in original form. Interpretative decision is an specific form of rejected decisions that contains binding instruction regading the interpretation of the norm, as a condition. This interpretation is given by Constitutional Court in order to make the norm in accordance with the Constitution. By the analysis of the practice of the Constitutional Court of Serbia, the paper has a task to determine wheter, to what extend and in what form the interpretative decisions occur in the work of the Court as well as to determine is there a clear constitutional base for establishing the jurisdiction of their adoption in our legal system.
Pre-salt new regulatory mark and the economic order: constitutionality analysis

International Nuclear Information System (INIS)

Pinheiro, Marcela Brasil Pedrosa; Araujo, Mayara de Carvalho; Xavier, Yanko Marcius; Guimaraes, Patricia Borba Vilar

2010-01-01

The discovery of vast reserves of hydrocarbons in the pre-salt layer that extends from Espirito Santo to Santa Catarina, added with the economic and strategic value of oil and natural gas, has brought discussion about the reasonableness of the regulatory model adopted so far. Would be prudent to explore these resources through the concession model? From detailed analysis of the doctrinal and bills that aim to inaugurate the new regulatory bills, we sought to answer this question, based majorly on the principles of economic activity applied in our constitutional system. Motivated by the analysis of these constitutional principles proposed, the State is seen as a regulating agent of the economic activities, fulfilling its role to supervise, encourage and plan the direction of national economic system. The sharing model gives greater state involvement and is able to convert the wealth of pre-salt in citizenship, but only if well implemented. Thus, based on constitutional principles and the notion of development as freedom, the conclusion of this paperwork is for the constitutionality of the new regulatory bills. (author)
Behavioral control by striatal adenosine A2A -dopamine D2 receptor heteromers.

Science.gov (United States)

Taura, J; Valle-León, M; Sahlholm, K; Watanabe, M; Van Craenenbroeck, K; Fernández-Dueñas, V; Ferré, S; Ciruela, F

2018-04-01

G protein-coupled receptors (GPCR) exhibit the ability to form receptor complexes that include molecularly different GPCR (ie, GPCR heteromers), which endow them with singular functional and pharmacological characteristics. The relative expression of GPCR heteromers remains a matter of intense debate. Recent studies support that adenosine A 2A receptors (A 2A R) and dopamine D 2 receptors (D 2 R) predominantly form A 2A R-D 2 R heteromers in the striatum. The aim of the present study was evaluating the behavioral effects of pharmacological manipulation and genetic blockade of A 2A R and D 2 R within the frame of such a predominant striatal heteromeric population. First, in order to avoid possible strain-related differences, a new D 2 R-deficient mouse with the same genetic background (CD-1) than the A 2A R knock-out mouse was generated. Locomotor activity, pre-pulse inhibition (PPI) and drug-induced catalepsy were then evaluated in wild-type, A 2A R and D 2 R knock-out mice, with and without the concomitant administration of either the D 2 R agonist sumanirole or the A 2A R antagonist SCH442416. SCH442416-mediated locomotor effects were demonstrated to be dependent on D 2 R signaling. Similarly, a significant dependence on A 2A R signaling was observed for PPI and for haloperidol-induced catalepsy. The results could be explained by the existence of one main population of striatal postsynaptic A 2A R-D 2 R heteromers, which may constitute a relevant target for the treatment of Parkinson's disease and other neuropsychiatric disorders. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.
GPCRM: a homology modeling web service with triple membrane-fitted quality assessment of GPCR models.

Science.gov (United States)

Miszta, Przemyslaw; Pasznik, Pawel; Jakowiecki, Jakub; Sztyler, Agnieszka; Latek, Dorota; Filipek, Slawomir

2018-05-21

Due to the involvement of G protein-coupled receptors (GPCRs) in most of the physiological and pathological processes in humans they have been attracting a lot of attention from pharmaceutical industry as well as from scientific community. Therefore, the need for new, high quality structures of GPCRs is enormous. The updated homology modeling service GPCRM (http://gpcrm.biomodellab.eu/) meets those expectations by greatly reducing the execution time of submissions (from days to hours/minutes) with nearly the same average quality of obtained models. Additionally, due to three different scoring functions (Rosetta, Rosetta-MP, BCL::Score) it is possible to select accurate models for the required purposes: the structure of the binding site, the transmembrane domain or the overall shape of the receptor. Currently, no other web service for GPCR modeling provides this possibility. GPCRM is continually upgraded in a semi-automatic way and the number of template structures has increased from 20 in 2013 to over 90 including structures the same receptor with different ligands which can influence the structure not only in the on/off manner. Two types of protein viewers can be used for visual inspection of obtained models. The extended sortable tables with available templates provide links to external databases and display ligand-receptor interactions in visual form.

Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

DEFF Research Database (Denmark)

Bartkova, J; Hamerlik, P; Stockhausen, Marie

2010-01-01

damage signalling in low- and high-grade human gliomas, and analyze the sources of such endogenous genotoxic stress. Based on analyses of human glioblastoma multiforme (GBM) cell lines, normal astrocytes and clinical specimens from grade II astrocytomas (n=41) and grade IV GBM (n=60), we conclude...... that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal...... brain and grade II astrocytomas, despite the degree of DDR activation was higher in grade II tumors. Markers indicative of ongoing DNA replication stress (Chk1 activation, Rad17 phosphorylation, replication protein A foci and single-stranded DNA) were present in GBM cells under high- or low...
78 FR 57777 - Constitution Day and Citizenship Day, Constitution Week, 2013

Science.gov (United States)

2013-09-19

... strength of our common ideals. In a document that has endured for more than two and a quarter centuries, the Framers put forth their vision for a more perfect Union. Our Constitution was signed on September... citizenship, recognize the enduring strength of our Constitution, and reaffirm our commitment to the rights...
Change of position of constitutional judiciary

Directory of Open Access Journals (Sweden)

Orlović Slobodan P.

2013-01-01

Full Text Available Constitutional judiciary is the youngest branch of authority in the horizontal level of state power. Constitutional judiciary has, during its existence - during two centuries as an ordinary court and during one century as a special authority, changed its position, role and importance. Those characteristics of constitutional judiciary had an increasing way - the position became better, in political and law sense, its role has expanded and the importance has increased. Today, constitutional judiciary is an inevitable subject of constitutional regimes in huge number of states (between them are almost all federations but, in the same time, constitutional judiciary is an authority which is at least limited by the constitution. The constitution is "soft" to the constitutional judiciary because the judiciary interpreted the constitution in accordance to its political and law attitudes, hidden by the guise of protection. Different separation of power, a rise of executive power, requests for better protections of fundamental human rights, a changed role of state and executive power, altogether, have influenced to change of position of constitutional judiciary.
alpha-Adducin mutations increase Na/K pump activity in renal cells by affecting constitutive endocytosis: implications for tubular Na reabsorption.

Science.gov (United States)

Torielli, Lucia; Tivodar, Simona; Montella, Rosa Chiara; Iacone, Roberto; Padoani, Gloria; Tarsini, Paolo; Russo, Ornella; Sarnataro, Daniela; Strazzullo, Pasquale; Ferrari, Patrizia; Bianchi, Giuseppe; Zurzolo, Chiara

2008-08-01

Genetic variation in alpha-adducin cytoskeletal protein is implicated in the polymerization and bundling of actin and alteration of the Na/K pump, resulting in abnormal renal sodium transport and hypertension in Milan hypertensive rats and humans. To investigate the molecular involvement of alpha-adducin in controlling Na/K pump activity, wild-type or mutated rat and human alpha-adducin forms were, respectively, transfected into several renal cell lines. Through multiple experimental approaches (microscopy, enzymatic assays, coimmunoprecipitation), we showed that rat and human mutated forms increased Na/K pump activity and the number of pump units; moreover, both variants coimmunoprecipitate with Na/K pump. The increased Na/K pump activity was not due to changes in its basolateral localization, but to an alteration of Na/K pump residential time on the plasma membrane. Indeed, both rat and human mutated variants reduced constitutive Na/K pump endocytosis and similarly affected transferrin receptor trafficking and fluid-phase endocytosis. In fact, alpha-adducin was detected in clathrin-coated vesicles and coimmunoprecipitated with clathrin. These results indicate that adducin, besides its modulatory effects on actin cytoskeleton dynamics, might play a direct role in clathrin-dependent endocytosis. The constitutive reduction of the Na/K pump endocytic rate induced by mutated adducin variants may be relevant in Na-dependent hypertension.
Contested constitutions: Legitimacy of constitution-making and constitutional conflict in Central Europe

NARCIS (Netherlands)

de Raadt, J.B.

2009-01-01

What were the effects of constitution-making procedures on the acceptance of the new "rules of the political game" in postcommunist Central Europe? This article sets out to scrutinise the increasingly popular claim among politicians and scholars of democratisation that inclusiveness and popular
Constitutional Referendums:A Theoretical Enquiry

OpenAIRE

Tierney, Stephen

2009-01-01

In recent decades the use of referendums to settle major constitutional questions has increased dramatically. Addressing this phenomenon as a case study in the relationship between democracy and constitutional sovereignty, this article has two aims.The first is to argue that these constitutional referendums are categorically different from ordinary, legislative referendums, and that this has important implications for theories of constitutional sovereignty. Secondly, the article suggests that...
Development of novel ligands for peptide GPCRs.

Science.gov (United States)

Moran, Brian M; McKillop, Aine M; O'Harte, Finbarr Pm

2016-12-01

Incretin based glucagon-like peptide-1 receptor (GLP-1R) agonists which target a G-protein coupled receptor (GPCR) are currently used in the treatment of type 2 diabetes. This review focuses on GPCRs from pancreatic β-cells, including GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucagon, somatostatin, pancreatic polypeptide (PP), cholecystokinin (CCK), peptide YY (PYY), oxyntomodulin (OXM) and ghrelin receptors. In addition, fatty acids GPCRs are thought to have an increasing role in regulating peptide secretions namely short fatty acids GPCR (GPR41, GPR43), medium chain fatty acid GPCR (GPR84), long chain fatty acid GPCR (GPR40, GPR120) and cannabinoid-like GPCR (GPR55, GPR119). Several pre-clinical and clinical trials are currently ongoing in peptide GPCR based therapies, including dual and triple agonist peptides which activate two or more GPCRs simultaneously. Copyright © 2016 Elsevier Ltd. All rights reserved.
Constitutional reform as process

OpenAIRE

Schultze, Rainer-Olaf (Prof.)

2000-01-01

Constitutional reform as process. - In: The politics of constitutional reform in North America / Rainer-Olaf Schultze ... (eds.). - Opladen : Leske + Budrich, 2000. - S. 11-31. - (Politikwissenschaftliche paperbacks ; 30)
CINPA1 binds directly to constitutive androstane receptor and inhibits its activity.

Science.gov (United States)

Cherian, Milu T; Chai, Sergio C; Wright, William C; Singh, Aman; Alexandra Casal, Morgan; Zheng, Jie; Wu, Jing; Lee, Richard E; Griffin, Patrick R; Chen, Taosheng

2018-03-31

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that regulate the expression of drug-metabolizing enzymes and efflux transporters. CAR activation promotes drug elimination, thereby reducing therapeutic effectiveness, or causes adverse drug effects via toxic metabolites. CAR inhibitors could be used to attenuate these adverse drug effects. CAR and PXR share ligands and target genes, confounding the understanding of the regulation of receptor-specific activity. We previously identified a small-molecule inhibitor, CINPA1, that inhibits CAR (without activating PXR at lower concentrations) by altering CAR-coregulator interactions and reducing CAR recruitment to DNA response elements of regulated genes. However, solid evidence was not presented for the direct binding of CINPA1 to CAR. In this study, we demonstrate direct interaction of CINPA1 with the CAR ligand-binding domain (CAR-LBD) and identify key residues involved in such interactions through a combination of biophysical and computational methods. We found that CINPA1 resides in the ligand-binding pocket to stabilize the CAR-LBD in a more rigid, less fluid state. Molecular dynamics simulations, together with our previously reported docking model, enabled us to predict which CAR residues were critical for interactions with CINPA1. The importance of these residues for CINPA1 binding were then validated by directed mutations and testing the mutant CAR proteins in transcription reporter and coregulatory interaction assays. We demonstrated strong hydrogen bonding of CINPA1 with N165 and H203 and identified other residues involved in hydrophobic contacts with CINPA1. Overall, our data confirm that CINPA1 directly binds to CAR. Copyright © 2018 Elsevier Inc. All rights reserved.
G protein-coupled receptor mutations and human genetic disease.

Science.gov (United States)

Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

2014-01-01

Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common
Post-Revolution Constitutionalism: The Impact of Drafting Processes on the Constitutional Documents in Tunisia and Egypt

DEFF Research Database (Denmark)

Elsayed, Ahmed Mohamed Abdelfattah

2014-01-01

This paper seeks to address the constitutional paths that followed the Arab awakening in both Tunisia and Egypt. The Tunisian constitutional process, despite some tensions, was largely peaceful and consensual. On the other hand, the process in Egypt of establishing a new constitutional arrangement...... at identifying the factors have impacted both the constitutional drafting process and the popular perception of the produced constitutions in each of Tunisia and Egypt....
Water permeation through the internal water pathway in activated GPCR rhodopsin.

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Katsufumi Tomobe

Full Text Available Rhodopsin is a light-driven G-protein-coupled receptor that mediates signal transduction in eyes. Internal water molecules mediate activation of the receptor in a rhodopsin cascade reaction and contribute to conformational stability of the receptor. However, it remains unclear how internal water molecules exchange between the bulk and protein inside, in particular through a putative solvent pore on the cytoplasmic. Using all-atom molecular dynamics simulations, we identified the solvent pore on cytoplasmic side in both the Meta II state and the Opsin. On the other hand, the solvent pore does not exist in the dark-adapted rhodopsin. We revealed two characteristic narrow regions located within the solvent pore in the Meta II state. The narrow regions distinguish bulk and the internal hydration sites, one of which is adjacent to the conserved structural motif "NPxxY". Water molecules in the solvent pore diffuse by pushing or sometimes jumping a preceding water molecule due to the geometry of the solvent pore. These findings revealed a total water flux between the bulk and the protein inside in the Meta II state, and suggested that these pathways provide water molecules to the crucial sites of the activated rhodopsin.
Constitutional Adjudication in Colombia: Avant-Garde or Case law Transplant? A Literature Review

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Liliana Lizarazo-Rodríguez

2011-03-01

Full Text Available Colombia is mentioned, together with the US, Uruguay, Argentina and Mexico, as one of the first countries worldwide to adopt the judicial review as a means for adjudicating on the constitutionality of legislation. In recent years, and particularly since the enactment of the Political Constitution of 1991, the Colombian Constitutional Court is also mentioned as a notorious example of judicial activism in terms of legislating through the constitutional adjudication process. This article presents a literature review on the globalization of judicial review and the contemporary methods of constitutional adjudication (including the balancing method, in order to assess the uniqueness and avantgarde nature of constitutional adjudication in Colombia in the global context. Brief reference is also made to the literature on the institutional limitations faced by less developed countries, inasmuch as they affect the way constitutional adjudication is applied and perceived.
The diffusion of constitutional rights

NARCIS (Netherlands)

Goderis, B.V.G.; Versteeg, M.

Constitutions are commonly regarded as uniquely national products, shaped by domestic ideals and politics. This paper develops and empirically investigates a novel hypothesis, which is that constitutions are also shaped by transnational influence, or “diffusion.” Constitutional rights can diffuse
Evodia alkaloids suppress gluconeogenesis and lipogenesis by activating the constitutive androstane receptor.

Science.gov (United States)

Yu, Lushan; Wang, Zhangting; Huang, Minmin; Li, Yingying; Zeng, Kui; Lei, Jinxiu; Hu, Haihong; Chen, Baian; Lu, Jing; Xie, Wen; Zeng, Su

2016-09-01

The constitutive androstane receptor (CAR) is a key sensor in xenobiotic detoxification and endobiotic metabolism. Increasing evidence suggests that CAR also plays a role in energy metabolism by suppressing the hepatic gluconeogenesis and lipogenesis. In this study, we investigated the effects of two evodia alkaloids, rutaecarpine (Rut) and evodiamine (Evo), on gluconeogenesis and lipogenesis through their activation of the human CAR (hCAR). We found that both Rut and Evo exhibited anti-lipogenic and anti-gluconeogenic effects in the hyperlipidemic HepG2 cells. Both compounds can potently activate hCAR, and treatment of cells with hCAR antagonists reversed the anti-lipogenic and anti-gluconeogenic effects of Rut and Evo. The anti-gluconeogenic effect of Rut and Evo was due to the CAR-mediated inhibition of the recruitment of forkhead box O1 (FoxO1) and hepatocyte nuclear factor 4α (HNF4α) onto the phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) gene promoters. In vivo, we showed that treatment of mice with Rut improved glucose tolerance in a CAR-dependent manner. Our results suggest that the evodia alkaloids Rut and Evo may have a therapeutic potential for the treatment of hyperglycemia and type 2 diabetes. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. Copyright © 2015 Elsevier B.V. All rights reserved.
Great Constitutional Ideas: Justice, Equality, and Property.

Science.gov (United States)

Starr, Isidore

1987-01-01

Examines the ideas of justice, equality, and property as they are represented in the Declaration of Independence, the U.S. Constitution and the Bill of Rights. Discusses how these ideas affect the way public schools operate and the lessons educators teach or don't teach about our society. Includes ideas for classroom activities. (JDH)
Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor

Science.gov (United States)

Cherian, Milu T.; Yang, Lei; Chai, Sergio C.; Lin, Wenwei

2016-01-01

The constitutive androstane receptor (CAR) regulates the expression of genes involved in drug metabolism and other processes. A specific inhibitor of CAR is critical for modulating constitutive CAR activity. We recently described a specific small-molecule inhibitor of CAR, CINPA1 (ethyl (5-(diethylglycyl)-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)carbamate), which is capable of reducing CAR-mediated transcription by changing the coregulator recruitment pattern and reducing CAR occupancy at the promoter regions of its target genes. In this study, we showed that CINPA1 is converted to two main metabolites in human liver microsomes. By using cell-based reporter gene and biochemical coregulator recruitment assays, we showed that although metabolite 1 was very weak in inhibiting CAR function and disrupting CAR-coactivator interaction, metabolite 2 was inactive in this regard. Docking studies using the CAR ligand-binding domain structure showed that although CINPA1 and metabolite 1 can bind in the CAR ligand-binding pocket, metabolite 2 may be incapable of the molecular interactions required for binding. These results indicate that the metabolites of CINPA1 may not interfere with the action of CINPA1. We also used in vitro enzyme assays to identify the cytochrome P450 enzymes responsible for metabolizing CINPA1 in human liver microsomes and showed that CINPA1 was first converted to metabolite 1 by CYP3A4 and then further metabolized by CYP2D6 to metabolite 2. Identification and characterization of the metabolites of CINPA1 enabled structure-activity relationship studies of this family of small molecules and provided information to guide in vivo pharmacological studies. PMID:27519550
An atlas of Caenorhabditis elegans chemoreceptor expression.

Directory of Open Access Journals (Sweden)

Berta Vidal

2018-01-01

Full Text Available One goal of modern day neuroscience is the establishment of molecular maps that assign unique features to individual neuron types. Such maps provide important starting points for neuron classification, for functional analysis, and for developmental studies aimed at defining the molecular mechanisms of neuron identity acquisition and neuron identity diversification. In this resource paper, we describe a nervous system-wide map of the potential expression sites of 244 members of the largest gene family in the C. elegans genome, rhodopsin-like (class A G-protein-coupled receptor (GPCR chemoreceptors, using classic gfp reporter gene technology. We cover representatives of all sequence families of chemoreceptor GPCRs, some of which were previously entirely uncharacterized. Most reporters are expressed in a very restricted number of cells, often just in single cells. We assign GPCR reporter expression to all but two of the 37 sensory neuron classes of the sex-shared, core nervous system. Some sensory neurons express a very small number of receptors, while others, particularly nociceptive neurons, coexpress several dozen GPCR reporter genes. GPCR reporters are also expressed in a wide range of inter- and motorneurons, as well as non-neuronal cells, suggesting that GPCRs may constitute receptors not just for environmental signals, but also for internal cues. We observe only one notable, frequent association of coexpression patterns, namely in one nociceptive amphid (ASH and two nociceptive phasmid sensory neurons (PHA, PHB. We identified GPCRs with sexually dimorphic expression and several GPCR reporters that are expressed in a left/right asymmetric manner. We identified a substantial degree of GPCR expression plasticity; particularly in the context of the environmentally-induced dauer diapause stage when one third of all tested GPCRs alter the cellular specificity of their expression within and outside the nervous system. Intriguingly, in a number of
Legal theology in imposed constitutionalism

DEFF Research Database (Denmark)

Abat Ninet, Antoni

2018-01-01

The focus of this paper is the question of legitimacy, and how can we consider legitimate an imposed constitution and the subsequent constitutional principles, practices and values that go hand-in-hand with the legal and political acculturation. Constitutional texts around the world are good...
Modeling and Deorphanization of Orphan GPCRs.

Science.gov (United States)

Diaz, Constantino; Angelloz-Nicoud, Patricia; Pihan, Emilie

2018-01-01

Despite tremendous efforts, approximately 120 GPCRs remain orphan. Their physiological functions and their potential roles in diseases are poorly understood. Orphan GPCRs are extremely important because they may provide novel therapeutic targets for unmet medical needs. As a complement to experimental approaches, molecular modeling and virtual screening are efficient techniques to discover synthetic surrogate ligands which can help to elucidate the role of oGPCRs. Constitutively activated mutants and recently published active structures of GPCRs provide stimulating opportunities for building active molecular models for oGPCRs and identifying activators using virtual screening of compound libraries. We describe the molecular modeling and virtual screening process we have applied in the discovery of surrogate ligands, and provide examples for CCKA, a simulated oGPCR, and for two oGPCRs, GPR52 and GPR34.

Constitutional Principles of State Control in the Republic of Armenia

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Gabriel K. Balayan

2015-12-01

Full Text Available In the present article some of the provision of the RA Constitution’s basic principles of state control, which, in my opinion, should be guided by regulatory authorities during the inspection are discussed. These principles are considered by the author as the basis of the constitutionality of state control. Some of the control principles are divided by the author into positive and negative. Positive defines the principles that provide the freedom of action of man and citizen. Negative same principles are for government agencies, since they restrict their activities. A separate analysis is subject to the presidency on the basis of the principle of separation and balance of powers. In conclusion, author concludes that, based on the interpretation of the constitutional principles discussed, the term "law" applies only to natural and legal persons on the other hand can not be used as an analogue of the terms "authority" and "authority." According to the author, the fundamental constitutional principles of state control are expressed formally in a single system as defined in the first chapter of the Constitution of the Republic of Armenia. Compliance and reform of constitutional principles provide the synergy and the constitutionality of the process of governance.
Structural motifs of importance for the constitutive activity of the orphan 7TM receptor EBI2: analysis of receptor activation in the absence of an agonist

DEFF Research Database (Denmark)

Benned-Jensen, Tau; Rosenkilde, Mette M

2008-01-01

were identified by a systematic mutational analysis of 29 residues in EBI2. The cAMP response element-binding protein transcription factor was used as a measure of receptor activity and was correlated to the receptor surface expression. PheVI:13 (Phe257), and the neighboring CysVI:12 (Cys256......, but not to Lys, decreased the constitutive activity more than 7-fold compared with wt EBI2. IleIII:03 (Ile106) is located only 4 A from ArgII:20, and a favorable electrostatic interaction with ArgII:20 was created by introduction of Glu in III:03, given that the activity increased to 4.4-fold of that wt EBI2...
The Normative Constitution of Agency

OpenAIRE

Korsgaard, Christine M.

2014-01-01

On a naturalistic conception of agency, someone is an agent when certain of his own mental states are the cause of his movements. On a normative conception, a person constitutes his agency by following certain principles or laws. In Kant’s conception, for example, a movement counts as the agent’s own when it is chosen autonomously, that is, in accordance with the categorical imperative. To say that someone acts is to imply that his movements are a manifestation of his own activity and that it...
Political dimension of European constitutionalism

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Kaplánová Patricia

2015-04-01

Full Text Available Author in the article tries to analyse different elements of document called European Constitution. Analysis is supported with theoretical framework of federalism, presented by Brezovšek. Authors is playing with idea of (confederal and international organization elements of European Constitution and their mix. They are also trying to set some connections between so called common European identity as necessary condition to give legitimacy to the European Constitution. This became important question after „votes of non-confidence“ to the European Constitution in France, despite it should be addressed already before. However, European Constitution is important document on the path of European integration and lack of support to it will slow down this process of widening and deepening European ties.
Human pregnane X receptor is activated by dibenzazepine carbamate-based inhibitors of constitutive androstane receptor.

Science.gov (United States)

Jeske, Judith; Windshügel, Björn; Thasler, Wolfgang E; Schwab, Matthias; Burk, Oliver

2017-06-01

Unintentional activation of xenosensing nuclear receptors pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR) by clinical drug use is known to produce severe side effects in patients, which may be overcome by co-administering antagonists. However, especially antagonizing CAR is hampered by the lack of specific inhibitors, which do not activate PXR. Recently, compounds based on a dibenzazepine carbamate scaffold were identified as potent CAR inhibitors. However, their potential to activate PXR was not thoroughly investigated, even if the lead compound was named "CAR inhibitor not PXR activator 1" (CINPA1). Thus, we performed a comprehensive analysis of the interaction of CINPA1 and four analogs with PXR. Cellular assays were used to investigate intra- and intermolecular interactions and transactivation activity of PXR as a function of the compounds. Modulation of PXR target gene expression was analyzed in primary human hepatocytes. Ligand binding to PXR was investigated by molecular docking and limited proteolytic digestion. We show here that CINPA1 induced the assembly of the PXR ligand-binding domain, released co-repressors from and recruited co-activators to the receptor. CINPA1 and its analogs induced the PXR-dependent activation of a CYP3A4 reporter gene and CINPA1 induced the expression of endogenous cytochrome P450 genes in primary hepatocytes, while not consistently inhibiting CAR-mediated induction. Molecular docking revealed favorable binding of CINPA1 and analogs to the PXR ligand-binding pocket, which was confirmed in vitro. Altogether, our data provide consistent evidence that compounds with a dibenzazepine carbamate scaffold, such as CINPA1 and its four analogs, bind to and activate PXR.
Constitutive Activity of the Arabidopsis MAP Kinase 3 Confers Resistance to Pseudomonas syringae and Drives Robust Immune Responses

KAUST Repository

Lang, Julien

2017-08-02

Mitogen Activated Protein Kinases (MAPKs) are known to be important mediators of plant responses to biotic and abiotic stresses. In a recent report, we enlarged the understanding of the Arabidopsis thaliana MPK3 functions showing that the expression of a constitutively active (CA) form of the protein led to auto-immune phenotypes. CA-MPK3 plants are dwarf and display defense responses that are characterized by the accumulation of salicylic acid and phytoalexins as well as by the upregulation of several defense genes. Consistently with these data, we present here results demonstrating that, compared to wild type controls, CA-MPK3 plants are more resistant to the hemibiotrophic pathogen Pseudomonas syringae DC3000. Based on our previous work, we also discuss the mechanisms of robust plant immunity controlled by sustained MPK3 activity, focusing especially on the roles of disease resistance proteins.
The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration

DEFF Research Database (Denmark)

Cornelia Peeters, Miriam; Fokkelman, Michiel; Boogaard, Bob

2015-01-01

Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which...
Influence of zemstvo self-government on process of national constitutionalism development in late XIX – early XX centuries

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Анатолій Іванович Козаченко

2016-04-01

Full Text Available Problem Setting. The article describes the activities of the zemstvo self-government which had a significant impact on the process of national constitutionalism development in the late XIX – early XX centuries. The actuality of the topic. Zemstvo self-government got significant practical experience of legal oppositional activity, which is an important element of constitutionalism. Within the territory of ethnicUkraine, zemstvo constitutionalism combined two components: All-Russian and national. The purpose of the article. All-Russian zemstvo constitutionalism had features of legal, semi-legal and illegal activities, which were represented by liberal and radical wing of zemstvo liberal-democratic movement respectively. In the All-Russian context, zemstvo radicals advocated the idea of establishing constitutional order in the Russian Empire, which involved constitutional recognition of democratic rights and freedoms, equality, parliamentary system and establishment of a constitutional monarchy, decentralization of power, political pluralism. The national component of zemstvo constitutionalism, which was formed at the beginning of the bourgeois-democratic revolution of 1905–1907, had illegal character and its essence can be described by the fact that some zemstvo liberals insisted on the need to restore the Ukrainian state in the form of autonomy within democratic Russia. Representatives of both components of zemstvo constitutionalism aimed to implement their constitutional ideas through reforms. Zemstvo constitutionalism reached its highest peak at the beginning of the bourgeois-democratic revolution of 1905–1907. In early XX century zemstvo liberals took active part in activities of bourgeois-democratic political parties. Development of Russian and Ukrainian political parties led to a split in the radical wing of zemstvo liberal-democratic movement on the national basis. Some zemstvo leaders joined the All-Russian Party of Octobrists and
GPCR-I-TASSER: A Hybrid Approach to G Protein-Coupled Receptor Structure Modeling and the Application to the Human Genome.

Science.gov (United States)

Zhang, Jian; Yang, Jianyi; Jang, Richard; Zhang, Yang

2015-08-04

Experimental structure determination remains difficult for G protein-coupled receptors (GPCRs). We propose a new hybrid protocol to construct GPCR structure models that integrates experimental mutagenesis data with ab initio transmembrane (TM) helix assembly simulations. The method was tested on 24 known GPCRs where the ab initio TM-helix assembly procedure constructed the correct fold for 20 cases. When combined with weak homology and sparse mutagenesis restraints, the method generated correct folds for all the tested cases with an average Cα root-mean-square deviation 2.4 Å in the TM regions. The new hybrid protocol was applied to model all 1,026 GPCRs in the human genome, where 923 have a high confidence score and are expected to have correct folds; these contain many pharmaceutically important families with no previously solved structures, including Trace amine, Prostanoids, Releasing hormones, Melanocortins, Vasopressin, and Neuropeptide Y receptors. The results demonstrate new progress on genome-wide structure modeling of TM proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.
Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

Science.gov (United States)

Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

2011-01-01

Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661
Regulator of G-protein signaling-5 is a marker of hepatic stellate cells and expression mediates response to liver injury.

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Arya J Bahrami

Full Text Available Liver fibrosis is mediated by hepatic stellate cells (HSCs, which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR-mediated signaling, via endothelin-1 (ET-1 and angiotensin II (AngII, increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5, an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs. Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl4-induced acute and chronic liver injury in Rgs5LacZ/LacZ reporter mice. Furthermore, CCl4 treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl4 and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.
Identification and Characterization of CINPA1 Metabolites Facilitates Structure-Activity Studies of the Constitutive Androstane Receptor.

Science.gov (United States)

Cherian, Milu T; Yang, Lei; Chai, Sergio C; Lin, Wenwei; Chen, Taosheng

2016-11-01

The constitutive androstane receptor (CAR) regulates the expression of genes involved in drug metabolism and other processes. A specific inhibitor of CAR is critical for modulating constitutive CAR activity. We recently described a specific small-molecule inhibitor of CAR, CINPA1 (ethyl (5-(diethylglycyl)-10,11-dihydro-5H-dibenzo[b,f]azepin-3-yl)carbamate), which is capable of reducing CAR-mediated transcription by changing the coregulator recruitment pattern and reducing CAR occupancy at the promoter regions of its target genes. In this study, we showed that CINPA1 is converted to two main metabolites in human liver microsomes. By using cell-based reporter gene and biochemical coregulator recruitment assays, we showed that although metabolite 1 was very weak in inhibiting CAR function and disrupting CAR-coactivator interaction, metabolite 2 was inactive in this regard. Docking studies using the CAR ligand-binding domain structure showed that although CINPA1 and metabolite 1 can bind in the CAR ligand-binding pocket, metabolite 2 may be incapable of the molecular interactions required for binding. These results indicate that the metabolites of CINPA1 may not interfere with the action of CINPA1. We also used in vitro enzyme assays to identify the cytochrome P450 enzymes responsible for metabolizing CINPA1 in human liver microsomes and showed that CINPA1 was first converted to metabolite 1 by CYP3A4 and then further metabolized by CYP2D6 to metabolite 2. Identification and characterization of the metabolites of CINPA1 enabled structure-activity relationship studies of this family of small molecules and provided information to guide in vivo pharmacological studies. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

Science.gov (United States)

Morre, D. James; Morre, Dorothy M.; Ternes, Philipp

2003-01-01

The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.
On the Constitution and the Constitutions in Contemporary Egypt (2011-2014: Analysis and Early Thoughts

Directory of Open Access Journals (Sweden)

José Carlos Castañeda Reyes

2016-01-01

world witnessed, a testament to what massive popular participation can attain. We believe the Constitution has true potential to guide the forces of change in the country. Therefore, the struggle for “bread, freedom, social justice and human dignity” will continue and is actually continuing in the Nile country via constitutional and non-constitutional means.
The constitutional court review of judicial decisions

Directory of Open Access Journals (Sweden)

Stojanović Dragan M.

2016-01-01

Full Text Available In principle, the constitutional precepts envisage that judicial decisions are not subject to extrajudicial control. However, in the course of deciding on constitutional complaints, the Constitutional Court reviews the compliance of individual legal acts and actions of state authorities with the Constitution, including court decisions on cases involving the constitutionally guaranteed rights. Hence, in order to eliminate tension or even contradiction between the constitutional precepts, the constitutional review of judicial decisions should be considered as a special form of judicial control, regardless of the fact that the Constitutional Court is not part of the judicial structure in the strict organizational sense. Thus, unlike the cases where the Court is involved in the normative control of the applicable law, in the process of reviewing judicial decision of lower courts the constitutional judiciary acts in the capacity of a specific judicial authority. According to another possible interpretation of the aforementioned constitutional norms, the direct constitutional protection of the constitutionally guaranteed rights may only be pursued in the process of reviewing individual legal acts and actions of state authorities, but not by pursuing a judicial review of court decisions which the Constitutional Court has no jurisdiction to decide upon. Thus, the dogma of judicial independence would prevail over the dogma of direct protection of fundamental rights. The third interpretation of this relationship maintains that that judicial decisions may be subject to control but, in this specific case, the Constitutional Court may only issue an opinion (a statement rather than a binding decision which would cancel the lower court judgment. Then, it is up to the judicial authorities of the lower instance to adjust their judicial decision, which in the opinion of the Constitutional Court constitutes a violation of the constitutionally guaranteed rights
Rac1-mediated membrane raft localization of PI3K/p110β is required for its activation by GPCRs or PTEN loss

Science.gov (United States)

Cizmecioglu, Onur; Ni, Jing; Xie, Shaozhen; Zhao, Jean J; Roberts, Thomas M

2016-01-01

We aimed to understand how spatial compartmentalization in the plasma membrane might contribute to the functions of the ubiquitous class IA phosphoinositide 3-kinase (PI3K) isoforms, p110α and p110β. We found that p110β localizes to membrane rafts in a Rac1-dependent manner. This localization potentiates Akt activation by G-protein-coupled receptors (GPCRs). Thus genetic targeting of a Rac1 binding-deficient allele of p110β to rafts alleviated the requirement for p110β-Rac1 association for GPCR signaling, cell growth and migration. In contrast, p110α, which does not play a physiological role in GPCR signaling, is found to reside in nonraft regions of the plasma membrane. Raft targeting of p110α allowed its EGFR-mediated activation by GPCRs. Notably, p110β dependent, PTEN null tumor cells critically rely upon raft-associated PI3K activity. Collectively, our findings provide a mechanistic account of how membrane raft localization regulates differential activation of distinct PI3K isoforms and offer insight into why PTEN-deficient cancers depend on p110β. DOI: http://dx.doi.org/10.7554/eLife.17635.001 PMID:27700986
High content screening for G protein-coupled receptors using cell-based protein translocation assays

DEFF Research Database (Denmark)

Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne

2005-01-01

G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...
BILF1 Mediated Transformation Correlates with Constitutive Signaling

DEFF Research Database (Denmark)

Lyngaa, Rikke Birgitte

2009-01-01

BIFL1 is a G protein-coupled receptor encoded by human EBV. It signals constitutively through G_alpha_i and is an orphan receptor known to down regulate MHCI expression. BILF1 also engage in dimerization with several chemokine receptors and it induced the activity of NF-kappa beta and inhibits...
Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor

DEFF Research Database (Denmark)

Holliday, Nicholas D; Holst, Birgitte; Rodionova, Elena A

2007-01-01

. Furthermore the interaction between phosphorylated receptors and beta-arrestin adaptor proteins has been examined. Replacement of the FLAG-tagged GhrelinR C tail with the equivalent GPR39 domain (GhR-39 chimera) preserved G(q) signaling. However in contrast to the GhrelinR, GhR-39 receptors exhibited no basal......,9), Leu(11)] substance P and a naturally occurring mutant GhrelinR (A204E) with eliminated constitutive activity inhibited basal GhrelinR internalization. Surprisingly, we found that noninternalizing GPR39 was highly phosphorylated and that basal and agonist-induced phosphorylation of the GhR-39 chimera......, but the high levels of GPR39 phosphorylation, and of the GhR-39 chimera, are not sufficient to drive endocytosis. In addition, basal GhrelinR internalization occurs independently of beta-arrestins....
PAFR activation of NF-?B p65 or p105 precursor dictates pro- and anti-inflammatory responses during TLR activation in murine macrophages

OpenAIRE

Ishizuka, Edson K.; Filgueiras, Luciano Ribeiro; Rios, Francisco J.; Serezani, Carlos H.; Jancar, Sonia

2016-01-01

Platelet-activating factor receptor (PAFR) is a G protein-coupled receptor (GPCR) implicated in many diseases. Toll-like receptors (TLRs) play a critical role in shaping innate and adaptive immune responses. In this study, we investigated whether PAFR signaling changes the macrophages responsiveness to agonists of TLR2 (Pam3Cys), TLR4 (LPS), and TLR3 agonist Poly(I:C). Exogenous PAF inhibited the production of pro-inflammatory cytokines (IL-12p40, IL-6, and TNF-α) and increased anti-inflammat...

Gpr124 is essential for blood-brain barrier integrity in central nervous system disease.

Science.gov (United States)

Chang, Junlei; Mancuso, Michael R; Maier, Carolina; Liang, Xibin; Yuki, Kanako; Yang, Lu; Kwong, Jeffrey W; Wang, Jing; Rao, Varsha; Vallon, Mario; Kosinski, Cynthia; Zhang, J J Haijing; Mah, Amanda T; Xu, Lijun; Li, Le; Gholamin, Sharareh; Reyes, Teresa F; Li, Rui; Kuhnert, Frank; Han, Xiaoyuan; Yuan, Jenny; Chiou, Shin-Heng; Brettman, Ari D; Daly, Lauren; Corney, David C; Cheshier, Samuel H; Shortliffe, Linda D; Wu, Xiwei; Snyder, Michael; Chan, Pak; Giffard, Rona G; Chang, Howard Y; Andreasson, Katrin; Kuo, Calvin J

2017-04-01

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.
A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells

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Huang, Peng; Chiu, Yi-Ting; Chen, Chongguang; Wang, Yujun; Liu-Chen, Lee-Yuan

2013-01-01

Introduction In contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs available, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability. Methods We fused tdT to the KOPR C-terminus. The KOPR-tdT cDNA construct was transfected into Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking. Results KOPR-tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR-tdT enables visualization of time course of agonist-induced internalization of KOPR in real time for 60 min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4 min and plateaued at about 30 min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H. Discussion tdT is an alternative to, or even a better tool than, GFPs for fusing to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance. PMID:23856011
ECHR and national constitutional courts

OpenAIRE

Nastić, Maja

2015-01-01

Comprising fundamental rights and freedoms and establishing the effective control system, the European Convention on Human Rights (ECHR) encroaches upon the area that is traditional reserved for constitutional law. Although built on the doctrine reserved for international treaty law, the Convention goes beyond the traditional boundaries that exist between international and constitutional law. It has gradually infiltrated into the national legal systems. Constitutional courts have had the cruc...
Constitutively active Notch1 induces growth arrest of HPV-positive cervical cancer cells via separate signaling pathways

International Nuclear Information System (INIS)

Talora, Claudio; Cialfi, Samantha; Segatto, Oreste; Morrone, Stefania; Kim Choi, John; Frati, Luigi; Paolo Dotto, Gian; Gulino, Alberto; Screpanti, Isabella

2005-01-01

Notch signaling plays a key role in cell-fate determination and differentiation in different organisms and cell types. Several reports suggest that Notch signaling may be involved in neoplastic transformation. However, in primary keratinocytes, Notch1 can function as a tumor suppressor. Similarly, in HPV-positive cervical cancer cells, constitutively active Notch1 signaling was found to cause growth suppression. Activated Notch1 in these cells represses viral E6/E7 expression through AP-1 down-modulation, resulting in increased p53 expression and a block of pRb hyperphosphorylation. Here we show that in cervical cancer cell lines in which Notch1 ability to repress AP-1 activity is impaired, Notch1-enforced expression elicits an alternative pathway leading to growth arrest. Indeed, activated Notch1 signaling suppresses activity of the helix-loop-helix transcription factor E47, via ERK1/2 activation, resulting in inhibition of cell cycle progression. Moreover, we found that RBP-Jκ-dependent Notch signaling is specifically repressed in cervical cancer cells and this repression could provide one such mechanism that needs to be activated for cervical carcinogenesis. Finally, we show that inhibition of endogenous Notch1 signaling, although results in a proliferative advantage, sensitizes cervical cancer cell lines to drug-induced apoptosis. Together, our results provide novel molecular insights into Notch1-dependent growth inhibitory effects, counteracting the transforming potential of HPV
The U.S. Constitution in Today's World.

Science.gov (United States)

Patrick, John J.

A comparative study of constitutions and governments in world history is a key to deeper understanding of the U.S. Constitution. While many countries have constitutions, the United States is among a minority of nations in today's world that has a constitutional government. Many nations' constitutions truly guarantee few protections of individual…
G protein-coupled receptor internalization assays in the high-content screening format.

Science.gov (United States)

Haasen, Dorothea; Schnapp, Andreas; Valler, Martin J; Heilker, Ralf

2006-01-01

High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.
Transitional processes: Territorial organization of authorities and the future constitution of Serbia comparative analysis of five constitutional models

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Despotović Ljubiša M.

2004-01-01

Full Text Available In this paper the authors give a comparative analysis of territorial organization of authorities in five constitutional models for Serbia. The paper consists of the following chapters: Introduction, Outline of the Constitution of Kingdom of Serbia, Basic Principles of the New Constitution of Serbia - DSS, Outline of Constitution of Republic of Serbia - DS Constitutional Solutions for Serbia - BCLJP, Project of Constitution of Republic of Serbia - Forum iuris, Conclusion. The analysis of territorial organization of authorities has been seen in the context of the processes of transition and archiving the important principles of civil society and civil autonomies.
Constitution and religiosity of/in the constitutional order of the National Socialist Empire

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Velez, Pedro

2017-05-01

Full Text Available In this article, we will analyse the National Socialist regime as a politico-constitutional reality. We will do it from a new way of looking at politico-constitutional phenomena, interpreting them as registered in a religious grounding. It seeks to show that the National Socialist regime was characterised by having identified the political community – a racially interpreted and raised community to the Absolute – with an empirical historic personality regarded as eminently communitarian. It suggests that the regime constitutes a sui generis case, either in a context of regimes conventionally classified as "right-wing authoritarian and/or totalitarian" or in a larger context of contemporary politics.
Activation of PKCβII and PKCθ is essential for LDL-induced cell proliferation of human aortic smooth muscle cells via Gi-mediated Erk1/2 activation and Egr-1 upregulation

International Nuclear Information System (INIS)

Heo, Kyung-Sun; Kim, Dong-Uk; Kim, Lila; Nam, Miyoung; Baek, Seung-Tae; Park, Song-Kyu; Park, Youngwoo; Myung, Chang-Seon; Hwang, Sung-Ook; Hoe, Kwang-Lae

2008-01-01

Native LDL may be a mitogenic stimulus of VSMC proliferation in lesions where endothelial disruption occurs. Recent studies have demonstrated that the mitogenic effects of LDL are accompanied by Erk1/2 activation via an unknown G-protein-coupled receptor (GPCR). In this article, we report that LDL translocated PKCβ II and PKCθ from cytosol to plasma membrane, and inhibition of PKCβ II and PKCθ decreased LDL effects via the deactivation of Erk1/2. Moreover, pertussis toxin, but not cholera toxin or heparin, inhibited LDL-induced translocation of PKCβ II and PKCθ, suggesting that Gi protein plays a role in LDL effects. Of LPA, S1P, and LDL, whose signaling is conveyed via Gi/o proteins, only LDL induced translocation of PKCβ II and PKCθ. Inhibition of PKCβ II or PKCθ, as well as of Erk1/2 and GPCR, decreases LDL-induced upregulation of Egr-1, which is critical for cell proliferation. This is the first report, to our knowledge, that the participation of PKCθ in VSMC proliferation is unique
Dynamic nanoplatforms in biosensor and membrane constitutional systems.

Science.gov (United States)

Mahon, Eugene; Aastrup, Teodor; Barboiu, Mihail

2012-01-01

Molecular recognition in biological systems occurs mainly at interfacial environments such as membrane surfaces, enzyme active sites, or the interior of the DNA double helix. At the cell membrane surface, carbohydrate-protein recognition principles apply to a range of specific non-covalent interactions including immune response, cell proliferation, adhesion and death, cell-cell interaction and communication. Protein-protein recognition meanwhile accounts for signalling processes and ion channel structure. In this chapter we aim to describe such constitutional dynamic interfaces for biosensing and membrane transport applications. Constitutionally adaptive interfaces may mimic the recognition capabilities intrinsic to natural recognition processes. We present some recent examples of 2D and 3D constructed sensors and membranes of this type and describe their sensing and transport capabilities.
CONSTITUTIONAL INTERPRETATION OF ROMANIA: POST MODERNITY

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P. RATHNASWAMY

2017-06-01

Full Text Available Austria had in 1920 Constitutional Court followed by Italy in 1946, Germany in 1949, Romania in 1989, South Africa in 1991, and Ethiopia in 1995. Each Constitution has its provisions on the constitutional interpretation. Romania has its own provisions and it is considered here its legality and the best possible measures and recommendations for future. Judicial power is vested in judiciary to interpret constitution, laws, and actions of other organs of government. Judicial review is the function resulted upon judicial power. Political body joins through the appointment of its members in the judicial review and it limits the independence of judiciary. It also reduces the values of separation of powers. Challenges and opportunities of growth and development do influence the spirit of separation of powers and judicial independence. The principle of inherent judicial power in judiciary inducts upon the constitutional interpretation. Thus, the principles of constitutional interpretation are varying in Romania and other similar constitutional courts of Germany, Ethiopia, and Italy but not in South Africa.
Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype

Science.gov (United States)

Murakami, Shunichi; Balmes, Gener; McKinney, Sandra; Zhang, Zhaoping; Givol, David; de Crombrugghe, Benoit

2004-01-01

We generated transgenic mice that express a constitutively active mutant of MEK1 in chondrocytes. These mice showed a dwarf phenotype similar to achondroplasia, the most common human dwarfism, caused by activating mutations in FGFR3. These mice displayed incomplete hypertrophy of chondrocytes in the growth plates and a general delay in endochondral ossification, whereas chondrocyte proliferation was unaffected. Immunohistochemical analysis of the cranial base in transgenic embryos showed reduced staining for collagen type X and persistent expression of Sox9 in chondrocytes. These observations indicate that the MAPK pathway inhibits hypertrophic differentiation of chondrocytes and negatively regulates bone growth without inhibiting chondrocyte proliferation. Expression of a constitutively active mutant of MEK1 in chondrocytes of Fgfr3-deficient mice inhibited skeletal overgrowth, strongly suggesting that regulation of bone growth by FGFR3 is mediated at least in part by the MAPK pathway. Although loss of Stat1 restored the reduced chondrocyte proliferation in mice expressing an achondroplasia mutant of Fgfr3, it did not rescue the reduced hypertrophic zone, the delay in formation of secondary ossification centers, and the achondroplasia-like phenotype. These observations suggest a model in which Fgfr3 signaling inhibits bone growth by inhibiting chondrocyte differentiation through the MAPK pathway and by inhibiting chondrocyte proliferation through Stat1. PMID:14871928
Economic Reforms and Constitutional Transition

OpenAIRE

Jeffrey D. Sachs; Wing Thye Woo; Xiaokai Yang

2000-01-01

This paper investigates the relationship between economic reforms and constitutional transition, which has been neglected by many transition economists. It is argued that assessment of reform performance might be very misleading if it is not recognized that economic reforms are just a small part of large scale of constitutional transition. Rivalry and competition between states and between political forces within each country are the driving forces for constitutional transition. We use Russia...
A PLEADING IN FAVOUR OF THE CONSTITUTIONAL COURT

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Valentina BĂRBĂŢEANU

2016-05-01

Full Text Available Most of the European countries have chosen the centralized system of constitutional review, performed by a unique authority empowered with the competence of removing from the normative ensemble those legal provisions that do not comply with the principles and rules comprised in the Basic Law. This „European model” has proved to be more appropriate than the so-called „American model” in what concerns the compatibility with the European jurisdictional mechanism. Romania has adopted the same European trend and the Constitutional Court has become a very important actor in the Romanian legal landscape. From the very beginning of its activity, it has influenced in a great measure the national normative system. It has been sometimes criticized and accused that it interferes in an excessive way in the legislative process. Due to its competence to regulate the juridical conflicts between the public authorities and its possibility to repeal laws before their promulgation, it has been many times in the centre of heavy attacks, mostly from different political forces, often driven through mass media. Nevertheless, despite of its detractors, the Constitutional Court has proven, over the years, its ability to develop the Romanian normative system. The present paper intends to display the most significant contribution of the Romanian Constitutional Court in improving various legal regulations. In the same time and much more important, using concrete examples from the Court’s case-law, the paper also intends to demonstrate that the Constitutional Court of Romania has been a major factor of improving peoples’ life, removing unconstitutional obstacles set in front of the unimpeded exercise of their fundamental rights and freedoms.
State Constitution Revision: An Exercise in Federalism and Sovereignty.

Science.gov (United States)

Myers, J. A.

1988-01-01

Discusses classroom activities designed to enhance student learning about federalism and sovereignty. Describes classroom discussions on jurisdiction, federalism, and sovereignty generated by asking students to clarify the constitutional section referring to the Writ of Habeas Corpus. Concludes that this exercise clarifies the concepts and…
The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils from patients with polycythemia vera

Science.gov (United States)

Pieri, Lisa; Bogani, Costanza; Guglielmelli, Paola; Zingariello, Maria; Rana, Rosa Alba; Bartalucci, Niccolò; Bosi, Alberto; Vannucchi, Alessandro M.

2009-01-01

Background The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes. Design and Methods We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63+ basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor. Results We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63+ basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63+ basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden. Conclusions These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells’ abnormal function and, putatively, in the pathogenesis of pruritus. PMID:19608683
Constitutions of Nature by Teacher Practice and Discourse in Ontario Grade 9 and 10 Academic Science

Science.gov (United States)

Hoeg, Darren Glen

This thesis presents an ethnographic study, based broadly on principles and methods of institutional ethnography, on the constitution of nature by nine Ontario Grade 9 and 10 Academic Science teachers. The intent of this methodological approach is to examine how the daily practice of participants works toward constituting nature in specific ways that are coordinated by the institution (Ontario public school and/or school science). Critical Discourse Analysis and general inductive analysis were performed on interview transcripts, texts related to teaching science selected by participants, and policy documents (i.e. curriculum; assessment policy) that coordinate science teacher practice. Findings indicate specific, dominant, and relatively uniform ontological and epistemological constitutions of nature. Nature was frequently constituted as a remote object, distant from and different than students studying it. More complex representations included constituting nature as a model, machine, or mathematical algorithm. Epistemological constitutions of nature were enacted through practices that engaged students in manipulating nature; controlling nature, and dominating nature. Relatively few practices that allow students to construct different constitutions of nature than those prioritized by the institution were observed. Dominant constitutions generally assume nature is simply the material to study, from which scientific knowledge can be obtained, with little ethical or moral consideration about nature itself, or how these constitutions produce discourse and relationships that may be detrimental to nature. Dominant constitutions of nature represent a type of objective knowledge that is prioritized, and made accessible to students, through science activities that attain a position of privilege in local science teacher cultures. The activities that allow students to attain the requisite knowledge of nature are collected, collated, and shared among existing science teachers
Defective interleukin-4/Stat6 activity correlates with increased constitutive expression of negative regulators SOCS-3, SOCS-7, and CISH in colon cancer cells.

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Liu, Xiao Hong; Xu, Shuang Bing; Yuan, Jia; Li, Ben Hui; Zhang, Yan; Yuan, Qin; Li, Pin Dong; Li, Feng; Zhang, Wen Jie

2009-12-01

Interleukin-4 (IL-4)-induced Stat6 activities (phenotypes) vary among human cancer cells, of which the HT-29 cell line carries an active Stat6(high) phenotype, while Caco-2 carries a defective Stat6(null) phenotype, respectively. Cancer cells with Stat6(high) show resistance to apoptosis and exaggerated metastasis, suggesting the clinical significance of Stat6 phenotypes. We previously showed that Stat6(high) HT-29 cells exhibited low constitutive expression of Stat6-negative regulators SOCS-1 and SHP-1 because of gene hypermethylation. This study further examined the constitutive expression of other closely related SOCS family numbers including SOCS-3, SOCS-5, SOCS-7, and CISH using RT-PCR. Similar to SOCS-1 and SHP-1, Stat6(high) HT-29 cells expressed low constitutive mRNA of SOCS-3, SOCS-7, and CISH than Stat6(null) Caco-2 cells. Interestingly, DNA demethylation using 5-aza-2'-deoxycytidine in HT-29 cells up-regulated mRNA expression of the above genes, indicating a hypermethylation status, which was confirmed by methylation-specific sequencing in selected SOCS-3 gene. Furthermore, defective Stat6(null) Caco-2 exhibited impaired phosphorylation of Stat6 after IL-4 stimulation by flow cytometry, in keeping with the notion of an over-performed negative regulation. The findings that IL-4/Stat6 phenotypes show differential expression of multiple negative regulators suggest a model that a collective force of powerful negative regulators, directly and indirectly, acts on Stat6 activation, which may result in differential Stat6 phenotypes.
Constitutive Activation of the Fission Yeast Pheromone-Responsive Pathway Induces Ectopic Meiosis and Reveals Ste11 as a Mitogen-Activated Protein Kinase Target

DEFF Research Database (Denmark)

Kjærulff, Søren; Lautrup-Larsen, I.; Truelsen, S.

2005-01-01

In the fission yeast Schizosaccharomyces pombe, meiosis normally takes place in diploid zygotes resulting from conjugation of haploid cells. In the present study, we report that the expression of a constitutively activated version of the pheromone-responsive mitogen-activated protein kinase kinase...... found that haploid meiosis was dramatically reduced when Ste11 was mutated to mimic phosphorylation by Pat1. The mutation of two putative MAPK sites in Ste11 also dramatically reduced the level of haploid meiosis, suggesting that Ste11 is a direct target of Spk1. Supporting this, we show that Spk1 can...... interact physically with Ste11 and also phosphorylate the transcription factor in vitro. Finally, we demonstrate that ste11 is required for pheromone-induced G1 arrest. Interestingly, when we mutated Ste11 in the sites for Pat1 and Spk1 phosphorylation simultaneously, the cells could still arrest in G1...
The constitutional momentum of European contract law (II): The DCFR and the European constitutional order

OpenAIRE

Mak, C.

2009-01-01

This paper analyses the potential impact of the recently published Draft Common Frame of Reference for European contract law (DCFR) on the European constitutional process. Looking at the combination of characteristics of codification and aspects of constitutionalism reflected in the DCFR, it is submitted that the further harmonisation of European contract law may contribute to the definition of the European constitutional order both on the institutional level (regarding the forms in which Eur...

Constitutional relevance of atomic energy law

International Nuclear Information System (INIS)

Lettow, S.

1980-01-01

In a decision publicized on December 20, 1979 the German Federal Constitutional Court rejected a claim of unconstitutionality in connection with the licensing procedure of the Muelheim-Kaerlich Nuclear Power Station currently under construction. This constitutes confirmation, by the 1st Department of the Court, of a decision in 1978 by the 2nd Department about the Kalkar fast breeder power plant, in which the peaceful utilization of nuclear energy had been found to be constitutional. However, the new decision by the Federal Constitutional Court particularly emphasizes the constitutional relevance of the rules of procedure under the Atomic Energy Act and their function with respect to the protection of civil rights. (orig.) [de
The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

DEFF Research Database (Denmark)

Yao, Xiao Jie; Vélez Ruiz, Gisselle; Whorton, Matthew R

2009-01-01

G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs...... of an agonist, the beta(2)AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents...... formation of the beta(2)AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the beta(2)AR and the structural basis for ligand efficacy....
Regulation of G Protein-Coupled Receptors by Ubiquitination

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Kamila Skieterska

2017-04-01

Full Text Available G protein-coupled receptors (GPCRs comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.
Constitutional aneuploidy and cancer predisposition.

Science.gov (United States)

Ganmore, Ithamar; Smooha, Gil; Izraeli, Shai

2009-04-15

Constitutional aneuploidies are rare syndromes associated with multiple developmental abnormalities and the alterations in the risk for specific cancers. Acquired somatic chromosomal aneuploidies are the most common genetic aberrations in sporadic cancers. Thus studies of these rare constitutional aneuploidy syndromes are important not only for patient counseling and clinical management, but also for deciphering the mechanisms by which chromosomal aneuploidy affect cancer initiation and progression. Here we review the major constitutional aneuploidy syndromes and suggest some general mechanisms for the associated cancer predisposition.
[Comparative research into the process of forming the theory of constitution in ancient western medicine and that of four trigrams constitution in Korean medicine and contents of two theories of constitution].

Science.gov (United States)

Park, Joo-Hong

2009-06-01

After conducting comparative research into the process of forming the Theory of Constitution in Ancient Western Medicine and that of Four Trigrams Constitution(Sasang Constitution) in Korean Medicine and contents of two Theories of Constitution in terms of medical history, both theories were found to be formed by an interaction between philosophy and medicine, followed by a combination of the two, on a philosophical basis. The Theory of Constitution in Ancient Western Medicine began with the Theory of Four Elements presented by Empedocles, followed by the Theory of Four Humors presented by Hippocrates and the Theory of Four Temperaments by Galenos, forming and developing the Theory of Constitution. After the Middle Ages, there was no significant advance in the Theory of Constitution by modern times ; however, it developed into the theory of constitution type of Kretschmer and others after the 19th century and into the scientific theory of constitution based on genetics presented by Garrod and others early in the 20th century. The Theory of Four Trigrams Constitution began with the Theory of Constitution in Huangdi Neijing, followed by developments and influences of existing medicine called beginning, restoration, and revival periods and DongeuisoosebowonSaSangChoBonGwon based on the original philosophy of Four Trigrams presented by Lee Je-ma, which is found in GyeokChiGo, DongMuYuGo and so on, ultimately forming and developing into the Theory of Four Trigrams Constitution in Dongeuisoosebowon. Recently, a lot of research is being conducted into making it objective in order to achieve reproducibility in diagnosis and so forth of Four Trigrams Constitution.
Constitutional aneuploidy and cancer predisposition†

Science.gov (United States)

Ganmore, Ithamar; Smooha, Gil; Izraeli, Shai

2009-01-01

Constitutional aneuploidies are rare syndromes associated with multiple developmental abnormalities and the alterations in the risk for specific cancers. Acquired somatic chromosomal aneuploidies are the most common genetic aberrations in sporadic cancers. Thus studies of these rare constitutional aneuploidy syndromes are important not only for patient counseling and clinical management, but also for deciphering the mechanisms by which chromosomal aneuploidy affect cancer initiation and progression. Here we review the major constitutional aneuploidy syndromes and suggest some general mechanisms for the associated cancer predisposition. PMID:19297405
Culture (and religion in constitutional adjudication

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C Rautenbach

2003-10-01

Full Text Available The faculty of law of the Potchefstroom University for Christian Higher Education in corroboration with the Konrad-Adenauer-Stifttung embarked on a study on Politics, Socio-Economic Issues and Culture in Constitutional Adjudication. The aim of the project is twofold. The first aim is to analyse the influence of political, socio-economic and cultural considerations on the constitutional court’s interpretation and application of the Bill of Rights. The second aim is to develop practical guidelines (based on the findings during the analysing process for South African courts confronted with issues of a political, socio-economic and cultural nature. This article is concerned with initiating discussions of the decisions of the constitutional court with regard to cultural and religious rights.Before we can explore the role of political, socio-economic and cultural (and religious rights in the decisions of the constitutional court it is important to discuss a few preliminary issues. In this article the meaning of culture and religion within the South African context receives some attention. Secondly, some preliminary comments regarding constitutional protection of culturally and religiously based rights will be made.We are well aware that this is a daunting task, not only in view of the seemingly abysmal gap between the applicable constitutional rights and values enshrined in the 1996 Constitution that, in some instances over centuries, brought about customs and practices within “traditional” communities which, seemingly, infringe on certain constitutional values and rights.
Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression.

Science.gov (United States)

DeSmet, Marsha L; Fleet, James C

2017-10-01

High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH) 2 D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH) 2 D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH) 2 D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (-300bp), an enhancer region at -6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH) 2 D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention. Copyright © 2017 Elsevier Ltd. All rights reserved.
VALUATION IN THE CONSTITUTIONAL ERA

African Journals Online (AJOL)

Brimer

16 ..... stem from the pre-constitutional era, and the constitutional framework and its legitimate reform efforts. A decision on what is just ...... Carroll L Alice's Adventures in Wonderland (Digital Scanning Scituate MA. 2007). Dagan 1999 Va L Rev.
A survey on constitutional justice

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Kheirollah Parvin

2015-05-01

Full Text Available The idea of supervising the conformity of statutory law with constitutional law, is due to necessities rooted in two essential principles: the supremacy of constitution and the hierarchy of the law. Constitution as the supreme law in the sense of status and legal value , is placed at the top of the legal pyramid of every political system and therefore requires a special organization and discipline that will act as the sanction of the principles and the main content incorporated in this legal instrument. this special organization and discipline known as Constitutional justice in legal Literature now and have studing in two main patterns. Firs pattern based on Supervision of courts on rules and other pattern is Apply by Emphasis on role of Political Institutions in Supervision on rules. This two patterns have common purpose but have different backgrounds and methods.
How do the Constitutional Courts decide?

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Pasquale Pasquino

2016-12-01

Full Text Available The purpose of this article is to explore the mode of production of judicial sentences drafted by constitutional courts in Europe. The natural object of study of the constitutional theory is the analysis of this final product of judicial creation of Law by Constitutional Courts. However, the doctrine has not given sufficient attention –from a comparative law perspective– to the mechanisms and procedures that lead to the decisions of these institutions. Thus, this document will classify the different types of decision-making processes in the courts, analyzing the stages that make up the «mode of production», from the study of the decisions of the Supreme Court of the United States, the Constitutional Council of the French Republic, The Constitutional Court of Italy and the Federal Constitutional Court of Germany. At the end of the paper, some conclusions are made about the period of the magistrates, their party affiliation, the temporary restrictions of deliberation and institutional factors such as the number of attendees or the personalization of its members.
Constitutionalism and Development in Nigeria: The 1999 Constitution

African Journals Online (AJOL)

Fr. Ikenga

the 3 arms of government the Executive, Legislative, Judiciary, Public Service and Marketing .... Rotation principle of a presidency position in Nigeria which will cultivate a sense of ... the issues survived inclusion in the 1999 Constitution of the Federal Republic of Nigeria. 13. S. 2 of Decree No. ..... Oxford, 5th Edition p.245.
Overexpression of OLE1 enhances stress tolerance and constitutively activates the MAPK HOG pathway in Saccharomyces cerevisiae.

Science.gov (United States)

Nasution, Olviyani; Lee, Young Mi; Kim, Eunjung; Lee, Yeji; Kim, Wankee; Choi, Wonja

2017-03-01

OLE1 of Saccharomyces cerevisiae encodes the sole and essential Δ-9 desaturase catalyzing the conversion of saturated to unsaturated fatty acids. Upon ectopic overexpression of OLE1 in S. cerevisiae, significant increases in the membrane oleic acid content were observed. OLE1-overexpressing strains displayed enhanced tolerance to various stresses, better proton efflux, lower membrane permeability, and lessened internal hydrogen peroxide content. The OLE1-mediated enhanced stress tolerance was considerably diminished upon deletion of HOG1, which encodes the mitogen-activated protein kinase (MAPK) Hog1 of the high osmolarity glycerol (HOG) pathway. Furthermore, OLE1 overexpression constitutively activated Hog1, which remained in the cytoplasm. Hog1 activation was accomplished through the MAPK kinase kinase (MAPKKK) Ssk2, but not Ste11 and Ssk22, the other MAPKKKs of the HOG pathway. Despite its cytoplasmic location, activated Hog1 was able to activate the expression of its canonical targets, including CTT1, HSP12, and STL1, and further, the cAMP and stress response elements present in the promoter. OLE1 overexpression neither caused nor relieved endoplasmic reticulum stress. Individually or in combination, the physiological and molecular changes caused by OLE1 overexpression may contribute to enhanced tolerance to various types of stress. Biotechnol. Bioeng. 2017;114: 620-631. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
National simple: Constitutionality Analysis of Exclusions Sectorial

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Guilherme Adolfo dos Santos Mendes

2016-06-01

Full Text Available The Constitution defines the favored legal treatment for small businesses without making any explicit exception, including and especially for tax obligations. Nevertheless, all the laws, which have introduced tax benefits guided by this higher provision, have discriminated small companies due to the economic sector of activity. Known as “National Simple” and introduced by the Complementary Law No. 123/06, the current legislation did not extend its benefits to small production units of a number of industries, such as the automotive industry, the passenger transport industry, the energy industry and the industry of manufacture of weapons, beverages and tobacco products. By demonstrating the mistakes of the arguments in favor of such exclusions, the article holds up that none of these provisions meets constitutional standards. Furthermore, based on a critical analysis of the Positive Law, it is shown that the hidden desire behind the exclusions was to keep the economic sectors of high profitability under control of big corporations to the detriment of smaller initiatives.
La légitimité de la Constitution dans la doctrine constitutionnelle japonaise The Legality of the Constitution in the Japanese constitutional Doctrine

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Simon Serverin

2010-07-01

Full Text Available La Constitution japonaise, promulguée en novembre 1946 et entrée en vigueur en mai 1947, pose à la théorie constitutionnelle un certain nombre de problèmes qui ne sont toujours pas résolus. Adoptée sous occupation américaine, rédigée par les services du GHQ dirigés par le général MacArthur, la Constitution a en outre été promulguée comme une simple révision de la Charte impériale de 1889, dite Constitution de Meiji, alors que par les principes démocratiques nouveaux qu’elle instaurait, elle p...
The limits of authority of the Constitutional Court of Bosnia and Herzegovina in the procedure for the assessment of compliance of laws with the Constitution of Bosnia and Herzegovina

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Simović Miodrag N.

2014-01-01

Full Text Available The Constitutional Court of Bosnia and Herzegovina is one of the pillars of rule of law and legal security as well as guarantee for preservation and development of democratic order in the constitutional framework of Bosnia and Herzegovina. It is not legislative, neither executive nor classical court authority, but a special kind of sui generis authority, acting as corrective factor for all three authority branches. In such a situation, the relationship between the Constitutional Court and legislative authority has a special significance, having in mind that legislative authority regulates, primarily through the law, legal order and, thereby, also defines social and political system of one state and that, on the other side, the Constitutional Court ensures that those laws are in accordance with the Constitution of Bosnia and Herzegovina and that, if it finds such a law has gone out of the framework of the Constitution, it may intervene by declaring the whole law or parts of it unconstitutional and put them out of force. Does the Constitutional Court in such a situation takes the role of legislator and what kind of legislator? What if the legislative authority does not comply with the decision of the Constitutional Court? Should Constitutional Court take the role of positive legislator? It is less problematic activity of the Constitutional Court as negative legislator in theory and practice. In such legal situation, the Constitutional Court in its decision finds unconstitutionality of a law provision (or the whole law and eliminates it from legal system generally after expiration of certain period of time when such provisions cease to be valid and the legislator replaces unconstitutional provisions with new ones within set time limit. However, we have a much more problematic situation when the Constitutional Court acts as positive legislation, i.e. when it makes a decision declaring validity of certain provisions of the law or instructing the
Constitutively Active MAVS Inhibits HIV-1 Replication via Type I Interferon Secretion and Induction of HIV-1 Restriction Factors.

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Sachin Gupta

Full Text Available Type I interferon is known to inhibit HIV-1 replication through the induction of interferon stimulated genes (ISG, including a number of HIV-1 restriction factors. To better understand interferon-mediated HIV-1 restriction, we constructed a constitutively active form of the RIG-I adapter protein MAVS. Constitutive MAVS was generated by fusion of full length MAVS to a truncated form of the Epstein Barr virus protein LMP1 (ΔLMP1. Supernatant from ΔLMP1-MAVS-transfected 293T cells contained high levels of type I interferons and inhibited HIV replication in both TZM-bl and primary human CD4+ T cells. Supernatant from ΔLMP1-MAVS-transfected 293T cells also inhibited replication of VSV-G pseudotyped single cycle SIV in TZM-bl cells, suggesting restriction was post-entry and common to both HIV and SIV. Gene array analysis of ΔLMP1-MAVS-transfected 293T cells and trans-activated CD4+ T cells showed significant upregulation of ISG, including previously characterized HIV restriction factors Viperin, Tetherin, MxB, and ISG56. Interferon blockade studies implicated interferon-beta in this response. In addition to direct viral inhibition, ΔLMP1-MAVS markedly enhanced secretion of IFN-β and IL-12p70 by dendritic cells and the activation and maturation of dendritic cells. Based on this immunostimulatory activity, an adenoviral vector (Ad5 expressing ΔLMP1-MAVS was tested as a molecular adjuvant in an HIV vaccine mouse model. Ad5-Gag antigen combined with Ad5-ΔLMP1-MAVS enhanced control of vaccinia-gag replication in a mouse challenge model, with 4/5 animals showing undetectable virus following challenge. Overall, ΔLMP1-MAVS is a promising reagent to inhibit HIV-1 replication in infected tissues and enhance vaccine-mediated immune responses, while avoiding toxicity associated with systemic type I interferon administration.
Constitutive Modelling in Geomechanics Introduction

CERN Document Server

Puzrin, Alexander M

2012-01-01

The purpose of this book is to bridge the gap between the traditional Geomechanics and Numerical Geotechnical Modelling with applications in science and practice. Geomechanics is rarely taught within the rigorous context of Continuum Mechanics and Thermodynamics, while when it comes to Numerical Modelling, commercially available finite elements or finite differences software utilize constitutive relationships within the rigorous framework. As a result, young scientists and engineers have to learn the challenging subject of constitutive modelling from a program manual and often end up with using unrealistic models which violate the Laws of Thermodynamics. The book is introductory, by no means does it claim any completeness and state of the art in such a dynamically developing field as numerical and constitutive modelling of soils. The author gives basic understanding of conventional continuum mechanics approaches to constitutive modelling, which can serve as a foundation for exploring more advanced theories....
PKD1 mediates negative feedback of PI3K/Akt activation in response to G protein-coupled receptors.

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Yang Ni

Full Text Available We examined whether protein kinase D1 (PKD1 mediates negative feeback of PI3K/Akt signaling in intestinal epithelial cells stimulated with G protein-coupled receptor (GPCR agonists. Exposure of intestinal epithelial IEC-18 cells to increasing concentrations of the PKD family inhibitor kb NB 142-70, at concentrations that inhibited PKD1 activation, strikingly potentiated Akt phosphorylation at Thr(308 and Ser(473 in response to the mitogenic GPCR agonist angiotensin II (ANG II. Enhancement of Akt activation by kb NB 142-70 was also evident in cells with other GPCR agonists, including vasopressin and lysophosphatidic acid. Cell treatment with the structurally unrelated PKD family inhibitor CRT0066101 increased Akt phosphorylation as potently as kb NB 142-70 [corrected]. Knockdown of PKD1 with two different siRNAs strikingly enhanced Akt phosphorylation in response to ANG II stimulation in IEC-18 cells. To determine whether treatment with kb NB 142-70 enhances accumulation of phosphatidylinositol (3,4,5-trisphosphate (PIP3 in the plasma membrane, we monitored the redistribution of Akt-pleckstrin homology domain-green fluorescent protein (Akt-PH-GFP in single IEC-18 cells. Exposure to kb NB 142-70 strikingly increased membrane accumulation of Akt-PH-GFP in response to ANG II. The translocation of the PIP3 sensor to the plasma membrane and the phosphorylation of Akt was completed prevented by prior exposure to the class I p110α specific inhibitor A66. ANG II markedly increased the phosphorylation of p85α detected by a PKD motif-specific antibody and enhanced the association of p85α with PTEN. Transgenic mice overexpressing PKD1 showed a reduced phosphorylation of Akt at Ser(473 in intestinal epithelial cells compared to wild type littermates. Collectively these results indicate that PKD1 activation mediates feedback inhibition of PI3K/Akt signaling in intestinal epithelial cells in vitro and in vivo.
Post-Revolution Constitutionalism: The Impact of Drafting Processes on the Constitutional Documents in Tunisia and Egypt

OpenAIRE

El-Sayed, Ahmed

2014-01-01

This paper seeks to address the constitutional paths that followed the Arab awakening in both Tunisia and Egypt. The Tunisian constitutional process, despite some tensions, was largely peaceful and consensual. On the other hand, the process in Egypt of establishing a new constitutional arrangement had been tumultuous with repercussions that are likely to linger on for a protracted period of time. Therefore, despite apparent resemblance in socio-political actors in both countries, (political I...

Non-transactivational, dual pathways for LPA-induced Erk1/2 activation in primary cultures of brown pre-adipocytes

International Nuclear Information System (INIS)

Holmstroem, Therese E.; Mattsson, Charlotte L.; Wang, Yanling; Iakovleva, Irina; Petrovic, Natasa; Nedergaard, Jan

2010-01-01

In many cell types, G-protein-coupled receptor (GPCR)-induced Erk1/2 MAP kinase activation is mediated via receptor tyrosine kinase (RTK) transactivation, in particular via the epidermal growth factor (EGF) receptor. Lysophosphatidic acid (LPA), acting via GPCRs, is a mitogen and MAP kinase activator in many systems, and LPA can regulate adipocyte proliferation. The mechanism by which LPA activates the Erk1/2 MAP kinase is generally accepted to be via EGF receptor transactivation. In primary cultures of brown pre-adipocytes, EGF can induce Erk1/2 activation, which is obligatory and determinant for EGF-induced proliferation of these cells. Therefore, we have here examined whether LPA, via EGF transactivation, can activate Erk1/2 in brown pre-adipocytes. We found that LPA could induce Erk1/2 activation. However, the LPA-induced Erk1/2 activation was independent of transactivation of EGF receptors (or PDGF receptors) in these cells (whereas in transformed HIB-1B brown adipocytes, the LPA-induced Erk1/2 activation indeed proceeded via EGF receptor transactivation). In the brown pre-adipocytes, LPA instead induced Erk1/2 activation via two distinct non-transactivational pathways, one G i -protein dependent, involving PKC and Src activation, the other, a PTX-insensitive pathway, involving PI3K (but not Akt) activation. Earlier studies showing LPA-induced Erk1/2 activation being fully dependent on RTK transactivation have all been performed in cell lines and transfected cells. The present study implies that in non-transformed systems, RTK transactivation may not be involved in the mediation of GPCR-induced Erk1/2 MAP kinase activation.
CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor

Science.gov (United States)

Cherian, Milu T; Lin, Wenwei; Wu, Jing

2015-01-01

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. PMID:25762023
Radioactive waste disposal and constitution

International Nuclear Information System (INIS)

Stober, R.

1983-01-01

The radioactive waste disposal has many dimensions with regard to the constitutional law. The central problem is the corret delimitation between adequate governmental precautions against risks and or the permitted risk which the state can impose on the citizen, and the illegal danger which nobody has to accept. The solution requires to consider all aspects which are relevant to the constitutional law. Therefore, the following analysis deals not only with the constitutional risks and the risks of the nuclear energy, but also with the liberal, overall-economic, social, legal, and democratic aspects of radioactive waste disposal. (HSCH) [de
RECONSTRUCTION THE AUTHORITY OF CONSTITUTIONAL COURT ON IMPEACHMENT PROCESS OF PRESIDENT AND/OR VICE PRESIDENT IN INDONESIAN CONSTITUTIONAL SYSTEM

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Hezron Sabar Rotua Tinambunan

2016-06-01

Full Text Available In the process of impeachment, Constitutional Court has the obligation to give its judgement to House of Representatives’s opinion regarding allegation of violation by the President and/or Vice President. Constitutional Court checks and judges House of Representatives’s opinion on whether or not the President and/or Vice President works fulfill Article 7A of Constitution of Republic Indonesia 1945 (UUD NRI 1945. The inspection done by Constitutional Court is the judicial process whose decision is in the form of justisil. The result of this impeachment process heavily depends on the judgement of People's Consultative Assembly in its plenary meeting which is also a politics forum, where President and/or Vice President could be dismissed or not. Constitutional Court’s judgement does not apply to People's Consultative Assembly, hence, the difference of Constitutional Court and People's Consultative Assembly’s judgement in plenary meeting that is very political by its nature is very likely to happen. Involvement of Constitutional Court in the procss of impeachment is, of course, different in each country. It depends on governance system in that particular country, it also relies on how much authority that is given by Constitution to Constitutional Court in the process of impeachment itself.
The constitutional momentum of European contract law (II): The DCFR and the European constitutional order

NARCIS (Netherlands)

Mak, C.

2009-01-01

This paper analyses the potential impact of the recently published Draft Common Frame of Reference for European contract law (DCFR) on the European constitutional process. Looking at the combination of characteristics of codification and aspects of constitutionalism reflected in the DCFR, it is
Curcumin Modulates the Radiosensitivity of Colorectal Cancer Cells by Suppressing Constitutive and Inducible NF-κB Activity

International Nuclear Information System (INIS)

Sandur, Santosh K.; Deorukhkar, Amit; Pandey, Manoj K.; Pabon, Ana Maria B.S.; Shentu, Shujun; Guha, Sushovan; Aggarwal, Bharat B.; Krishnan, Sunil

2009-01-01

Purpose: Radiation therapy is an integral part of the preoperative treatment of rectal cancers. However, only a minority of patients achieve a complete pathologic response to therapy because of resistance of these tumors to radiation therapy. This resistance may be mediated by constitutively active pro-survival signaling pathways or by inducible/acquired mechanisms in response to radiation therapy. Simultaneous inhibition of these pathways can sensitize these tumors to radiation therapy. Methods and Materials: Human colorectal cancer cells were exposed to clinically relevant doses of gamma rays, and the mechanism of their radioresistance was investigated. We characterized the transcription factor nuclear factor-κB (NF-κB) activation as a mechanism of inducible radioresistance in colorectal cancer and used curcumin, the active ingredient in the yellow spice turmeric, to overcome this resistance. Results: Curcumin inhibited the proliferation and the post-irradiation clonogenic survival of multiple colorectal cancer cell lines. Radiation stimulated NF-κB activity in a dose- and time-dependent manner, whereas curcumin suppressed this radiation-induced NF-κB activation via inhibition of radiation-induced phosphorylation and degradation of inhibitor of κB alpha, inhibition of inhibitor of κB kinase activity, and inhibition of Akt phosphorylation. Curcumin also suppressed NF-κB-regulated gene products (Bcl-2, Bcl-x L , inhibitor of apoptosis protein-2, cyclooxygenase-2, and cyclin D1). Conclusions: Our results suggest that transient inducible NF-κB activation provides a prosurvival response to radiation that may account for development of radioresistance. Curcumin blocks this signaling pathway and potentiates the antitumor effects of radiation therapy.
Light Controls Cytokinin Signaling via Transcriptional Regulation of Constitutively Active Sensor Histidine Kinase CKI1.

Science.gov (United States)

Dobisova, Tereza; Hrdinova, Vendula; Cuesta, Candela; Michlickova, Sarka; Urbankova, Ivana; Hejatkova, Romana; Zadnikova, Petra; Pernisova, Marketa; Benkova, Eva; Hejatko, Jan

2017-05-01

In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL ( LPH ) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 ( CKI1 ), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 ( HY1 ) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph / hy1 - 7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development. © 2017 American Society of Plant Biologists. All Rights Reserved.
The constitutional view

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Roberto Horácio Sá Pereira

2016-05-01

Full Text Available http://dx.doi.org/10.5007/1808-1711.2016v20n2p165 This brief paper is devoted to criticizing the widespread reading of Kant’s first Critique, according to which reference to subject-independent objects is “constituted” by higherorder cognitive abilities (concepts. Let us call this the “constitutional view”. In this paper, I argue that the constitutional reading confuses the un-Kantian problem of how we come to represent objects (which I call the intentionality thesis, with the quite different problem of how we cognize (erkennen (which I call the “cognition thesis” that we do represent objects, that is, things that exist independently of the subject.
Latitudinal Gradients in Induced and Constitutive Resistance against Herbivores.

Science.gov (United States)

Anstett, Daniel N; Chen, Wen; Johnson, Marc T J

2016-08-01

Plants are hypothesized to evolve increased defense against herbivores at lower latitudes, but an increasing number of studies report evidence that contradicts this hypothesis. Few studies have examined the evolution of constitutive and induced resistance along latitudinal gradients. When induction is not considered, underlying patterns of latitudinal clines in resistance can be obscured because plant resistance represents a combination of induced and constitutive resistance, which may show contrasting patterns with latitude. Here, we asked if there are latitudinal gradients in constitutive versus induced resistance by using genotypes of Oenothera biennis (Onagraceae) sampled along an 18° latitudinal gradient. We conducted two bioassay experiments to compare the resistance of plant genotypes against one generalist (Spodoptera exigua) and one specialist (Acanthoscelidius acephalus) herbivore. These insects were assayed on: i) undamaged control plants, ii) plants that had been induced with jasmonic acid, and iii) plants induced with herbivore damage. Additionally, we examined latitudinal gradients of constitutive and induced chemical resistance by measuring the concentrations of total phenolics, the concentration of oxidized phenolics, and the percentage of phenolics that were oxidized. Spodoptera exigua showed lower performance on plants from lower latitudes, whereas A. acephalus showed no latitudinal pattern. Constitutive total phenolics were greater in plants from lower latitudes, but induced plants showed higher total phenolics at higher latitudes. Oxidative activity was greatest at higher latitudes regardless of induction. Overall, both latitude and induction have an impact on different metrics of plant resistance to herbivory. Further studies should consider the effect of induction and herbivore specialization more explicitly, which may help to resolve the controversy in latitudinal gradients in herbivory and defense.
Essential Medicines in National Constitutions: Progress Since 2008.

Science.gov (United States)

Katrina Perehudoff, S; Toebes, Brigit; Hogerzeil, Hans

2016-06-01

A constitutional guarantee of access to essential medicines has been identified as an important indicator of government commitment to the progressive realization of the right to the highest attainable standard of health. The objective of this study was to evaluate provisions on access to essential medicines in national constitutions, to identify comprehensive examples of constitutional text on medicines that can be used as a model for other countries, and to evaluate the evolution of constitutional medicines-related rights since 2008. Relevant articles were selected from an inventory of constitutional texts from WHO member states. References to states' legal obligations under international human rights law were evaluated. Twenty-two constitutions worldwide now oblige governments to protect and/or to fulfill accessibility of, availability of, and/or quality of medicines. Since 2008, state responsibilities to fulfill access to essential medicines have expanded in five constitutions, been maintained in four constitutions, and have regressed in one constitution. Government commitments to essential medicines are an important foundation of health system equity and are included increasingly in state constitutions.
Federal Constitutional Court. Decision of July 8, 1982 (Wyhl)

International Nuclear Information System (INIS)

Anon.

1983-01-01

With the decision of July 8, 1982, the second senate of the Federal Constitutional Court judged the action on constitutional grounds, brought in by the municipality of Sassbach near Kaiserstuhl to achieve annulment of the construction permit for Wyhl nuclear power plant, to be inadmissible and also partly unfounded. This decision was taken unanimously. In its statement the Court explains basic ideas on the applicability of the Basic Law with regard to juristic persons within the purview of public law and activities on their part outside the scope of fulfilment of public tasks, as well as on the compatibility of material regulations of preclusion in administrative procedures with the Basic Law, especially with article 19, sub-section (4) of the Basic Law. The Court decided that a municipality is not in the position to claim the right of property as laid down in article 14, para. (1) no. 1 of the Basic Law, even if it becomes active outside the scope of fulfilment of public tasks. (CB) [de
Unanimous Constitutional Consent and the Immigration Problem

OpenAIRE

Josten, Stefan D.; Zimmermann, Klaus W.

2004-01-01

This paper utilizes the cross-cutting cleavages approach to evaluate the probability of a unanimous constitutional consent and, based on these results, discusses the implications of immigration on an existing constitutional consent. It is shown that previous conclusions of beneficial effects stemming from a multitude of political dimensions for a unanimous constitutional consent crucially depend on the assumption of an extreme mode of intrapersonal compensation of constitutional majority and ...
Analysis on constitution of American college republicans

Directory of Open Access Journals (Sweden)

Minghua Su

2017-02-01

Full Text Available This paper, based on internet survey and comparative analysis, according to the firsthand materials, comprehensively and systematically probes the formation of the constitution form and structure, and analyzes its contents of Constitution of American College Republicans among 15 colleges respectively, which includes the illustration of constitution, membership, personnel, meeting, financial amendment, etc. Finally, this essay analyzes the characteristics of constitution of college republicans and its advantages.
Crushed Salt Constitutive Model

International Nuclear Information System (INIS)

Callahan, G.D.

1999-01-01

The constitutive model used to describe the deformation of crushed salt is presented in this report. Two mechanisms -- dislocation creep and grain boundary diffusional pressure solution -- are combined to form the basis for the constitutive model governing the deformation of crushed salt. The constitutive model is generalized to represent three-dimensional states of stress. Upon complete consolidation, the crushed-salt model reproduces the Multimechanism Deformation (M-D) model typically used for the Waste Isolation Pilot Plant (WIPP) host geological formation salt. New shear consolidation tests are combined with an existing database that includes hydrostatic consolidation and shear consolidation tests conducted on WIPP and southeastern New Mexico salt. Nonlinear least-squares model fitting to the database produced two sets of material parameter values for the model -- one for the shear consolidation tests and one for a combination of the shear and hydrostatic consolidation tests. Using the parameter values determined from the fitted database, the constitutive model is validated against constant strain-rate tests. Shaft seal problems are analyzed to demonstrate model-predicted consolidation of the shaft seal crushed-salt component. Based on the fitting statistics, the ability of the model to predict the test data, and the ability of the model to predict load paths and test data outside of the fitted database, the model appears to capture the creep consolidation behavior of crushed salt reasonably well
Dissecting signaling and functions of adhesion G protein-coupled receptors

DEFF Research Database (Denmark)

Araç, Demet; Aust, Gabriela; Calebiro, Davide

2012-01-01

G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix...... contacts in a variety of organ systems, adhesion-GPCRs are by far the most poorly understood GPCR class. Adhesion-GPCRs possess a unique molecular structure, with extended N-termini containing various adhesion domains. In addition, many adhesion-GPCRs are autoproteolytically cleaved into an N......-terminal fragment (NTF, NT, α-subunit) and C-terminal fragment (CTF, CT, β-subunit) at a conserved GPCR autoproteolysis-inducing (GAIN) domain that contains a GPCR proteolysis site (GPS). These two features distinguish adhesion-GPCRs from other GPCR classes. Though active research on adhesion-GPCRs in diverse areas...
Molecular evolution of a peptide GPCR ligand driven by artificial neural networks.

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Sebastian Bandholtz

Full Text Available Peptide ligands of G protein-coupled receptors constitute valuable natural lead structures for the development of highly selective drugs and high-affinity tools to probe ligand-receptor interaction. Currently, pharmacological and metabolic modification of natural peptides involves either an iterative trial-and-error process based on structure-activity relationships or screening of peptide libraries that contain many structural variants of the native molecule. Here, we present a novel neural network architecture for the improvement of metabolic stability without loss of bioactivity. In this approach the peptide sequence determines the topology of the neural network and each cell corresponds one-to-one to a single amino acid of the peptide chain. Using a training set, the learning algorithm calculated weights for each cell. The resulting network calculated the fitness function in a genetic algorithm to explore the virtual space of all possible peptides. The network training was based on gradient descent techniques which rely on the efficient calculation of the gradient by back-propagation. After three consecutive cycles of sequence design by the neural network, peptide synthesis and bioassay this new approach yielded a ligand with 70fold higher metabolic stability compared to the wild type peptide without loss of the subnanomolar activity in the biological assay. Combining specialized neural networks with an exploration of the combinatorial amino acid sequence space by genetic algorithms represents a novel rational strategy for peptide design and optimization.
The Constitution of the Republic of Estonia

Index Scriptorium Estoniae

2009-01-01

Sisaldab ka: The Constitution of the Republic of Estonia Amendment Act. The Constitution of the Republic of Estonia Implementation Act. Act to Amend the Constitution of the Republic of Estonia for Election of Local Government Councils for Term of Four Years
CHAPTER FOUR LİBERTY AND TURKISH CONSTITUTIONS:

OpenAIRE

FENDOĞLU, Doç.Dr.Hasan Tahsin

2002-01-01

CHAPTER FOUR LIBERTY AND TURKISH CONSTITUTIONS: Doç.Dr.Hasan Tahsin FENDOĞLU ABSTRACT: Turkish Constitution of 1982 is the first and only Turkish Constitution that has a main purpose on strengthening the political power not the liberty or democr...
Ultima Ratio as a Constitutional Principle

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Kaarlo Tuori

2013-01-01

Full Text Available The paper argues the criminal law notion of ultima ratio is an instance of a broader constitutional law principle of proportionality. However, ultima ratio is not the only principle relevant in a constitutional assessment of criminalization. The role of ultima ratio is to impose limitations on criminalization. But constitutional doctrines also exist which call for criminalization and might even be seen as establishing a criminalization obligation. The paper examines three constitutional counter weights to ultima ratio. The first of these is discussed in the context of state constitutions. This is the cluster of the interrelated constitutional doctrines of the horizontal effect of fundamental rights and the protective duty of the state, as well as the understanding of collective security as a basic right. These doctrines are analysed in the light of the praxis of the German Constitutional Court and the Finnish Constitutional Law Committee. The two other constitutional counterweights are discussed at the level of the transnational, European constitution. These are the principles of precaution and effectiveness. Este artículo defiende que el concepto de ultima ratio es una instancia más amplia del principio de proporcionalidad dentro del derecho constitucional. Sin embargo, el ultima ratio no es el único principio relevante en la valoración constitucional de la criminalización. El papel del ultima ratio es imponer límites a la criminalización. Pero también existen doctrinas constitucionales que exigen la criminalización e incluso dan pie a entender que obligan a establecer una pena. El documento examina tres contrapesos constitucionales al ultima ratio. En primer lugar, se analiza en el contexto de las constituciones estatales. Este es el conjunto de las doctrinas constitucionales interrelacionadas entre el efecto horizontal de los derechos fundamentales y el deber de protección del Estado, así como la asunción de la seguridad colectiva
Human Rights in Indonesian Constitutional Amendments

OpenAIRE

Kharlie, Ahmad Tholabi

2013-01-01

Human Rights in Indonesian Constitutional Amendments. Indonesian constitutional amendments incorporated human rights principles into the Constitution of the Republic of Indonesia 1945 (UUD NRI), especially in the second amendment in 2000. Under that amendment, the UUD NRI currently stipulates human rights principles as provided for in the Universal Declaration of Human Rights (UDHR). However, there are some important notes, which at its core is a lack of emphasis on the vision and mission of ...

The right to work in the Constitution of the Republic of Serbia

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Kulidžan Milica Z.

2016-01-01

Full Text Available The right to work is a fundamental human right which is a challenge for every country to accomplish achieve. Importance of guaranteeing the right to work in a constitution of a country does not come into question. Constitutional guarantee of the right to work does not imply the obligation for a country to provide employment for its every citizen, but rather to increase country's activities in order to make the conditions for achieving the right to work, that is, to conduct the policy of full employment. The Constitution of the Republic of Serbia, passed in 2006, continues to guarantee the right to work, which was firstly guaranteed by the Constitutional Law of 1953. Considering the achieved level of recognized human rights, principles and values, the Constitution of the Republic of Serbia of2006 has guaranteed the right to work and other rights that essentially constitute the right to work from a wider perspective, such as the right to limited working hours, safe and healthy working conditions, the right to daily, weekly interval for rest and a paid annual holiday, the right to a fair remuneration for work done and a legal protection in case of termination of working relations. The main characteristics of guaranteed right to work are the right to choose one's occupation freely and the equal opportunity for gaining employment under equal conditions.
Generation of Trichoderma atroviride mutants with constitutively activated G protein signaling by using geneticin resistance as selection marker

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Gruber Sabine

2012-11-01

Full Text Available Abstract Background Species of the fungal genus Trichoderma are important industrial producers of cellulases and hemicellulases, but also widely used as biocontrol agents (BCAs in agriculture. In the latter function Trichoderma species stimulate plant growth, induce plant defense and directly antagonize plant pathogenic fungi through their mycoparasitic capabilities. The recent release of the genome sequences of four mycoparasitic Trichoderma species now forms the basis for large-scale genetic manipulations of these important BCAs. Thus far, only a limited number of dominant selection markers, including Hygromycin B resistance (hph and the acetamidase-encoding amdS gene, have been available for transformation of Trichoderma spp. For more extensive functional genomics studies the utilization of additional dominant markers will be essential. Results We established the Escherichia coli neomycin phosphotransferase II-encoding nptII gene as a novel selectable marker for the transformation of Trichoderma atroviride conferring geneticin resistance. The nptII marker cassette was stably integrated into the fungal genome and transformants exhibited unaltered phenotypes compared to the wild-type. Co-transformation of T. atroviride with nptII and a constitutively activated version of the Gα subunit-encoding tga3 gene (tga3Q207L resulted in a high number of mitotically stable, geneticin-resistant transformants. Further analyses revealed a co-transformation frequency of 68% with 15 transformants having additionally integrated tga3Q207L into their genome. Constitutive activation of the Tga3-mediated signaling pathway resulted in increased vegetative growth and an enhanced ability to antagonize plant pathogenic host fungi. Conclusion The neomycin phosphotransferase II-encoding nptII gene from Escherichia coli proved to be a valuable tool for conferring geneticin resistance to the filamentous fungus T. atroviride thereby contributing to an enhanced genetic
Resveratrol suppresses constitutive activation of AKT via generation of ROS and induces apoptosis in diffuse large B cell lymphoma cell lines.

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Azhar R Hussain

Full Text Available BACKGROUND: We have recently shown that deregulation PI3-kinase/AKT survival pathway plays an important role in pathogenesis of diffuse large B cell lymphoma (DLBCL. In an attempt to identify newer therapeutic agents, we investigated the role of Resveratrol (trans-3,4', 5-trihydroxystilbene, a naturally occurring polyphenolic compound on a panel of diffuse large B-cell lymphoma (DLBCL cells in causing inhibition of cell viability and inducing apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the action of Resveratrol on DLBCL cells and found that Resveratrol inhibited cell viability and induced apoptosis by inhibition of constitutively activated AKT and its downstream targets via generation of reactive oxygen species (ROS. Simultaneously, Resveratrol treatment of DLBCL cell lines also caused ROS dependent upregulation of DR5; and interestingly, co-treatment of DLBCL with sub-toxic doses of TRAIL and Resveratrol synergistically induced apoptosis via utilizing DR5, on the other hand, gene silencing of DR5 abolished this effect. CONCLUSION/SIGNIFICANCE: Altogether, these data suggest that Resveratrol acts as a suppressor of AKT/PKB pathway leading to apoptosis via generation of ROS and at the same time primes DLBCL cells via up-regulation of DR5 to TRAIL-mediated apoptosis. These data raise the possibility that Resveratrol may have a future therapeutic role in DLBCL and possibly other malignancies with constitutive activation of the AKT/PKB pathway.
The Constitution of the Republic of Estonia

Index Scriptorium Estoniae

2005-01-01

Raamat sisaldab ka: The Constitution of the Republic of Estonia amendment act ; The Constitution of the Republic of Estonia implementation act ; Act to amend the Constitution of the Republic of Estonia for election of local government councils for term of four years
Urgensi Pengaturan Perkara Constitutional Complaint Dalam Kewenangan Mahkamah Konstitusi

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Rahmat Muhajir Nugroho

2016-02-01

Full Text Available This study aimed to evaluate the mechanism for settling disputes in the Constitutional Court containing elements of constitutional complaint. In particular, this study focused on two things: first assessing the urgency/ importance of the idea of setting constitutional complaint in the Constitutional Court's authority. Secondly, to formulate recommendations on setting constitutional complaint in the Constitutional Court's authority. The method used in this research is descriptive qualitative. This type of research is a doctrinal law. This research studied the concept and implementation of the judicial settlement of cases that contain elements of constitutional complaint (complaint constitution by the Constitutional Court. The conclusion of this study is an important constitutional complaint mechanism is contained within the competence of the Constitutional Court to solve problems of injustice experienced by citizens as a result of public policies undertaken by the government in a broad sense, namely the executive, legislature and judiciary. Setting constitutional complaint within the competence of the Constitutional Court does not have to be explicitly stipulated in the constitution, but enough in the explanation of the Constitutional Court Act. That is not to add direct authority of the Court, but expand the meaning of the authority of the Court in testing the law.
Childrens' Rights in the South African Constitution

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JA Robinson

2003-10-01

Full Text Available Children were in many respects defenceless victims of discriminatory practices in ‘apartheid South Africa’. In fact, discrimination on the basis of gender, race and other inequalities were inscribed in the social fabric of the previous constitutional dispensation. The constitutional dispensation that came into effect on the 27th April 1994 was therefore designed to innovate social, political and legal structures that would be radically different from those of the country’s past history.In this contribution the impact of the Constitution upon the rights of children are considered. In order to fathom the impact. a general overview of constitutional principles and provisions necessary for the comprehension of the rights of children is provided. Thereafter the rights of children expressly mentioned in the Constitution will be addressed. Attention is also paid to the equal protection and non-discrimination provisions of the Constitution, albeit only indirectly.
CINPA1 is an inhibitor of constitutive androstane receptor that does not activate pregnane X receptor.

Science.gov (United States)

Cherian, Milu T; Lin, Wenwei; Wu, Jing; Chen, Taosheng

2015-05-01

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
Constitutional Conservatism

Science.gov (United States)

Berkowitz, Peter

2009-01-01

After their dismal performance in election 2008, conservatives are taking stock. As they examine the causes that have driven them into the political wilderness and as they explore paths out, they should also take heart. After all, election 2008 shows that America's constitutional order is working as designed. Indeed, while sorting out their errors…
The constitutional control system in Colombia

OpenAIRE

Luis Javier Moreno Ortiz

2010-01-01

The system of constitutional control in Colombia designed in the Legislative Act n.º 3 of 1910 is a milestone in a long and fruitful political and constitutional tradition owes much to his Hispanic roots and its American developments. Both the public action of unconstitutionality as the plea of unconstitutionality have clear precedents in the constitutionalism of Spain and the Colony were prepared by a Constituent Assembly acted with knowledge and faithfulness to that tradition and have been ...
Receptor oligomerization in family B1 of G-protein-coupled receptors

DEFF Research Database (Denmark)

Roed, Sarah Norklit; Ørgaard, Anne; Jørgensen, Rasmus

2012-01-01

, the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this......, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality......The superfamily of the seven transmembrane G-protein-coupled receptors (7TM/GPCRs) is the largest family of membrane-associated receptors. GPCRs are involved in the pathophysiology of numerous human diseases, and they constitute an estimated 30-40% of all drug targets. During the last two decades...
Constitutive Effects of Performance Indicators

DEFF Research Database (Denmark)

Dahler-Larsen, Peter

2014-01-01

that are demonstrably problematic. Based on a distinction between trivial and advanced measure fixation, an argument is made for constitutive effects that are based on less problematic assumptions. Through this conceptual move, the political dimension of performance indicators is appreciated. The conceptual dimensions...... of constitutive effects are carved out, empirical illustrations of their applicability are offered and implications discussed....
Constitutive law for thermally-activated plasticity of recrystallized tungsten

Science.gov (United States)

Zinovev, Aleksandr; Terentyev, Dmitry; Dubinko, Andrii; Delannay, Laurent

2017-12-01

A physically-based constitutive law relevant for ITER-specification tungsten grade in as-recrystallized state is proposed. The material demonstrates stages III and IV of the plastic deformation, in which hardening rate does not drop to zero with the increase of applied stress. Despite the classical Kocks-Mecking model, valid at stage III, the strain hardening asymptotically decreases resembling a hyperbolic function. The material parameters are fitted by relying on tensile test data and by requiring that the strain and stress at the onset of diffuse necking (uniform elongation and ultimate tensile strength correspondingly) as well as the yield stress be reproduced. The model is then validated in the temperature range 300-600 °C with the help of finite element analysis of tensile tests which confirms the reproducibility of the experimental engineering curves up to the onset of diffuse necking, beyond which the development of ductile damage accelerates the material failure. This temperature range represents the low temperature application window for tungsten as divertor material in fusion reactor ITER.
STAT3-mediated constitutive expression of SOCS-3 in cutaneous T-cell lymphoma

DEFF Research Database (Denmark)

Brender, C; Nielsen, M; Kaltoft, K

2001-01-01

) obtained from affected skin from a patient with mycosis fungoides (MF) and from peripheral blood from a patient with Sezary syndrome (SS). In contrast, constitutive SOCS-3 expression is not found in the leukemic Jurkat T-cell line, the MOLT-4 acute lymphoblastic leukemia cell line, and the monocytic......, it has been hypothesized that an aberrant SOCS expression plays a role in neoplastic transformation. This study reports on a constitutive SOCS-3 expression in cutaneous T-cell lymphoma (CTCL) cell lines. SOCS-3 protein is constitutively expressed in tumor cell lines (but not in nonmalignant T cells...... leukemic cell line U937. Expression of SOCS-3 coincides with a constitutive activation of STAT3 in CTCL tumor cells, and stable transfection of CTCL tumor cells with a dominant negative STAT3 strongly inhibits SOCS-3 expression, whereas transfection with wild-type STAT3 does not. Moreover, the reduced SOCS...
Constitutional compatibility of energy systems

International Nuclear Information System (INIS)

Rossnagel, A.

1983-01-01

The paper starts from the results of the Enquiry Commission on 'Future Nuclear Energy Policy' of the 8th Federal German Parliament outlining technically feasible energy futures in four 'pathways'. For the purpose of the project, which was to establish the comparative advantages and disadvantages of different energy systems, these four scenarios were reduced to two alternatives: cases K (= nuclear energy) and S (= solar energy). The question to Ge put is: Which changes within our legal system will be ushered in by certain technological developments and how do these changes relate to the legal condition intended so far. Proceeding in this manner will not lead to the result of a nuclear energy system or a solar energy system being in conformity or in contradiction with the constitutional law, but will provide a catalogue of implications orientated to the aims of legal standards: a person deciding in favour of a nuclear energy system or a solar energy system supports this or that development of constitutional policy, and a person purishing this or that aim of legal policy should be consistent and decide in favour of this or that energy system. The investigation of constitutional compatibility leads to the question what effects different energy systems will have on the forms of political intercourse laid down in the constitutional law, which are orientated to models of a liberal constitutional tradition of citizens. (orig./HSCH) [de
The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis

DEFF Research Database (Denmark)

Waldhoer, Maria; Casarosa, Paola; Rosenkilde, Mette M

2003-01-01

are separable entities in this viral chemokine receptor. We generated chimeric and mutant US28 proteins that were altered in either their constitutive endocytic (US28 Delta 300, US28 Delta 317, US28-NK1-ctail, and US28-ORF74-ctail) or signaling properties (US28R129A). By using this series of mutants, we show...... further show that the constitutive endocytic property of US28 affects the action of its chemokine ligand fractalkine/CX3CL1 and show that in the absence of the US28 C terminus, fractalkine/CX3CL1 acts as an agonist on US28. This demonstrates for the first time that the endocytic properties of a 7TM......US28 is one of four 7 transmembrane (7TM) chemokine receptors encoded by human cytomegalovirus and has been shown to both signal and endocytose in a ligand-independent, constitutively active manner. Here we show that the constitutive activity and constitutive endocytosis properties of US28...
On the constitutive law of environment assisted fatigue: The physical meaning of the Paris type equations. Pt. 1

International Nuclear Information System (INIS)

Krausz, K.; Wu Xijia; Krausz, A.S.; Lian Zhiwen

1992-01-01

Environment assisted fatigue crack growth is a complex of thermally activated processes. Accordingly, the framework for the expression of rational constitutive law is developed from fracture kinetics theory. The correlation of the constitutive law with the Paris equation is discussed and the empirical parameters in the Paris equation are expressed explicitly in terms of activation energy, stress intensity factor range, temperature, stress ratio, and other physically rigorous engineering quantities. The theory assures and facilitates, the rigorous quantitative evaluation of the effects of the microstructure: the constitutive law gives guidance to its measurement and expresses it in terms of energy-related quantities. (orig.) [de
Inverse agonism of SQ 29,548 and Ramatroban on Thromboxane A2 receptor.

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Raja Chakraborty

Full Text Available G protein-coupled receptors (GPCRs show some level of basal activity even in the absence of an agonist, a phenomenon referred to as constitutive activity. Such constitutive activity in GPCRs is known to have important pathophysiological roles in human disease. The thromboxane A2 receptor (TP is a GPCR that promotes thrombosis in response to binding of the prostanoid, thromboxane A2. TP dysfunction is widely implicated in pathophysiological conditions such as bleeding disorders, hypertension and cardiovascular disease. Recently, we reported the characterization of a few constitutively active mutants (CAMs in TP, including a genetic variant A160T. Using these CAMs as reporters, we now test the inverse agonist properties of known antagonists of TP, SQ 29,548, Ramatroban, L-670596 and Diclofenac, in HEK293T cells. Interestingly, SQ 29,548 reduced the basal activity of both, WT-TP and the CAMs while Ramatroban was able to reduce the basal activity of only the CAMs. Diclofenac and L-670596 showed no statistically significant reduction in basal activity of WT-TP or CAMs. To investigate the role of these compounds on human platelet function, we tested their effects on human megakaryocyte based system for platelet activation. Both SQ 29,548 and Ramatroban reduced the platelet hyperactivity of the A160T genetic variant. Taken together, our results suggest that SQ 29,548 and Ramatroban are inverse agonists for TP, whereas, L-670596 and Diclofenac are neutral antagonists. Our findings have important therapeutic applications in the treatment of TP mediated pathophysiological conditions.
Constitutionalism and good governance in Nigeria (1999-2014 ...

African Journals Online (AJOL)

While the country, at present, has a written constitution which in reality, was derived from a military decree, there has been constant debate as to whether what the country has is really a constitution and whether the subsequent civilian regimes are constitutional governments. This paper attempts to analyse the constitutional ...
75 FR 57835 - Constitution Day and Citizenship Day, Constitution Week, 2010

Science.gov (United States)

2010-09-22

... four short months, delegates to the Constitutional Convention in Philadelphia established a... government, a protector of liberties, and a guarantee that we are all free to shape our own destiny. As we...
Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

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Yuichi Ikeda

Full Text Available Identification of cognate ligands for G protein-coupled receptors (GPCRs provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS. In a reporter cell, complementary fragments of β-lactamase (α and ω were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω, and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3. We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR and neuromedin B receptor (NMBR. Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

Power: Constitutional Update. Bar/School Partnership Programs Series.

Science.gov (United States)

American Bar Association, Chicago, IL. Special Committee on Youth Education for Citizenship.

The fourth in a special series of handbooks dealing with constitutional themes, this document looks at power in the context of the U.S. Constitution. "The Constitution's Prescription for Freedom" (L. Peach) examines the separation of powers provided for in the Constitution. "The Concept of Power" (C. Roach) is a series of…
Spatiotemporal Control of GPR37 Signaling and Its Behavioral Effects by Optogenetics

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Wu Zheng

2018-03-01

Full Text Available Despite the progress in deorphanization of G Protein-Coupled Receptors (GPCRs, ≈100 GPCRs are still classified as orphan receptors without identified endogenous ligands and with unknown physiological functions. The lack of endogenous ligands triggering GPCR signaling has hampered the study of orphan GPCR functions. Using GPR37 as an example, we provide here the first demonstration of the channelrhodopsin 2 (ChR2-GPCR approach to bypass the endogenous ligand and selectively activate the orphan GPCR signal by optogenetics. Inspired by the opto-XR approach, we designed the ChR2-GPR37 chimera, in which the corresponding parts of GPR37 replaced the intracellular portions of ChR2. We showed that optogenetic activation of ChR2/opto-GPR37 elicited specific GPR37 signaling, as evidenced by reduced cAMP level, enhanced ERK phosphorylation and increased motor activity, confirming the specificity of opto-GPR37 signaling. Besides, optogenetic activation of opto-GPR37 uncovered novel aspects of GPR37 signaling (such as IP-3 signaling and anxiety-related behavior. Optogenetic activation of opto-GPR37 permits the causal analysis of GPR37 activity in the defined cells and behavioral responses of freely moving animals. Importantly, given the evolutionarily conserved seven-helix transmembrane structures of ChR2 and orphan GPCRs, we propose that opto-GPR37 approach can be readily applied to other orphan GPCRs for their deorphanization in freely moving animals.
The Spanish Constitution, the Constitutional Court and the Catalan Referendum

DEFF Research Database (Denmark)

Abat Ninet, Antoni

2015-01-01

and politeia that Aristotle defined now clashes between two powerful symbolic and romantic phenomena. In the Spanish-Catalan binomial scenario, there are some elements that need to be analysed to obtain a complete picture of the constitutional possibilities to of accommodating a Catalan self...
Separation of powers and constitutional loyalty

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Marieta SAFTA

2013-06-01

Full Text Available The complexity and dynamics of political life leads to developments and reconsiderations in terms of classical theories of constitutional law. Such a process occurs also in the case of separation of powers. Many factors have a bearing on how this theory is currently translated into practice, which requires additional perspectives of analysis in order to develop improved models of cooperation and balance of powers, according to new political realities. This study aims at examining the principle of separation and balance of powers in terms of mutual respect and loyal cooperation between institutions, or, in a broader sense, of constitutional loyalty, an intrinsic value-principle of all constitutions, without which no fundamental law, no matter of how democratic it might be, could function properly2. Based on examination of concrete cases drawn from the case-law of the Constitutional Court of Romania, the study demonstrates that, in lack of constitutional loyalty, the objective pursued by enshrining the principle of separation of powers cannot be achieved effectively, i.e. compliance of public authorities and political actors with constitutional provisions is purely formal and the alleged collaboration between them is a "dialogue of the deaf" at the expense of democracy. The seriousness of the consequences of this type of behaviour requires identification of remedies. What are the limits and what solutions can be identified in this regard are questions that also we aim to answer.
Nuclear energy and the constitutional state

International Nuclear Information System (INIS)

Saladin, P.

1984-01-01

This article puts the main emphasis on the problems of the constitutional principles of democracy, federalism, peaceful living together of peoples and constitutional state, i.e. problems caused by the development of nuclear energy. The fact that these problems are explained by way of the example of Switzerland, does not reduce the validity of the findings also for the German constitutional system, since the problems are identical and comparable. A long-term goal is a state theory which helps to define the aims and tasks of the state under technical, social, economic and cultural conditions of the end of the 20th and perhaps of the 21st century. Nuclear technology challenges the modern Western state and puts to the test the firmness of its legitimacy basis and the efficiency of its principles. It was conceived in a time which is separated from the present by technological revolutions. Safeguarding of humanity is aim and obligation of the modern constitutional state; the constitutional state stipulates the rules of conduct and, if the state remains true to its claim, it sets the procedures and the organization which give due priority order to the development of modern technology. (orig./HSCH) [de
Loading method of core constituting elements

International Nuclear Information System (INIS)

Kasai, Shigeo

1976-01-01

Purpose: To provide a remote-controlled replacing method for core constituting elements in a liquid-metal cooling fast breeder, wherein particularly, the core constituting elements are prevented from being loaded on the core position other than as designated. Constitution: The method comprises a first step which determines a position of a suitable neutron shielding body in order to measure a reference level of complete insertion of the core constituting elements, a second step which inserts a gripper for a fuel exchanger, a third step which decides stroke dimensions of the complete insertion, and a fourth step which discriminates the core constituting elements to begin handling of fuel rods. The method further comprises a fifth step which determines a loading position of fuel rod, and a sixth step which inserts and loads fuel rods into the core. The method still further comprises a seventh step which compares and judges the dimension of loading stroke and the dimension of complete inserting stroke so that when coincided, loading is completed, and when not coincided, loading is not completed and then the cycle of the fourth step is repeated. (Kawakami, Y.)
76 FR 58705 - Constitution Day and Citizenship Day, Constitution Week, 2011

Science.gov (United States)

2011-09-21

... vision, we resolve to stay true to their spirit of patriotism and unity. In remembrance of the signing of... citizenship, recognize the enduring strength of our Constitution, and reaffirm our commitment to the rights...
Constitutional Verbosity and Social Trust

DEFF Research Database (Denmark)

Bjørnskov, Christian; Voigt, Stefan

2014-01-01

A common argument in the trust literature is that high-trust cultures allow efficient commercial contracts to be shorter, covering fewer contingencies. We take this idea to the topic of social contracts. Specifically, we ask whether social trust affects the length and detail of constitutions. Cross......-country estimates suggest that national trust levels are indeed robustly and negatively associated with the length of countries’ constitutions....
The constitutional control system in Colombia

Directory of Open Access Journals (Sweden)

Luis Javier Moreno Ortiz

2010-12-01

Full Text Available The system of constitutional control in Colombia designed in the Legislative Act n.º 3 of 1910 is a milestone in a long and fruitful political and constitutional tradition owes much to his Hispanic roots and its American developments. Both the public action of unconstitutionality as the plea of unconstitutionality have clear precedents in the constitutionalism of Spain and the Colony were prepared by a Constituent Assembly acted with knowledge and faithfulness to that tradition and have been and are institutions of our capital social and democratic state of law.
The negotiation of The Constitution of Romania of 1923 and The Constitution of Romania of 1991

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Ivona-Arina Raef

2012-12-01

Full Text Available In the present study the author is trying to find if there was a negotiation between main parties regarding The Constitution of Romania of 1923 and The Constitution Of Romania of 1991 or if there was a project imposed by one party to the others.
PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior

Science.gov (United States)

Wang, Han; Sieburth, Derek

2013-01-01

Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels. PMID:24086161
PKA controls calcium influx into motor neurons during a rhythmic behavior.

Directory of Open Access Journals (Sweden)

Han Wang

Full Text Available Cyclic adenosine monophosphate (cAMP has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.
Constitutive androstane receptor activation promotes bilirubin clearance in a murine model of alcoholic liver disease.

Science.gov (United States)

Wang, Xiuyan; Zheng, Liyu; Wu, Jinming; Tang, Binbin; Zhang, Mengqin; Zhu, Debin; Lin, Xianfan

2017-06-01

Increased plasma levels of bilirubin have been reported in rat models and patients with alcoholic liver disease (ALD). The constitutive androstane receptor (CAR) is a known xenobiotic receptor, which induces the detoxification and transport of bilirubin. In the present study, the bilirubin transport regulatory mechanisms, and the role of CAR activation in hepatic and extrahepatic bilirubin clearance were investigated in a murine model of ALD. The mice were fed a Lieber-DeCarli ethanol diet or an isocaloric control diet for 4 weeks, followed by the administration of CAR agonists, 1,4-bis-[2‑(3,5-dichlorpyridyloxy)]benzene (TCPOBOP) and phenobarbital (PB), and their vehicles to examine the effect of the pharmacological activation of CAR on serum levels of bilirubin and on the bilirubin clearance pathway in ALD by serological survey, western blotting and reverse transcription‑quantitative polymerase chain reaction. The results showed that chronic ethanol ingestion impaired the nuclear translocation of CAR, which was accompanied by elevated serum levels of bilirubin, suppression of the expression of hepatic and renal organic anion transporting polypeptide (OATP) 1A1 and hepatic multidrug resistance‑associated protein 2 (MRP2), and induction of the expression of UDP-glucuronosyltransferase (UGT) 1A1. The activation of CAR by TCPOBOP and PB resulted in downregulation of the serum levels of bilirubin followed by selective upregulation of the expression levels of OATP1A1, OATP1A4, UGT1A1 and MRP2 in ALD. These results revealed the bilirubin transport regulatory mechanisms and highlighted the importance of CAR in modulating the bilirubin clearance pathway in the ALD mouse model.
Functional importance of the Ala(116)-Pro(136) region in the calcium-sensing receptor. Constitutive activity and inverse agonism in a family C G-protein-coupled receptor

DEFF Research Database (Denmark)

Jensen, Anders A.; Spalding, T A; Burstein, E S

2000-01-01

The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca(2+) have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found......, suppressed the elevated basal response of the constitutively activated Ca/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala(116)-Pro(136) region is of key importance for the maintenance of the inactive conformation of CaR....
Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs.

Science.gov (United States)

Navarro, Gemma; Cordomí, Arnau; Zelman-Femiak, Monika; Brugarolas, Marc; Moreno, Estefania; Aguinaga, David; Perez-Benito, Laura; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Pardo, Leonardo; García-Sáez, Ana J; McCormick, Peter J; Franco, Rafael

2016-04-05

G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.
G Protein-Coupled Receptor-G-Protein βγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

Science.gov (United States)

Kamal, Fadia A; Travers, Joshua G; Schafer, Allison E; Ma, Qing; Devarajan, Prasad; Blaxall, Burns C

2017-01-01

Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein βγ-subunit (Gβγ), GPCR-kinase 2, and β-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gβγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gβγ signaling and ET system expression. Notably, systemic pharmacologic Gβγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gβγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gβγ in AKI and suggest GPCR-Gβγ inhibition as a novel therapeutic approach for treating CRS2 and AKI. Copyright © 2016 by the American Society of Nephrology.
Optodynamic simulation of β-adrenergic receptor signalling.

Science.gov (United States)

Siuda, Edward R; McCall, Jordan G; Al-Hasani, Ream; Shin, Gunchul; Il Park, Sung; Schmidt, Martin J; Anderson, Sonya L; Planer, William J; Rogers, John A; Bruchas, Michael R

2015-09-28

Optogenetics has provided a revolutionary approach to dissecting biological phenomena. However, the generation and use of optically active GPCRs in these contexts is limited and it is unclear how well an opsin-chimera GPCR might mimic endogenous receptor activity. Here we show that a chimeric rhodopsin/β2 adrenergic receptor (opto-β2AR) is similar in dynamics to endogenous β2AR in terms of: cAMP generation, MAP kinase activation and receptor internalization. In addition, we develop and characterize a novel toolset of optically active, functionally selective GPCRs that can bias intracellular signalling cascades towards either G-protein or arrestin-mediated cAMP and MAP kinase pathways. Finally, we show how photoactivation of opto-β2AR in vivo modulates neuronal activity and induces anxiety-like behavioural states in both fiber-tethered and wireless, freely moving animals when expressed in brain regions known to contain β2ARs. These new GPCR approaches enhance the utility of optogenetics and allow for discrete spatiotemporal control of GPCR signalling in vitro and in vivo.
Elastic-plastic constitutive modeling of concrete

International Nuclear Information System (INIS)

Takahashi, Y.

1983-03-01

The need to understand concrete behavior under high temperatures in the nuclear industry has become rather accute. For this purpose, a constitutive model of concrete especially developed for this severe environment is indispensable. This report reviews the presently available constitutive models of concrete at standard-temperature conditions and considers their advantages and drawbacks. A rather simple but effective approach is selected to treat concrete behavior at high temperatures. Special emphasis is devoted to the modeling of concrete up to and including failure. The derived constitutive model is checked with biaxial and triaxial benchmark experimental results. Very good agreement is obtained
Atypical Rulings of the Indonesian Constitutional Court

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Bisariyadi

2016-08-01

Full Text Available In deciding judicial review cases, the Court may issue rulings that is not in accordance to what is stipulated in the Constitutional Court Law (Law Number 8 Year 2011. Atypical rulings means that the court may reconstruct a provision, delay the legislation/rulings enactment or give instruction to lawmakers. In addition, the court also introduce the “conditionally (unconstitutional” concept. This essay attempts to identify and classify these atypical rulings, including conditionally (un constitutional rulings, by examined the constitutional court judicial review rulings from 2003 to 2015. This study will provide a ground work for advance research on typical rulings by the Indonesian constitutional court.
GPCR Interaction: 80 [GRIPDB[Archive

Lifescience Database Archive (English)

Full Text Available o permit dimerization, and the corresponding monomers were characterized in a fun...ctional melanocyte dispersion assay. Peptide dimers have enhanced functional activity; monospecific dimers h

Screening for MPL mutations in essential thrombocythemia and primary myelofibrosis: normal Mpl expression and absence of constitutive STAT3 and STAT5 activation in MPLW515L-positive platelets.

Science.gov (United States)

Glembotsky, Ana C; Korin, Laura; Lev, Paola R; Chazarreta, Carlos D; Marta, Rosana F; Molinas, Felisa C; Heller, Paula G

2010-05-01

To evaluate the frequency of MPL W515L, W515K and S505N mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) and to determine whether MPLW515L leads to impaired Mpl expression, constitutive STAT3 and STAT5 activation and enhanced response to thrombopoietin (TPO). Mutation detection was performed by allele-specific PCR and sequencing. Platelet Mpl expression was evaluated by flow cytometry, immunoblotting and real-time RT-PCR. Activation of STAT3 and STAT5 before and after stimulation with increasing concentrations of TPO was studied by immunoblotting. Plasma TPO was measured by ELISA. MPLW515L was detected in 1 of 100 patients with ET and 1 of 11 with PMF. Platelets from the PMF patient showed 100% mutant allele, which was Mpl surface and total protein expression were normal, and TPO levels were mildly increased in the MPLW515L-positive ET patient, while MPL transcripts did not differ from controls in both MPLW515L-positive patients. Constitutive STAT3 and STAT5 phosphorylation was absent and dose response to TPO-induced phosphorylation was not enhanced. The low frequency of MPL mutations in this cohort is in agreement with previous studies. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for myeloproliferative neoplasms. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models.
Constitution, 5 October 1988.

Science.gov (United States)

1989-01-01

This document contains major provisions of the constitution adopted by Brazil on 5 October 1988. This constitution seeks to promote the welfare of all citizens without discrimination. The equality of all citizens is guaranteed, and the equal rights of women are specifically mentioned. Property rights are also guaranteed and defined. Female inmates are granted the right to remain with their children while breast feeding. Workers are guaranteed a minimum wage, a family allowance for dependents, maternity/paternity leave, specific incentives to protect the labor market for women, retirement benefits, free day care for preschool-age children, pay equity, and equal rights between tenured and sporadically employed workers. Agrarian reform provisions are given, including the authority to expropriate land. Social and economic policies to promote health are called for, and public health services are to be decentralized, to be integrated, and to foster community participation. Pension plan and social assistance provisions are outlined as are duties of the state in regard to education. The amount of money to be dedicated to education is set out, and a national educational plan is called for to achieve such goals as the eradication of illiteracy, the universalization of school attendance, the improvement of instruction, and the provision of vocational training. Specific measures are set out to protect and preserve the environment. Family policy deals with issues of marriage, the definition of a family, divorce, the right to family planning services, and the deterrence of domestic violence. Social protection provisions cover mothers and children, handicapped persons, and protection of minors. Finally, the customs and rights of Indians are protected, with special provisions given to protect land tenure and to protect the rights of Indians in water resource development and prospecting and mining activities.
Constitutional orders in multinational firms

DEFF Research Database (Denmark)

Hull Kristensen, Peer; Morgan, Glenn

Multinationals are faced with the problem of how to coordinate different actors and stop `fiefdoms' emerging that inhibits the achievement of transnational cooperation? We identify this as a problem of `constitutional ordering' in the firm. Drawing on Varieties of Capitalism approaches, we explore...... how multinationals from different contexts seek to create constitutional orders. We argue that the models which exist appear to be destructive of coordination. We explore the implications for MNCs....
Desensitization of menthol-activated cold receptors in lower extremities during local cooling in young women with a cold constitution.

Science.gov (United States)

Yamazaki, Fumio; Sone, Ryoko

2017-03-01

To test the hypothesis that topical menthol-induced reactivity of cold sensation and cutaneous vasoconstriction to local cooling is augmented in individuals with a cold constitution, we examined thermal sensation and cutaneous vasoconstrictor responses at menthol-treated and untreated sites in the legs during local skin cooling in young women complaining of chilliness (C group) and young women with no complaint as a normal control group (N group). During local skin cooling, the sensitivity to cold sensation was greater in the C group than in the N group. The application of menthol enhanced the cold sensation at a low temperature in the N group, but not in the C group. Cutaneous vasoconstrictor responses to local skin cooling were not altered by menthol treatment in either of the two groups. These findings suggest the desensitization of menthol-activated cold receptors in the legs of C group subjects, and a minor role of cold receptor activity in cutaneous vasoconstrictor response to local cooling.
Relationships between Endogenous Plasma Biomarkers of Constitutive Cytochrome P450 3A Activity and Single-Time-Point Oral Midazolam Microdose Phenotype in Healthy Subjects.

Science.gov (United States)

Woolsey, Sarah J; Beaton, Melanie D; Choi, Yun-Hee; Dresser, George K; Gryn, Steven E; Kim, Richard B; Tirona, Rommel G

2016-04-01

Due to high basal interindividual variation in cytochrome P450 3A (CYP3A) activity and susceptibility to drug interactions, there has been interest in the application of efficient probe drug phenotyping strategies, as well as endogenous biomarkers for assessment of in vivo CYP3A activity. The biomarkers 4β-hydroxycholesterol (4βHC) and 6β-hydroxycortisol (6βHCL) are sensitive to CYP3A induction and inhibition. However, their utility for the assessment of constitutive CYP3A activity remains uncertain. We investigated whether endogenous plasma biomarkers (4βHC and 6βHCL) are associated with basal CYP3A metabolic activity in healthy subjects assessed by a convenient single-time-point oral midazolam (MDZ) phenotyping strategy. Plasma 4βHC and 6βHCL metabolic ratios (MRs) were analysed in 51 healthy adult participants. CYP3A activity was determined after administration of an oral MDZ microdose (100 μg). Simple linear and multiple linear regression analyses were performed to assess relationships between MDZ oral clearance, biomarkers and subject covariates. Among study subjects, basal MDZ oral clearance, 4βHC and 6βHCL MRs ranged 6.5-, 10- and 13-fold, respectively. Participant age and alcohol consumption were negatively associated with MDZ oral clearance (p = 0.03 and p = 0.045, respectively), while weight and female sex were associated with lower plasma 4βHC MR (p = 0.0003 and p = 0.032, respectively). Neither 4βHC nor 6βHCL MRs were associated with MDZ oral clearance. Plasma 4βHC and 6βHCL MRs do not relate to MDZ single-time-point metabolic phenotype in the assessment of constitutive CYP3A activity among healthy individuals. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).
Territory in the Constitutional Standards of Unitary States

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Marina V. Markhgeym

2017-06-01

Full Text Available The article is based on the analysis of the constitutions of seven European countries (Albania, Hungary, Greece, Spain, Malta, Poland, Sweden. The research allows to reveal general and specific approaches to consolidation of norms on territories in a state and give the characteristic of the corresponding constitutional norms. Given the authors ' comprehensive approach to the definition of the territory of the state declared constitutional norms were assessed from the perspective of the fundamental principles and constituent elements of the territory. Considering the specifics of the constitutional types of state territories authors suggest typical and variative models and determine the constitutions of unitary states, distinguished by their originality in the declared group of legal relations. The original constitutional language areas associated with the introduction at the state level, these types of areas that are not typical for other countries.
Constitutionalism and Democracy in Contemporary International Community

OpenAIRE

Padjen, Ivan

1992-01-01

Starting from the insight that jurisprudence of legal theory should be concerned primarily with,on the one hand, international law, and, on the other, constitutional developments, the paper; analyzes some prominent conceptions of constitutionalism and democracy in international community and municipal legal orders; formulates a new set of criteria for the analysis of constitutionalism and democracy in international law; and argues that Laswell and McDougal's policy oriented jurisprudence offe...
Constitutive phosphorylation of ATM in lymphoblastoid cell lines from patients with ICF syndrome without downstream kinase activity.

Science.gov (United States)

Goldstine, Jimena V; Nahas, Shareef; Gamo, Kristin; Gartler, Stanley M; Hansen, R Scott; Roelfsema, Jeroen H; Gatti, Richard A; Marahrens, York

2006-04-08

Double strand DNA breaks in the genome lead to the activation of the ataxia-telangiectasia mutated (ATM) kinase in a process that requires ATM autophosphorylation at serine-1981. ATM autophosphorylation only occurs if ATM is previously acetylated by Tip60. The activated ATM kinase phosphorylates proteins involved in arresting the cell cycle, including p53, and in repairing the DNA breaks. Chloroquine treatment and other manipulations that produce chromatin defects in the absence of detectable double strand breaks also trigger ATM phosphorylation and the phosphorylation of p53 in primary human fibroblasts, while other downstream substrates of ATM that are involved in the repair of DNA double strand breaks remain unphosphorylated. This raises the issue of whether ATM is constitutively activated in patients with genetic diseases that display chromatin defects. We examined lymphoblastoid cell lines (LCLs) generated from patients with different types of chromatin disorders: Immunodeficiency, Centromeric instability, Facial anomalies (ICF) syndrome, Coffin Lowry syndrome, Rubinstein Taybi syndrome and Fascioscapulohumeral Muscular Dystrophy. We show that ATM is phosphorylated on serine-1981 in LCLs derived from ICF patients but not from the other syndromes. The phosphorylated ATM in ICF cells did not phosphorylate the downstream targets NBS1, SMC1 and H2AX, all of which require the presence of double strand breaks. We demonstrate that ICF cells respond normally to ionizing radiation, ruling out the possibility that genetic deficiency in ICF cells renders activated ATM incapable of phosphorylating its downstream substrates. Surprisingly, p53 was also not phosphorylated in ICF cells or in chloroquine-treated wild type LCLs. In this regard the response to chromatin-altering agents differs between primary fibroblasts and LCLs. Our findings indicate that although phosphorylation at serine-1981 is essential in the activation of the ATM kinase, serine-1981 phosphorylation is
Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors.

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Shiva Kumar

Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR. Crystal structures of a number of Class B GPCR extracellular domains (ECD bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.
Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists.

Science.gov (United States)

Cheng, Han; Lear-Rooney, Calli M; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W; Olinger, Gene G; Rong, Lijun

2015-10-01

Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious
h5-HT(1B) receptor-mediated constitutive Galphai3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT.

Science.gov (United States)

Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

2003-03-01

1. Serotonin 5-HT(1B) receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5'-0-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding strategy, we characterised Galpha(i3) subunit activation by h5-HT(1B) receptors stably expressed in Chinese hamster ovary (CHO) cells. 2. The agonists, 5-HT, alniditan and BMS181,101, stimulated Galpha(i3), whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT(1B) receptor ligand, S18127, modestly stimulated Galpha(i3) and reversed the actions of both 5-HT and methiothepin. S18127 (1 micro M) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. 3. Isotopic dilution experiments ([(35)S]GTPgammaS versus GTPgammaS) revealed high-affinity [(35)S]GTPgammaS binding to Galpha(i3) subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [(35)S]GTPgammaS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPgammaS for Galpha(i3) subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. 4. h5-HT(1B) receptor-mediated Galpha(i3) subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [(35)S]GTPgammaS binding, basal Galpha(i3) activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Galpha(i3) activation. 5. In conclusion, at h5-HT(1B) receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Galpha(i3), and its reversal by S18127, reveals constitutive activation of this Galpha subunit; (ii) constitutive Galpha(i3) activation can be quantified by isotopic dilution [(35)S]GTPgammaS binding and (iii) decreasing NaCl concentrations enhances Galpha(i3
Youth political participation and gender constitution: a question for developmental psychology

Directory of Open Access Journals (Sweden)

Maria Cláudia Santos Lopes De Oliveira

2014-06-01

Full Text Available The predominant modes of subjectivity in contemporary youth, defined according to consumption, may collaborate for the preponderance of forms of subjective organization not committed with the social and political participation. This paper focuses on discussing the role of political participation to the subjective constitution and citizenship construction of adolescents and youth. The relationship between identity and political commitment are discussed considering two case studies extracted of data of a previous research project in the field of gender diversity. The focus of the analysis is to understand if and how the experience within political activism acts over developmental trajectories and constitution of subjectivity of activists, considering narratives of self-presentation, in interview settings.
Time and constitution of sense

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Pedro Gerardo Acosta

2014-06-01

Full Text Available This article proposes a reflection over our time-consciousness under the Phenomenology of Edmund Husserl. The idea is make a release the key role of the sense constitution like the fundament and development of the ongoing intentionality, a shape that make the possibility to catch sight of the sense of every life situation like conscience experience that displays itself over the time, and open the world of the Phenomenon World, constituted in the flux and flow of our live experience. The immanent time in which the things served in a lived-present inevitably displays to its own immediate-past of retentions, then of commemorations, constituting and enabling, not just the sense of ever present, but the sense of our own past like memory and our future like expectative. This reflection is based and supporter over the text “Phenomenology Lesson of the Internal Time-Consiusness” (Husserl, 2002.
GPCR Interaction: 59 [GRIPDB[Archive

Lifescience Database Archive (English)

Full Text Available required for their anti-HIV-1 activity. 11854425, 14716309, 10725362, 12089144 Point mutation, bioinforma...tics approach BRET Western blot Cross-linking in the presence of agonist, immunoprecipitation, western blot NP_000570.1 ...
Receptor-G Protein Interaction Studied by Bioluminescence Resonance Energy Transfer: Lessons From Protease-Activated Receptor 1

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Mohammed Akli eAYOUB

2012-06-01

Full Text Available Since its development, the bioluminescence resonance energy transfer (BRET approach has been extensively applied to study G protein-coupled receptors (GPCRs in real time and in live cells. One of the major aspects of GPCRs investigated in considerable details is their physical coupling to the heterotrimeric G proteins. As a result, new concepts have emerged, but few questions are still a matter of debate illustrating the complexity of GPCR-G protein interactions and coupling. Here, we summarized the recent advances on our understanding of GPCR-G protein coupling based on BRET approaches and supported by other FRET-based studies. We essentially focused on our recent studies in which we addressed the concept of preassembly versus the agonist-dependent interaction between the protease-activated receptor 1 (PAR1 and its cognate G proteins. We discussed the concept of agonist-induced conformational changes within the preassembled PAR1-G protein complexes as well as the critical question how the multiple coupling of PAR1 with two different G proteins, Gi1 and G12, but also -arrestin 1, can be regulated.
Stability of non-linear constitutive formulations for viscoelastic fluids

CERN Document Server

Siginer, Dennis A

2014-01-01

Stability of Non-linear Constitutive Formulations for Viscoelastic Fluids provides a complete and up-to-date view of the field of constitutive equations for flowing viscoelastic fluids, in particular on their non-linear behavior, the stability of these constitutive equations that is their predictive power, and the impact of these constitutive equations on the dynamics of viscoelastic fluid flow in tubes. This book gives an overall view of the theories and attendant methodologies developed independently of thermodynamic considerations as well as those set within a thermodynamic framework to derive non-linear rheological constitutive equations for viscoelastic fluids. Developments in formulating Maxwell-like constitutive differential equations as well as single integral constitutive formulations are discussed in the light of Hadamard and dissipative type of instabilities.
Cicero and the Mixed Constitution (res publica mixta

Directory of Open Access Journals (Sweden)

Mitja Sadek

2009-12-01

Full Text Available The story of the mixed constitution is the story of the most stable and just constitution. In theory, this is a combination of at least two of the three elementary forms of government (monarchy, aristocracy and democracy, with some advantages that elementary forms may lack. It originated with the deliberation of Greek philosophers, who wanted to draw up a constitution safeguarding against the permanent variation of elementary constitutional forms and against coups d’état. For both Plato and Aristotle, the mixed constitution was, above all, the reflection of a search for balance between the two extreme forms of government, direct (Athenian democracy on the one hand and the exclusion of the people from governing on the other. The Greek theory was applied by the historian Polybius to the traditional tripartite constitution of the Roman republic. In his view, the consuls were monarchic elements, the senate an aristocratic element, and the comitia a democratic one. Cicero’s introduction of the idea of the mixed constitution in De re publica can only be understood in the light of the author’s personal situation and contemporary political circumstances. His political engagement at a time when the republic was gradually transforming into a monarchy aimed at restoring the important role of the nobility, represented by the senate. For Cicero, the mixed constitution was mainly an instrument for restoring the lost balance between the consuls, the senate, and the comitia, a last chance to save the decaying republic. The concluding part of the article addresses Alois Riklin’s recent discussion of the modern reception of the mixed constitution idea, which advances the controversial thesis that the paradigm of power division, the foundation of modern representative democracy, originates directly from the mixed constitution.
Constitutive Activation of the G-Protein Subunit G[alpha]s within Forebrain Neurons Causes PKA-Dependent Alterations in Fear Conditioning and Cortical "Arc" mRNA Expression

Science.gov (United States)

Kelly, Michele P.; Cheung, York-Fong; Favilla, Christopher; Siegel, Steven J.; Kanes, Stephen J.; Houslay, Miles D.; Abel, Ted

2008-01-01

Memory formation requires cAMP signaling; thus, this cascade has been of great interest in the search for cognitive enhancers. Given that medications are administered long-term, we determined the effects of chronically increasing cAMP synthesis in the brain by expressing a constitutively active isoform of the G-protein subunit G[alpha]s…
Evaluation of potential crushed-salt constitutive models

International Nuclear Information System (INIS)

Callahan, G.D.; Loken, M.C.; Sambeek, L.L. Van; Chen, R.; Pfeifle, T.W.; Nieland, J.D.; Hansen, F.D.

1995-12-01

Constitutive models describing the deformation of crushed salt are presented in this report. Ten constitutive models with potential to describe the phenomenological and micromechanical processes for crushed salt were selected from a literature search. Three of these ten constitutive models, termed Sjaardema-Krieg, Zeuch, and Spiers models, were adopted as candidate constitutive models. The candidate constitutive models were generalized in a consistent manner to three-dimensional states of stress and modified to include the effects of temperature, grain size, and moisture content. A database including hydrostatic consolidation and shear consolidation tests conducted on Waste Isolation Pilot Plant and southeastern New Mexico salt was used to determine material parameters for the candidate constitutive models. Nonlinear least-squares model fitting to data from the hydrostatic consolidation tests, the shear consolidation tests, and a combination of the shear and hydrostatic tests produces three sets of material parameter values for the candidate models. The change in material parameter values from test group to test group indicates the empirical nature of the models. To evaluate the predictive capability of the candidate models, each parameter value set was used to predict each of the tests in the database. Based on the fitting statistics and the ability of the models to predict the test data, the Spiers model appeared to perform slightly better than the other two candidate models. The work reported here is a first-of-its kind evaluation of constitutive models for reconsolidation of crushed salt. Questions remain to be answered. Deficiencies in models and databases are identified and recommendations for future work are made. 85 refs
Crushed-salt constitutive model update

International Nuclear Information System (INIS)

Callahan, G.D.; Loken, M.C.; Mellegard, K.D.; Hansen, F.D.

1998-01-01

Modifications to the constitutive model used to describe the deformation of crushed salt are presented in this report. Two mechanisms--dislocation creep and grain boundary diffusional pressure solutioning--defined previously but used separately are combined to form the basis for the constitutive model governing the deformation of crushed salt. The constitutive model is generalized to represent three-dimensional states of stress. New creep consolidation tests are combined with an existing database that includes hydrostatic consolidation and shear consolidation tests conducted on Waste Isolation Pilot Plant and southeastern New Mexico salt to determine material parameters for the constitutive model. Nonlinear least-squares model fitting to data from the shear consolidation tests and a combination of the shear and hydrostatic consolidation tests produced two sets of material parameter values for the model. The change in material parameter values from test group to test group indicates the empirical nature of the model but demonstrates improvement over earlier work with the previous models. Key improvements are the ability to capture lateral strain reversal and better resolve parameter values. To demonstrate the predictive capability of the model, each parameter value set was used to predict each of the tests in the database. Based on the fitting statistics and the ability of the model to predict the test data, the model appears to capture the creep consolidation behavior of crushed salt quite well

Crushed-salt constitutive model update

Energy Technology Data Exchange (ETDEWEB)

Callahan, G.D.; Loken, M.C.; Mellegard, K.D. [RE/SPEC Inc., Rapid City, SD (United States); Hansen, F.D. [Sandia National Labs., Albuquerque, NM (United States)

1998-01-01

Modifications to the constitutive model used to describe the deformation of crushed salt are presented in this report. Two mechanisms--dislocation creep and grain boundary diffusional pressure solutioning--defined previously but used separately are combined to form the basis for the constitutive model governing the deformation of crushed salt. The constitutive model is generalized to represent three-dimensional states of stress. New creep consolidation tests are combined with an existing database that includes hydrostatic consolidation and shear consolidation tests conducted on Waste Isolation Pilot Plant and southeastern New Mexico salt to determine material parameters for the constitutive model. Nonlinear least-squares model fitting to data from the shear consolidation tests and a combination of the shear and hydrostatic consolidation tests produced two sets of material parameter values for the model. The change in material parameter values from test group to test group indicates the empirical nature of the model but demonstrates improvement over earlier work with the previous models. Key improvements are the ability to capture lateral strain reversal and better resolve parameter values. To demonstrate the predictive capability of the model, each parameter value set was used to predict each of the tests in the database. Based on the fitting statistics and the ability of the model to predict the test data, the model appears to capture the creep consolidation behavior of crushed salt quite well.
The Biased G-Protein-Coupled Receptor Agonism Bridges the Gap between the Insulin Receptor and the Metabolic Syndrome

Science.gov (United States)

Liauchonak, Iryna; Dawoud, Fady; Riat, Yatin; Sambi, Manpreet; Jain, Justin; Kalaydina, Regina-Veronicka; Mendonza, Nicole; Bajwa, Komal

2018-01-01

Insulin signaling, as mediated through the insulin receptor (IR), plays a critical role in metabolism. Aberrations in this signaling cascade lead to several pathologies, the majority of which are classified under the umbrella term “metabolic syndrome”. Although many of these pathologies are associated with insulin resistance, the exact mechanisms are not well understood. One area of current interest is the possibility of G-protein-coupled receptors (GPCRs) influencing or regulating IR signaling. This concept is particularly significant, because GPCRs have been shown to participate in cross-talk with the IR. More importantly, GPCR signaling has also been shown to preferentially regulate specific downstream signaling targets through GPCR agonist bias. A novel study recently demonstrated that this GPCR-biased agonism influences the activity of the IR without the presence of insulin. Although GPCR-IR cross-talk has previously been established, the notion that GPCRs can regulate the activation of the IR is particularly significant in relation to metabolic syndrome and other pathologies that develop as a result of alterations in IR signaling. As such, we aim to provide an overview of the physiological and pathophysiological roles of the IR within metabolic syndrome and its related pathologies, including cardiovascular health, gut microflora composition, gastrointestinal tract functioning, polycystic ovarian syndrome, pancreatic cancer, and neurodegenerative disorders. Furthermore, we propose that the GPCR-biased agonism may perhaps mediate some of the downstream signaling effects that further exacerbate these diseases for which the mechanisms are currently not well understood. PMID:29462993
GPCR Interaction: 188 [GRIPDB[Archive

Lifescience Database Archive (English)

Full Text Available ivation are reduced by activation of CXCR4 in advance. In addition, calcium mobilization assays show these cross-desensitization...ion is not responsible for desensitization between these receptors. 18533278 Calcium mobilization assay NP_000903.2 ...
GPCR Interaction: 187 [GRIPDB[Archive

Lifescience Database Archive (English)

Full Text Available tivation are reduced by activation of CXCR4 in advance. In addition, calcium mobilization assays show these cross-desensitization...tion is not responsible for desensitization between these receptors. 18533278 Calcium mobilization assay NP_001008540.1 ...
Human Rights under the Ethiopian Constitution: A Descriptive ...

African Journals Online (AJOL)

This article summarizes human rights under the Ethiopian Constitution (mainly surrounding Chapter 3 of Constitution and related constitutional provisions on human and democratic rights), and forwards some insights. It, inter alia, covers various aspects of the application and interpretation of human rights provisions, ...
Work Towards a (New) Definition of Peace Constitution

OpenAIRE

Gilliam, Jay R.; Jay R., Gilliam

2011-01-01

Current research on peace constitutions generally centers on Japan’s post-World War II pacifist constitution, specifically Article 9 where Japan renounces war, dissolves its military, and vows to work towards peace in the world. In fact, researching peace constitution (or 平和憲法 in Japanese) in books, academic journals, or on the Internet routinely returns only results about Japan and its Article 9. While a substantial body of work exists about Japan’s peace constitution, too often that body of...
Sonora Legislators and their Constitution, 1857-1861

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Zulema Trejo

2010-01-01

Full Text Available This paper describes the members of the Sonora constituent congress (1857-61, and analyzes the debates they held regarding the project for the state's Constitution, which would follow the lines estblished by the 1857 Federal Constitution. It also points out the relations between each legislator's trajectory and politicial affiliation (as far as available sources allow for this, and the proposals he presented during the legislative debates that gave place to the 1861 Constitution of Sonora.
Competition And Antitrust Law In Ecuadorian Constitution

OpenAIRE

Marcelo Marín Sevilla

2013-01-01

This work allows us to establish the Economic Constitution and the Competition Law (C.L) in the Constitution. Additionally, the paper analyzes whether the rules outlined in the C. L. and in doctrine are consistent and appropriate with the Constitution of Ecuador. The Competition and Antitrust Laws has rules for investigating and punishing the cartels, the abuses of power market (dominant position), the rules for merger control, the behaviors of Abuse of economic dependence, and unfair competi...
Complexities of Constitutional Change in the Philippines

OpenAIRE

Saunders, Cheryl; Yusingco, Michael Henry

2018-01-01

President Rodrigo Duterte assumed office in July 2016, His party, PDP-Laban, had campaigned under the slogan: “No to Drugs, Yes to Federalism”. Duterte thus is committed to shepherding the Philippines towards a federal form of government; an undertaking that would require an extensive overhaul of the country’s constitution. The future of constitutional change under Duterte in any event is uncertain for a series of constitutional and political reasons. Critically, some of the most pressing of ...
[Etiologic spectrum of solitary constitutional syndrome].

Science.gov (United States)

Hernández Hernández, J L; Matorras Galán, P; Riancho Moral, J A; González-Macías, J

2002-07-01

To know the spectrum of diseases responsible for the solitary constitutional syndrome in our setting. This syndrome was defined as a clinical picture characterized by the presence of asthenia, anorexia, and weight loss of at least 5% of body weight in the last six months, not associated with any other symptom or sign suggesting the diagnosis of an organ or system disease. All patients diagnosed of the solitary constitutional syndrome (328) in a tertiary-care level teaching hospital between January 1991 and December 1996. Fifty-two (170) percent of patients with solitary constitutional syndrome were males and 48% (158) females. The mean age was 65.4%, ranging from 15 to 97 years. The average of the monthly estimated weight loss was 3 to 4 kilograms. A total of 115 (35%) malignant neoplasms and 5 (1.5%) benign tumors were diagnosed. The most common malignant tumors corresponded to the digestive tract (51.3% of the total malignant tumors). The second cause in frequency of the solitary constitutional syndrome corresponded to psychiatric diseases, with a total of 80 patients (24.3%). A total of 116 non-neoplastic organic diseases were detected, with digestive tract diseases --mainly peptic disease-- being the most common cause in this group. After follow-up, only in twenty cases were we unable to detect the underlying disease responsible for the syndrome. In nine of these, the solitary constitutional syndrome was self-limited. Forty-four percent of patients had at least another concomitant disease and in 24% of patients more than one associated condition was found. The most common diseases responsible for the solitary constitutional syndrome were, by decreasing frequency, malignant tumors, psychiatric disorders, and non-malignant organic diseases located in the digestive tract. A better knowledge of the etiological spectrum of this syndrome might be useful for a more efficient management of these patients.
Constitutional provisions regarding juristic persons | Pienaar ...

African Journals Online (AJOL)

The interim Constitution of 1993 and the final Constitution of 1996 contain specific provisions applicable to juristic persons. Juristic persons are also entitled to the fundamental rights contained in the Bill of Rights to the extent that these rights are applicable to them. It must be kept in mind that juristic persons have peculiar ...
The Constitutional Debate: A One Man Show? Václav Klaus and the Constitutional Discourse in the Czech Republic

Czech Academy of Sciences Publication Activity Database

Rakušanová, Petra

2007-01-01

Roč. 8, č. 8 (2007), s. 342-373 ISSN 1570-5854 Grant - others:VW Stiftung(DE) 218036 Institutional research plan: CEZ:AV0Z70280505 Keywords : European Constitutional Treaty * European constitutional ratification * Czech Republic Subject RIV: AO - Sociology, Demography
Neuronal Functions of Activators of G Protein Signaling

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Man K. Tse

2012-05-01

Full Text Available G protein-coupled receptors (GPCRs are one of the most important gateways for signal transduction across the plasma membrane. Over the past decade, several classes of alternative regulators of G protein signaling have been identified and reported to activate the G proteins independent of the GPCRs. One group of such regulators is the activator of G protein signaling (AGS family which comprises of AGS1-10. They have entirely different activation mechanisms for G proteins as compared to the classic model of GPCR-mediated signaling and confer upon cells new avenues of signal transduction. As GPCRs are widely expressed in our nervous system, it is believed that the AGS family plays a major role in modulating the G protein signaling in neurons. In this article, we will review the current knowledge on AGS proteins in relation to their potential roles in neuronal regulations.
Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

Science.gov (United States)

Jones, Matthew L; Norman, Jane E; Morgan, Neil V; Mundell, Stuart J; Lordkipanidzé, Marie; Lowe, Gillian C; Daly, Martina E; Simpson, Michael A; Drake, Sian; Watson, Steve P; Mumford, Andrew D

2015-04-01

Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) < 0.05 %. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21 %) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF< 1 % and 22 with MAF≥ 1 %). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.
Genesis of the constitutionality and statehood of the Republic of Srpska: Constitutional acts in Bosnia and Herzegovina in 1990 and 1991

Directory of Open Access Journals (Sweden)

Nešković Radomir

2012-01-01

Full Text Available Constitutional (statehood or foundation acts are those on which the establishment of a state is founded. These acts determine its statehood entities (part-owners of the state in which the foundations of state organization are defined. All other authorities (legislative, executive and administrative, and judicial are extracted from constitutional acts which are synthesized in the Constitution as the highest political and legal act. Constitutional acts in Bosnia and Herzegovina in the abovementioned period influenced each other as the domino effect, since one act caused the adoption of another, thus, each state-building act had the cause and consequence feature. Constitutional act of representatives of a nation caused the adoption of a similar act among representatives of another nation, which resulted in the statehood authority losing its unique character and being 'torn apart' into three national state-building authorities. In this text we mention the basic constitutional acts preceding the establishment of the Assembly of the Serb People in Bosnia and Herzegovina.
James Madison's "Public" As Interpreter of the Constitution.

Science.gov (United States)

Dewey, Donald O.

James Madison's thoughts on various interpretations of the Constitution maintain that public opinion is the ultimate method of legitimizing the document. The Constitution must prevail against mere public opinion, but public opinion may be used to establish the meaning of the Constitution when conflicting interpretations exist. The public good and…
A Communicatively Constituted Online Crisis

DEFF Research Database (Denmark)

Valentini, Chiara; Romenti, Stefania; Kruckeberg, Dean

2017-01-01

into specific public crisis perceptions. Drawing from a communicative constitution perspective, the authors argue that if crises are perceptions or experiences of difficult situations that exceed a person’s current resources and coping mechanisms, and if perceptions and experiences in social media are typically...... by offering suggestions on how to study online critical conversations through the lens of a communicative constitution perspective that could inform how critical issues eventually transform and become crises and how crisis perceptions evolve and are discursively shaped by communicative practices occurring...
Isolation and structure–function characterization of a signaling-active rhodopsin–G protein complex

Science.gov (United States)

Gao, Yang; Westfield, Gerwin; Erickson, Jon W.; Cerione, Richard A.; Skiniotis, Georgios; Ramachandran, Sekar

2017-01-01

The visual photo-transduction cascade is a prototypical G protein–coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (GT). This results in the dissociation of GT into its component αT–GTP and β1γ1 subunit complex. Structural information for the Rho*–GT complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (GT*) comprising a GαT/Gαi1 chimera (αT*) and β1γ1. The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to GαT* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one GT*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β2-adrenergic receptor–GS complex, including a flexible αT* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex. PMID:28655769
Health care law versus constitutional law.

Science.gov (United States)

Hall, Mark A

2013-04-01

National Federation of Independent Business v. Sebelius, the Supreme Court's ruling on the Patient Protection and Affordable Care Act, is a landmark decision - both for constitutional law and for health care law and policy. Others will study its implications for constitutional limits on a range of federal powers beyond health care. This article considers to what extent the decision is also about health care law, properly conceived. Under one view, health care law is the subdiscipline that inquires how courts and government actors take account of the special features of medicine that make legal or policy issues especially problematic - rather than regarding health care delivery and finance more generically, like most any other economic or social enterprise. Viewed this way, the opinions from the Court's conservative justices are mainly about general constitutional law principles. In contrast, Justice Ruth Bader Ginsburg's dissenting opinion for the four more liberal justices is just as much about health care law as it is about constitutional law. Her opinion gives detailed attention to the unique features of health care finance and delivery in order to inform her analysis of constitutional precedents and principles. Thus, the Court's multiple opinions give a vivid depiction of the compelling contrasts between communal versus individualistic conceptions of caring for those in need, and between health care and health insurance as ordinary commodities versus ones that merit special economic, social, and legal status.
The Precedent 443 of the Superior Labor Court and Judicial Activism: The Defense of a Non Selective Constitutional Hermeneutics

Directory of Open Access Journals (Sweden)

Humberto Lima de Lucena Filho

2015-12-01

Full Text Available This paper is dedicated to the analysis of the Precedent 443 of the Superior Labor Court, which deals with the discriminatory dismissal presumption of those labor with social stigma diseases from the perspective of judicial activism in the brazilian labor law and its objective influence on law and labor procedure law from the from the perspective of a not univocal constitutional hermeneutics. It aims, also, to conceptualize the stability institute in order to configure the dangers and misconceptions arising from it. Therefore, it concludes that the analogy that embodied the repeated judicial position was excessive. The research points out the logical-deductive method operated by the aid of the literature review, case law and positioning applicable law.

Possibilities for a constitutional embodiment of environment protection

International Nuclear Information System (INIS)

Hattenberger, D.

1991-03-01

The main topic is the question of adequacy and desirability of a constitutional rule for the protection of the environment with regard to the specific Austrian situation and the extensive discussion about this topic in German. As variants are discussed a human right of environment, objective categories of constitutional rules (constitutional aims of state's policy) and procedural or organisational rules. It begins with an explanation of the general responsibility of states in the field of environment protection and a determination of the term 'environment '. In respect of a human right first already guaranteed rights are examined for their relevance in the field of conservation with regard to the change in the understanding of human rights. Then arguments pro and against a specific right to environment are mentioned and appraised. With regard to the counter-arguments a subjective right should not be incorporated. A revision of the present concept of human rights would be necessary. Similar arguments are mentioned against the constitution of an objective norm in respect of some judicial decisions of the Austrian Constitutional Court. The problem of the protection of the environment will not be solved with a constitutional rule. It would raise new problems, which would damage the constitution as a fundamental order of human living together. (author)
Constitutional Provisions on the Press: A World View.

Science.gov (United States)

Paraschos, Manny

A study examined the legal treatment of the press in constitutions or other basic legal institutional documents from around the world. Sixty-three constitutions or basic documents from the Western World, the Communist Bloc, the Middle East, Africa, Southeast Asia, and Latin America were analyzed. Analysis revealed that most constitutions open with…
Theoretical (dis-) position and strategic leitmotivs in constitutional ...

African Journals Online (AJOL)

This essay takes a look at the historic restoration that bequeathed this country and its people a prototypical, justiciable Constitution. The advent of constitutional democracy in South Africa went hand in hand with an about-turn in the interpretation of enacted law-texts (including the Constitution) and a critical interrogation of ...
Unconstitutional constitutional amendments in Ethiopia: the practice ...

African Journals Online (AJOL)

Haramaya Law Review ... The Constitution of the Federal Democratic Republic of Ethiopia (FDRE) under Article 104 and 105 sets ... that sets procedures to be observed in the process of constitutional amendments: both initiation and approval.
New Constitutionalism for Biosiversity vs. Neoconstitutionalism of Risk

Directory of Open Access Journals (Sweden)

Michele Carducci

2016-08-01

Full Text Available http://dx.doi.org/10.5007/2177-7055.2016v37n73p255 Based on an “eco-systemic” democracy that seeks to preserve biodiversity through the recognition of the co-evolutionary link between nature and culture, the Andean Constitutionalism emerges as the expression of a counter-hegemonic constitutionalism committed to the construction of a new institutional framework through the inclusion of new participatory and intercultural mechanisms. Departing from western constitutional paradigms, this groundbreaking constitutionalism revisits the “Gaia hypothesis” and legitimizes a real “social contract” among the people and nature, and instead of considering it as an “object” of ownership, exploitation, or conservation, it regards nature as a legal “subject” and primary source of society itself and the Constitution as its “legal grantor and protector”.
h5-HT1B receptor-mediated constitutive Gαi3-protein activation in stably transfected Chinese hamster ovary cells: an antibody capture assay reveals protean efficacy of 5-HT

Science.gov (United States)

Newman-Tancredi, Adrian; Cussac, Didier; Marini, Laetitia; Touzard, Manuelle; Millan, Mark J

2003-01-01

Serotonin 5-HT1B receptors couple to G-proteins of the Gi/o family. However, their activation of specific G-protein subtypes is poorly characterised. Using an innovative antibody capture/guanosine-5′-0-(3-[35S]thio)-triphosphate ([35S]GTPγS) binding strategy, we characterised Gαi3 subunit activation by h5-HT1B receptors stably expressed in Chinese hamster ovary (CHO) cells. The agonists, 5-HT, alniditan and BMS181,101, stimulated Gαi3, whereas methiothepin and SB224,289 behaved as inverse agonists. The selective 5-HT1B receptor ligand, S18127, modestly stimulated Gαi3 and reversed the actions of both 5-HT and methiothepin. S18127 (1 μM) also produced parallel, dextral shifts of the 5-HT and methiothepin isotherms. Isotopic dilution experiments ([35S]GTPγS versus GTPγS) revealed high-affinity [35S]GTPγS binding to Gαi3 subunits in the absence of receptor ligands indicating constitutive activity. High-affinity [35S]GTPγS binding was increased 2.8-fold by 5-HT with an increase in the affinity of GTPγS for Gαi3 subunits. In contrast, methiothepin halved the number of high-affinity binding sites and decreased their affinity. h5-HT1B receptor-mediated Gαi3 subunit activation was dependent on the concentration of NaCl. At 300 mM, 5-HT stimulated [35S]GTPγS binding, basal Gαi3 activation was low and methiothepin was inactive. In contrast, at 10 mM NaCl, basal activity was enhanced and the inverse agonist activity of methiothepin was accentuated. Under these conditions, 5-HT decreased Gαi3 activation. In conclusion, at h5-HT1B receptors expressed in CHO cells: (i) inverse agonist induced inhibition of Gαi3, and its reversal by S18127, reveals constitutive activation of this Gα subunit; (ii) constitutive Gαi3 activation can be quantified by isotopic dilution [35S]GTPγS binding and (iii) decreasing NaCl concentrations enhances Gαi3 activation and leads to protean agonist properties of 5-HT: that is a switch to inhibition of Gαi3. PMID:12684263
Constitutive activation of Nrf2 induces a stable reductive state in the mouse myocardium

Directory of Open Access Journals (Sweden)

Gobinath Shanmugam

2017-08-01

Full Text Available Redox homeostasis regulates key cellular signaling pathways in both physiology and pathology. The cell's antioxidant response provides a defense against oxidative stress and establishes a redox tone permissive for cell signaling. The molecular regulation of the well-known Keap1/Nrf2 system acts as sensor responding to changes in redox homeostasis and is poorly studied in the heart. Importantly, it is not yet known whether Nrf2 alone can serve as a master regulator of cellular redox homeostasis without compensation of the transcriptional regulation of antioxidant response element (ARE genes through alternate mechanisms. Here, we addressed this question using cardiac-specific transgenic expression at two different levels of constitutively active nuclear erythroid related factor 2 (caNrf2 functioning independently of Keap1. The caNrf2 mice showed augmentation of glutathione (GSH, the key regulator of the cellular thiol redox state. The Trans-AM assay for Nrf2-binding to the antioxidant response element (ARE showed a dose-dependent increase associated with upregulation of several major antioxidant genes and proteins. This was accompanied by a significant decrease in dihydroethidium staining and malondialdehyde (MDA in the caNrf2-TG mice myocardium. Interestingly, caNrf2 gene-dosage dependent redox changes were noted resulting in generation of a multi-stage model of pro-reductive and reductive conditions in the myocardium of TG-low and TG-high mice, respectively. These data clearly show that Nrf2 levels alone are capable of serving as the master regulator of the ARE. These models provide an important platform to investigate the impact of the Nrf2 system independent of the need to regulate the activity of Keap1 and the consequent exposure to pro-oxidants or electrophiles, which have numerous off-target effects.
The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens.

Science.gov (United States)

Ellis, C; Turner, J G

2001-05-01

Jasmonates (JAs) inhibit plant growth and induce plant defense responses. To define genes in the Arabidopsis JA signal pathway, we screened for mutants with constitutive expression of a luciferase reporter for the JA-responsive promoter from the vegetative storage protein gene VSP1. One mutant, named constitutive expression of VSP1 (cev1), produced plants that were smaller than wild type, had stunted roots with long root hairs, accumulated anthocyanin, had constitutive expression of the defense-related genes VSP1, VSP2, Thi2.1, PDF1.2, and CHI-B, and had enhanced resistance to powdery mildew diseases. Genetic evidence indicated that the cev1 phenotype required both COI1, an essential component of the JA signal pathway, and ETR1, which encodes the ethylene receptor. We conclude that cev1 stimulates both the JA and the ethylene signal pathways and that CEV1 regulates an early step in an Arabidopsis defense pathway.
Constitutional and acquired autosomal aneuploidy.

Science.gov (United States)

Jackson-Cook, Colleen

2011-12-01

Chromosomal imbalances can result from numerical or structural anomalies. Numerical chromosomal abnormalities are often referred to as aneuploid conditions. This article focuses on the occurrence of constitutional and acquired autosomal aneuploidy in humans. Topics covered include frequency, mosaicism, phenotypic findings, and etiology. The article concludes with a consideration of anticipated advances that might allow for the development of screening tests and/or lead to improvements in our understanding and management of the role that aneuploidy plays in the aging process and acquisition of age-related and constitutional conditions.
Urgency of Attorney Governed by the Constitution

Directory of Open Access Journals (Sweden)

Rommy Patra

2015-12-01

Full Text Available Attorney existence in the Indonesian constitutional structure has a dilemma for this position. On one side is the Prosecutor’s law enforcement agencies to exercise power independently prosecution while on the other hand is part of a government institution under Law No. 16 of 2004 regarding the Attorney. The position of Attorney as an institution of government has been led to the independence of the Prosecutor is not optimal so that it appears stigma that the Prosecutor merely as a tool of the ruling power. In addition the terms of the arrangement just under the Act, the Attorney General has no legal standing as a constitutional organ that has the constitutional authority so that the current position does not reflect the urgency of its duties and functions. In an effort to organize the next Attorney institutions should be regulated directly by the Constitution. It is intended to make the Attorney as part of the main state organs have the same legal standing as other law enforcement agencies, the police and the courts (Supreme Court and Constitutional Court. As well as to strengthen and clarify the position as a state institution, prosecution authorities are focusing on the Attorney as central of authority, to fix the institutional relations between the members of law enforcement and related agencies and strengthen the independence of the Prosecutor in performing the function of prosecution in the constitutional structure of Indonesia.
The foundational tenets of Johannes Althusius' constitutionalism ...

African Journals Online (AJOL)

Potchefstroom Electronic Law Journal/Potchefstroomse Elektroniese Regsblad ... sovereignty, in contrast to undivided (statist) sovereignty and his views on public office provided the framework for constitutionalism and limited government which could arguably improve on that of contemporary statist constitutionalism.
A constitutive theory of reacting electrolyte mixtures

Science.gov (United States)

Costa Reis, Martina; Wang, Yongqi; Bono Maurizio Sacchi Bassi, Adalberto

2013-11-01

A constitutive theory of reacting electrolyte mixtures is formulated. The intermolecular interactions among the constituents of the mixture are accounted for through additional freedom degrees to each constituent of the mixture. Balance equations for polar reacting continuum mixtures are accordingly formulated and a proper set of constitutive equations is derived with basis in the Müller-Liu formulation of the second law of thermodynamics. Moreover, the non-equilibrium and equilibrium responses of the reacting mixture are investigated in detail by emphasizing the inner and reactive structures of the medium. From the balance laws and constitutive relations, the effects of molecular structure of constituents upon the fluid flow are studied. It is also demonstrated that the local thermodynamic equilibrium state can be reached without imposing that the set of independent constitutive variables is time independent, neither spatially homogeneous nor null. The resulting constitutive relations presented throughout this work are of relevance to many practical applications, such as swelling of clays, developing of bio and polymeric membranes, and use of electrorheological fluids in industrial processes. The first author acknowledges financial support from National Counsel of Technological and Scientific Development (CNPq) and German Academic Exchange Service (DAAD).
GPCR Interaction: 40 [GRIPDB[Archive

Lifescience Database Archive (English)

Full Text Available at similar levels. A significant agonist-promoted internalization of the beta2AR activates ERK1/2 MAPK in be...expressing beta1AR and both receptors. Agonist-promoted internalization of the be...efore, heterodimerization between beta1AR and beta2AR inhibits the agonist-promoted internalization of the b
Nuclear phase-out a part of the German Constitution? On the political grammar of constitutional changes

International Nuclear Information System (INIS)

Gaerditz, Klaus Ferdinand

2016-01-01

The study examines whether and to what extent the simple legal effected nuclear phase-out could be safeguarded in the Basic Law, which regulatory methods would be available, the advantages and disadvantages of these and how their effects would be assessed. In addition to political and practical consequences, this also includes considerations of democracy and constitutional theory in an overall consideration, which ultimately touches the question of the regulatory function of constitutional changes. [de
Constitutive behavior of reconsolidating crushed salt

International Nuclear Information System (INIS)

Callahan, G.D.; Mellegard, K.D.; Hansen, F.D.

1998-02-01

The constitutive model used to describe deformation of crushed salt is presented in this paper. Two mechanisms--dislocation creep and grain boundary diffusional pressure solutioning--are combined to form the basis for the constitutive model governing deformation of crushed salt. The constitutive model is generalized to represent three-dimensional states of stress. Recently completed creep consolidation tests are combined with an existing database that includes hydrostatic consolidation and shear consolidation tests conducted on Waste Isolation Pilot Plant (WIPP) and southeastern New Mexico salt to determine material parameters for the constitutive model. Nonlinear least-squares model fitting to data from shear consolidation tests and a combination of shear and hydrostatic tests produces two sets of material parameter values for the model. Changes in material parameter values from test group to test group indicate the empirical nature of the model but show significant improvement over earlier work. To demonstrate the predictive capability of the model, each parameter value set was used to predict each of the tests in the database. Based on fitting statistics and ability of the model to predict test data, the model appears to capture the creep consolidation behavior of crushed salt quite well
Development of CINPA1 analogs as novel and potent inverse agonists of constitutive androstane receptor

OpenAIRE

Lin, Wenwei; Yang, Lei; Chai, Sergio C.; Lu, Yan; Chen, Taosheng

2015-01-01

Constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2) are master regulators of endobiotic and xenobiotic metabolism and disposition. Because CAR is constitutively active in certain cellular contexts, inhibiting CAR might reduce drug-induced hepatotoxicity and resensitize drug-resistant cancer cells to chemotherapeutic drugs. We recently reported a novel CAR inhibitor/inverse agonist CINPA1 (11). Here, we have obtained or designed 54 analogs of CINPA1 and used a ti...
The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors.

Science.gov (United States)

Bondar, Alexey; Lazar, Josef

2017-06-09

The G i/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of G i/o -GPCR coupling and the high rate of G i/o signal transduction have been hypothesized to be enabled by the existence of stable associates between G i/o proteins and their cognate GPCRs in the inactive state (G i/o -GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gα i1 subunits labeled with fluorescent proteins and four GPCRs: the α 2A -adrenergic receptor, GABA B , cannabinoid receptor type 1 (CB 1 R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gα i1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gα i1 subunits and α 2A -adrenergic receptor, GABA B , or dopamine receptor type 2 receptors did not reveal any interaction between the G i1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB 1 R exhibited an interaction with the G i1 protein. Further experiments revealed that this interaction is caused solely by CB 1 R basal activity; no preassembly between CB 1 R and the G i1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active G i1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and G i1 (basal coupling). These findings suggest that G i1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

Energy Technology Data Exchange (ETDEWEB)

Lynch, Caitlin; Pan, Yongmei; Li, Linhao [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Heyward, Scott; Moeller, Timothy [Bioreclamation In Vitro Technologies, Baltimore, MD 21227 (United States); Swaan, Peter W. [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Wang, Hongbing, E-mail: hwang@rx.umaryland.edu [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States)

2014-08-15

Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role
Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

International Nuclear Information System (INIS)

Lynch, Caitlin; Pan, Yongmei; Li, Linhao; Heyward, Scott; Moeller, Timothy; Swaan, Peter W.; Wang, Hongbing

2014-01-01

Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role
Insights into Basal Signaling Regulation, Oligomerization, and Structural Organization of the Human G-Protein Coupled Receptor 83.

Directory of Open Access Journals (Sweden)

Anne Müller

Full Text Available The murine G-protein coupled receptor 83 (mGPR83 is expressed in the hypothalamus and was previously suggested to be involved in the regulation of metabolism. The neuropeptide PEN has been recently identified as a potent GPR83 ligand. Moreover, GPR83 constitutes functionally relevant hetero-oligomers with other G-protein coupled receptors (GPCR such as the ghrelin receptor (GHSR or GPR171. Previous deletion studies also revealed that the long N-terminal extracellular receptor domain (eNDo of mGPR83 may act as an intra-molecular ligand, which participates in the regulation of basal signaling activity, which is a key feature of GPCR function. Here, we investigated particular amino acids at the eNDo of human GPR83 (hGPR83 by side-directed mutagenesis to identify determinants of the internal ligand. These studies were accompanied by structure homology modeling to combine functional insights with structural information. The capacity for hetero-oligomer formation of hGPR83 with diverse family A GPCRs such as the melanocortin-4 receptor (MC4R was also investigated, with a specific emphasis on the impact of the eNDo on oligomerization and basal signaling properties. Finally, we demonstrate that hGPR83 exhibits an unusual basal signaling for different effectors, which also supports signaling promiscuity. hGPR83 interacts with a variety of hypothalamic GPCRs such as the MC4R or GHSR. These interactions are not dependent on the ectodomain and most likely occur at interfaces constituted in the transmembrane regions. Moreover, several amino acids at the transition between the eNDo and transmembrane helix 1 were identified, where mutations lead also to biased basal signaling modulation.

GPCR Interaction: 39 [GRIPDB[Archive

Lifescience Database Archive (English)

Full Text Available s. A significant agonist-promoted internalization of the beta2AR activates ERK1/2 MAPK in beta2AR-expressing...AR and both receptors. Agonist-promoted internalization of the beta2AR was inhibi...erization between beta1AR and beta2AR inhibits the agonist-promoted internalization of the beta2AR and the a
National Constitutional Avenues for Further EU Integration

NARCIS (Netherlands)

Besselink, L.F.M.; Claes, M.; Imamovic, Š.; Reestman, J.H.

2014-01-01

This study investigates national constitutional limits to further EU integration and explores ways to overcome them. It includes an in-depth examination of the constitutional systems of 12 Member States (Croatia, the Czech Republic, Estonia, Finland, France, Germany, Hungary, Ireland, Italy, the
Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

Science.gov (United States)

Niczyj, Marta; Champagne, Antoine; Alam, Iftekhar; Nader, Joseph; Boutry, Marc

2016-11-01

Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H + -ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H + -ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H + -ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H + -ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.
Constitutive and ligand-induced TCR degradation

DEFF Research Database (Denmark)

von Essen, Marina; Bonefeld, Charlotte Menné; Siersma, Volkert

2004-01-01

Modulation of TCR expression levels is a central event during T cell development and activation, and it probably plays an important role in adjusting T cell responsiveness. Conflicting data have been published on down-regulation and degradation rates of the individual TCR subunits, and several di...... to the lysosomes. Similar results were obtained in studies of primary human Vbeta8+ T cells stimulated with superantigen. Based on these results, the simplest model for TCR internalization, sorting, and degradation is proposed.......Modulation of TCR expression levels is a central event during T cell development and activation, and it probably plays an important role in adjusting T cell responsiveness. Conflicting data have been published on down-regulation and degradation rates of the individual TCR subunits, and several...... divergent models for TCR down-regulation and degradation have been suggested. The aims of this study were to determine the rate constants for constitutive and ligand-induced TCR degradation and to determine whether the TCR subunits segregate or are processed as an intact unit during TCR down...
Neuronal Orphan G-Protein Coupled Receptor Proteins Mediate Plasmalogens-Induced Activation of ERK and Akt Signaling.

Directory of Open Access Journals (Sweden)

Md Shamim Hossain

Full Text Available The special glycerophospholipids plasmalogens (Pls are enriched in the brain and reported to prevent neuronal cell death by enhancing phosphorylation of Akt and ERK signaling in neuronal cells. Though the activation of Akt and ERK was found to be necessary for the neuronal cells survival, it was not known how Pls enhanced cellular signaling. To answer this question, we searched for neuronal specific orphan GPCR (G-protein coupled receptor proteins, since these proteins were believed to play a role in cellular signal transduction through the lipid rafts, where both Pls and some GPCRs were found to be enriched. In the present study, pan GPCR inhibitor significantly reduced Pls-induced ERK signaling in neuronal cells, suggesting that Pls could activate GPCRs to induce signaling. We then checked mRNA expression of 19 orphan GPCRs and 10 of them were found to be highly expressed in neuronal cells. The knockdown of these 10 neuronal specific GPCRs by short hairpin (sh-RNA lentiviral particles revealed that the Pls-mediated phosphorylation of ERK was inhibited in GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells. We further found that the overexpression of these GPCRs enhanced Pls-mediated phosphorylation of ERK and Akt in cells. Most interestingly, the GPCRs-mediated cellular signaling was reduced significantly when the endogenous Pls were reduced. Our cumulative data, for the first time, suggest a possible mechanism for Pls-induced cellular signaling in the nervous system.
Involvement of a putative intercellular signal-recognizing G protein-coupled receptor in the engulfment of Salmonella by the protozoan Tetrahymena

Directory of Open Access Journals (Sweden)

P.N. Agbedanu

2013-07-01

Full Text Available In an effort to investigate the molecular basis of protozoa engulfment-mediated hypervirulence of Salmonella in cattle, we evaluated protozoan G protein-coupled receptors (GPCRs as transducers of Salmonella engulfment by the model protozoan Tetrahymena. Our laboratory previously demonstrated that non-pathogenic protozoa (including Tetrahymena engulf Salmonella and then exacerbate its virulence in cattle, but the mechanistic details of the phenomenon are not fully understood. GPCRs were investigated since these receptors facilitate phagocytosis of particulates by Tetrahymena, and a GPCR apparently modulates bacterial engulfment for the pathogenic protozoan Entamoeba histolytica. A database search identified three putative Tetrahymena GPCRs, based on sequence homologies and predicted transmembrane domains, that were the focus of this study. Salmonella engulfment by Tetrahymena was assessed in the presence of suramin, a non-specific GPCR inhibitor. Salmonella engulfment was also assessed in Tetrahymena in which expression of putative GPCRs was knocked-down using RNAi. A candidate GPCR was then expressed in a heterologous yeast expression system for further characterization. Our results revealed that Tetrahymena were less efficient at engulfing Salmonella in the presence of suramin. Engulfment was reduced concordantly with a reduction in the density of protozoa. RNAi-based studies revealed that knock-down of one the Tetrahymena GPCRs caused diminished engulfment of Salmonella. Tetrahymena lysates activated this receptor in the heterologous expression system. These data demonstrate that the Tetrahymena receptor is a putative GPCR that facilitates bacterial engulfment by Tetrahymena. Activation of the putative GPCR seemed to be related to protozoan cell density, suggesting that its cognate ligand is an intercellular signaling molecule.
Competition And Antitrust Law In Ecuadorian Constitution

Directory of Open Access Journals (Sweden)

Marcelo Marín Sevilla

2013-01-01

Full Text Available This work allows us to establish the Economic Constitution and the Competition Law (C.L in the Constitution. Additionally, the paper analyzes whether the rules outlined in the C. L. and in doctrine are consistent and appropriate with the Constitution of Ecuador. The Competition and Antitrust Laws has rules for investigating and punishing the cartels, the abuses of power market (dominant position, the rules for merger control, the behaviors of Abuse of economic dependence, and unfair competition behaviors. Always the Antitrust Authority will analyze these behaviors in terms of welfare of both: the consumer and the market.
NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with μ-Opioid Agonist Activity.

Science.gov (United States)

Renton, Paul; Green, Brenda; Maddaford, Shawn; Rakhit, Suman; Andrews, John S

2012-03-08

A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the μ-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the μ-opioid GPCR was predicated on the modulatory role of nitric oxide on μ-opioid receptor function. Structure-activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 μM), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent μ-opioid binding affinity, K i = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 μM). This work represents a novel approach in the development of new analgesics for the treatment of pain.
High constitutive signaling of the ghrelin receptor--identification of a potent inverse agonist

DEFF Research Database (Denmark)

Holst, Birgitte; Cygankiewicz, Adam; Jensen, Tine Halkjaer

2003-01-01

Ghrelin is a GH-releasing peptide that also has an important role as an orexigenic hormone-stimulating food intake. By measuring inositol phosphate turnover or by using a reporter assay for transcriptional activity controlled by cAMP-responsive elements, the ghrelin receptor showed strong, ligand......-independent signaling in transfected COS-7 or human embryonic kidney 293 cells. Ghrelin and a number of the known nonpeptide GH secretagogues acted as agonists stimulating inositol phosphate turnover further. In contrast, the low potency ghrelin antagonist, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P was surprisingly...... found to be a high potency (EC50 = 5.2 nm) full inverse agonist as it decreased the constitutive signaling of the ghrelin receptor down to that observed in untransfected cells. The homologous motilin receptor functioned as a negative control as it did not display any sign of constitutive activity...
Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor.

Science.gov (United States)

Kleinau, Gunnar; Haas, Ann-Karin; Neumann, Susanne; Worth, Catherine L; Hoyer, Inna; Furkert, Jens; Rutz, Claudia; Gershengorn, Marvin C; Schülein, Ralf; Krause, Gerd

2010-07-01

The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.
SPECIFIC CONSTITUTIONAL FEATURES OF CHILDREN INFECTED WITH TUBERCULOSIS

Directory of Open Access Journals (Sweden)

Yu. A. Yarovaya

2017-01-01

Full Text Available In order to define specific constitutional features of the children infected with tuberculosis 222 children in the age from 1 to 14 years old have been examined: 106 children with active tuberculosis; 54 children with remaining post-tuberculosis changes; 62 children infected with tuberculous mycobacteria. The following types of diatheses were identified: lymphohypoplastic, allergic, neuroarthritic, exudative-catarrhal. It has been found out that among those with active tuberculosis the children suffering from lymphohypoplastic and neuroarthritic diatheses prevail (17.0 ± 3.7%, and allergic diathesis is less common (10.4 ± 3.0% cases. Children with lymphohypoplastic diathesis have a complicated course of tuberculosis (27.8 ± 10.6% and more intensive intoxication syndrome (55.6 ± 11.7%. The frequency of allergic diathesis is higher in the children with remaining post-tuberculosis changes (29.6 ± 6.2% and those infected with tuberculosis (33.8 ± 6.1% compared to children with active tuberculosis (10.4 ± 3.0%.
The European Constitution: sovereignty, legitimacy and constituent power

Directory of Open Access Journals (Sweden)

Signe Larsen

2014-06-01

Full Text Available On the basis of Hannah Arendt’s and Carl Schmitt’s writings on the constituent power, this article sets out to develop an interpretative framework which would aid the understanding of the legitimation crisis of European integration initiated by the EU constitutional failure of 2004. The question raised in this essay is whether the successful establishment of democratic constitutional legitimacy is conditional upon the existence of a federal state. From the perspective of the constituent power, two opposing answers are given based on two rivalling notions of the ultimate meaning of constitutional politics: freedom and security. The article concludes that even though the EU as a case remains undecided, it seems likely that democracy and constitutional politics have parted ways in the EU both in the Arendtian and in the Schmittian sense. If that is the case, the constitutional crisis is a serious problem for the future of democracy in the EU.
Public and Constitutional Support for Character Education.

Science.gov (United States)

Vessels, Gordon G.; Boyd, Stephen M.

1996-01-01

Character education thrives on an informed understanding of constitutional principles and an inclusive commitment-building process. U.S. Supreme Court opinions that clarify public school students' free speech rights have established values education as a constitutionally acceptable practice. Challenges might lie in possible violations of the First…
Constitutive equations for two-phase flows

International Nuclear Information System (INIS)

Boure, J.A.

1974-12-01

The mathematical model of a system of fluids consists of several kinds of equations complemented by boundary and initial conditions. The first kind equations result from the application to the system, of the fundamental conservation laws (mass, momentum, energy). The second kind equations characterize the fluid itself, i.e. its intrinsic properties and in particular its mechanical and thermodynamical behavior. They are the mathematical model of the particular fluid under consideration, the laws they expressed are so called the constitutive equations of the fluid. In practice the constitutive equations cannot be fully stated without reference to the conservation laws. Two classes of model have been distinguished: mixture model and two-fluid models. In mixture models, the mixture is considered as a single fluid. Besides the usual friction factor and heat transfer correlations, a single constitutive law is necessary. In diffusion models, the mixture equation of state is replaced by the phasic equations of state and by three consitutive laws, for phase change mass transfer, drift velocity and thermal non-equilibrium respectively. In the two-fluid models, the two phases are considered separately; two phasic equations of state, two friction factor correlations, two heat transfer correlations and four constitutive laws are included [fr
What constitutes information integrity?

Directory of Open Access Journals (Sweden)

S. Flowerday

2008-01-01

Full Text Available This research focused on what constitutes information integrity as this is a problem facing companies today. Moreover, information integrity is a pillar of information security and is required in order to have a sound security management programme. However, it is acknowledged that 100% information integrity is not currently achievable due to various limitations and therefore the auditing concept of reasonable assurance is adopted. This is in line with the concept that 100% information security is not achievable and the notion that adequate security is the goal, using appropriate countermeasures. The main contribution of this article is to illustrate the importance of and provide a macro view of what constitutes information integrity. The findings are in harmony with Samuel Johnson's words (1751: 'Integrity without knowledge is weak and useless, and knowledge without integrity is dangerous and dreadful.'
What constitutes information integrity?

Directory of Open Access Journals (Sweden)

S. Flowerday

2007-12-01

Full Text Available This research focused on what constitutes information integrity as this is a problem facing companies today. Moreover, information integrity is a pillar of information security and is required in order to have a sound security management programme. However, it is acknowledged that 100% information integrity is not currently achievable due to various limitations and therefore the auditing concept of reasonable assurance is adopted. This is in line with the concept that 100% information security is not achievable and the notion that adequate security is the goal, using appropriate countermeasures. The main contribution of this article is to illustrate the importance of and provide a macro view of what constitutes information integrity. The findings are in harmony with Samuel Johnson's words (1751: 'Integrity without knowledge is weak and useless, and knowledge without integrity is dangerous and dreadful.'
Endoplasmic reticulum stress pathway required for immune homeostasis is neurally controlled by arrestin-1.

Science.gov (United States)

Singh, Varsha; Aballay, Alejandro

2012-09-28

In response to pathogen infection, the host innate immune system activates microbial killing pathways and cellular stress pathways that need to be balanced because insufficient or excessive immune responses have deleterious consequences. Recent studies demonstrate that two G protein-coupled receptors (GPCRs) in the nervous system of Caenorhabditis elegans control immune homeostasis. To investigate further how GPCR signaling controls immune homeostasis at the organismal level, we studied arrestin-1 (ARR-1), which is the only GPCR adaptor protein in C. elegans. The results indicate that ARR-1 is required for GPCR signaling in ASH, ASI, AQR, PQR, and URX neurons, which control the unfolded protein response and a p38 mitogen-activated protein kinase signaling pathway required for innate immunity. ARR-1 activity also controlled immunity through ADF chemosensory and AFD thermosensory neurons that regulate longevity. Furthermore, we found that although ARR-1 played a key role in the control of immunity by AFD thermosensory neurons, it did not control longevity through these cells. However, ARR-1 partially controlled longevity through ADF neurons.
Constitutive androstane receptor upregulates Abcb1 and Abcg2 at the blood-brain barrier after CITCO activation.

Science.gov (United States)

Lemmen, Julia; Tozakidis, Iasson E P; Bele, Prachee; Galla, Hans-Joachim

2013-03-21

ATP-driven efflux transporters are considered to be the major hurdle in the treatment of central nervous system (CNS) diseases. Abcb1 (P-glycoprotein) and Abcg2 (breast cancer resistance protein/brain multidrug resistance protein) belong to the best known ABC-transporters. These ABC-transporters limit the permeability of the blood-brain barrier and protect the brain against toxic compounds in the blood but on the other hand they also reduce the efficacy of CNS pharmacotherapy. Even after 40 years of extensive research, the regulatory mechanisms of these efflux transporters are still not completely understood. To unravel the efflux transporter regulation, we analyzed the effect of the nuclear receptor CAR (constitutive androstane receptor) on the expression of Abcb1 and Abcg2 in primary cultures of porcine brain capillary endothelial cells (PBCEC). CAR is a xenobiotic-activated transcription factor, which is, like the other important nuclear receptor pregnane X receptor (PXR), highly expressed in barrier tissue and known to be a positive regulator of ABC-transporters. We demonstrate that activation of porcine CAR by the human CAR (hCAR) ligand CITCO (6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde) leads to an up-regulation of both transporters, whereas the mouse-specific CAR ligand TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene) had no effect on transporter expression. The stimulation of PBCEC with CITCO caused a significant up-regulation of both efflux-transporters on RNA-level, protein level and transport level. Furthermore the additional application of a CAR inhibitor significantly decreased the transporter expression to control niveau. In conclusion our data prove CAR activation only by the human ligand CITCO leading to an increased ABC-transporter expression and transport activity. Copyright © 2013 Elsevier B.V. All rights reserved.
[Women, gender, and the Constitution].

Science.gov (United States)

1993-12-01

Although all the constitutions of Latin America directly or indirectly acknowledge the juridical equality of the sexes, these patriarchal societies continue to maintain institutional power in male hands and to neutralize legal actions favoring women. International instruments such as the Convention on Elimination of All Forms of Discrimination Against Women, approved by the UN in 1979, have given a firmer basis to policies and actions to improve the status of women. Obstacles to full equality of Latin American women are rooted in economic and sociopolitical factors, but lack of true political will also plays a significant role. A number of new laws in the past several years as well as the new Constitution have improved the legal position of Colombian women. The new Constitution recognizes fundamental rights that may be claimed directly before a judge, and social, economic, and collective rights requiring legislative development. Article 43 of the new Constitution states that women will not be subjected to any form of discrimination. Another norm states that women will enjoy special assistance and protection before and after childbirth, in recognition of the social functions of maternity. Article 43 also states that women who are heads of households will receive special assistance, but the corresponding regulations have not yet been promulgated. The mechanism of tutelage has become an important recourse that has been used in several cases in which fundamental rights of women have been violated or threatened because of their sex. The order of tutelage has been used in cases of adolescents expelled from school for pregnancy and of abused wives, as well as to force recognition of the social and economic contributions of housework.
constitutional adjudication in ethiopia

African Journals Online (AJOL)

eliasn

2000-01-25

Jan 25, 2000 ... Thus the seeds of what some authors call. “federal .... Pre-WWII Europe trusted its legislature and led to .... European and Civil Law Forum v. 11 ...... on the sovereignty of nationalities and the fact that language constitutes one.

Islamization in Malaysia: the constitutional and legal dimensions

OpenAIRE

Choo, Kah Sing

2017-01-01

This thesis examines the legitimacy of the Islamization of the politics, society and law in Malaysia. The Islamization plan was carried out by the federal government in the early 1980s under the Mahathir administration. The legitimacy of the Islamization plan is measured against the original intent of the Independence Constitution, with particular reference to the position of Islam in the Constitution. Those constitutional provisions which have been claimed to have granted I...
Clinical study on constitutional herbal tea for treating chronic fatigue.

Science.gov (United States)

Park, Soo-Jung; Bae, Young-Chun; Choi, Na-Rae; Ryu, Seung-Yeob; Kwon, Young-Mi; Joo, Jong-Cheon

2014-12-01

This study was designed to evaluate the efficacy and the safety of constitutional herbal tea for treating chronic fatigue with no diagnosed cause, which is called Mibyeong in Korea. Males and females with ages between 40 and 59 years who had complained of fatigue for 1 month consistently or for 6 months intermittently without a definite cause were recruited. At the same time, a Chalder fatigue scale (CFS) score of 19 was essential for participation in this study. Sixty five subjects completed the entire process, including blood tests and tests with medical devices. Five assessments of health status were accomplished over 8 weeks by using the CFS and the visual analogue scale (VAS). To ensure that the constitutional herbal tea was being safely used, we conducted and analyzed renal function and liver function tests. For the diagnosis of the Sasang constitution, the Sasang Constitutional Analysis Tool (SCAT) was used, and a specialist in Sasang constitutional medicine made the final diagnosis based on the SCAT result. Constitutional herbal tea was served four weeks after the first visit. The subjects took the constitutional herbal tea twice a day for one month. The results are as follows: The CFS and the VAS scores were significantly improved for the subjects in the constitutional herbal tea. No abnormalities were found on the blood tests to evaluate safety after taking the constitutional herbal tea. The improvements in the CFS and the VAS scores due to the constitutional herbal tea had no significant differences according to the Sasang constitution. Constitutional herbal tea may be used to reduce fatigue and improve health and has no adverse effect on either the kidney or the liver.
Constitutive relations for nuclear reactor core materials

International Nuclear Information System (INIS)

Zaverl, F. Jr.; Lee, D.

1978-01-01

A strain rate dependent constitutive equation is proposed which is capable of describing inelastic deformation behavior of anisotropic metals, such as Zircaloys, under complex loading conditions. The salient features of the constitutive equations are that they describe history dependent inelastic deformation behaviour of anisotropic metals under three-dimensional stress states in the presence of fast neutron flux. It is shown that the general form of the constitutive relations is consistent with experimental observations made under both unirradiated and irradiated conditions. The utility of the model is demonstrated by examining the analytical results obtained for a segment of tubing undergoing different loading histories in a reactor. (Auth.)
Constitutional problems in the handling of plutonium

International Nuclear Information System (INIS)

Papier, H.J.

1989-01-01

In principle the decision between direct storage and reprocessing comes into the scope of regulation by the first or second power, resp., reserved for them according to the constitutional principle of necessity. The author thinks that a possibly increased hazard potential might result in increased protection requirements or protection measures as envisaged by the licensing preconditions of the Atomic Energy Law and those of other normative protection regulations. This is no issue of constitutional jurisdiction but of political intent and technical-economic capabilities whether or not certain technologies are precluded a prior or permitted and implemented at a safety level satisfying the specific constitutional protection requirements. (orig./HSCH) [de
Proposals for the Reform of Constitutional Regulations on Public Finances

OpenAIRE

Tibor András Hetei

2011-01-01

The main purpose of this paper is to make specific proposals in the areas of public finance and the budget process in the context of the reform of the Hungarian Constitution. In preparing the proposals the author has reviewed the prevailing constitutional regulations as well as the relevant practice of the Constitutional Court, and examined and compared the constitutional schemes of European countries. The paper finds that a number of aspects of the constitutional regulation of public finance...
A Federal Constitution for the European Union

DEFF Research Database (Denmark)

Sweeney, Richard J.

2003-01-01

A constitution is more likely to be accepted if it federalizes those issues that arewidely seen as needing complete harmonization. A constitution is more likely to endure if thefederal government does not have powers that are not vital to it but which may alienate somemember states to the point t...
Movimientos sociales, activismo constitucional y narrativa democrática en la Argentina contemporánea Social movements, constitutional activism and democratic narrative in contemporary Argentina

Directory of Open Access Journals (Sweden)

Gabriela Delamata

2013-04-01

Full Text Available El artículo analiza tres procesos recientes de movilización socio-legal en Argentina y subraya el rol de la Constitución y la comunidad legal garantista en su despliegue. El enfoque utilizado propone entender los procesos bajo la forma de una reactivación del patrón activista gestado en la transición democrática y señala sus efectos actuales sobre los procedimientos democráticos y la gramática de ciudadanía.The article analyzes three contemporary processes of socio-legal mobilization in Argentina and underlines the role of the Constitution and of the legal community in its enforcement. The adopted approach aims at understanding those processes as a sort of reactivation of the activist tradition born during the transition to democracy, and emphasizing the present effects of this constitutional activism on democratic procedures and the citizenship grammar.
Democracy, Citizen Sovereignty and Constitutional Economics

OpenAIRE

Vanberg, Viktor J.

2006-01-01

This paper is an exercise in conceptual clarification. Its purpose is to explore the contribution that constitutional economics can make to the theory of democracy. Constitutional economics as the economics of rules is concerned with the study of how the choice of rules in the social, economic and political realm affects the nature of the processes of human interaction that evolve within these rules. The theory of democracy is concerned with institutionalorganizational problems of self-govern...
Constitutive relation of concrete containing meso-structural characteristics

Directory of Open Access Journals (Sweden)

Li Guo

Full Text Available A constitutive model of concrete is proposed based on the mixture theory of porous media within thermodynamic framework. By treating concrete as a multi-phase multi-component mixture, we constructed the constitutive functions for elastic, interfacial, and plastic strain energy respectively. A constitutive law of concrete accommodating internal micro-cracks and interfacial boundaries was established. The peak stress predicted with the developed model depends primarily on the volume ratio of aggregate, and the results explain very well reported experimental phenomena. The strain-stress curve under uniaxial loading was found in a good agreement with experimental data for concrete with three different mixing proportions. Keywords: Constitutive model of concrete, Mixture theory of porous media, Meso-structure, Interfacial energy
Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex.

Science.gov (United States)

Gao, Yang; Westfield, Gerwin; Erickson, Jon W; Cerione, Richard A; Skiniotis, Georgios; Ramachandran, Sekar

2017-08-25

The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (G T ). This results in the dissociation of G T into its component α T -GTP and β 1 γ 1 subunit complex. Structural information for the Rho*-G T complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (G T *) comprising a Gα T /Gα i1 chimera (α T *) and β 1 γ 1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to Gα T * is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one G T *. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β 2 -adrenergic receptor-G S complex, including a flexible α T * helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Communicative Constitution of Organizations

DEFF Research Database (Denmark)

Schoeneborn, Dennis; Vasquez, Consuelo

2017-01-01

The notion of the communicative constitution of organizations (CCO) is at the center of a growing theoretical development within organizational communication studies. CCO scholarship is based on the idea that organization emerges in and is sustained and transformed by communication. This entry...
The constitutive sofa cushion

DEFF Research Database (Denmark)

Hanghøj, Sara

2009-01-01

personal values materialize through a hand-made everyday artefact, and how can the artefact constitute action and self-perception? The empirical research and analysis concerns how a former textile crafts teacher's subjective values and professional identity materialize through a hand-woven sofa cushion...
Land-restoration provisions of the Surface Mining Control and Reclamation Act: constitutional considerations

Energy Technology Data Exchange (ETDEWEB)

Eichbaum, W.M.; Buente, D.T.

1980-01-01

This examination of the land-capability requirements of SMCRA rested upon the proposition that a proper application of constitutional principles requires an appreciation of changing national conditions. Accordingly, this article examined the possible Fifth and Tenth Amendment challenges to SMCRA's land-capability provisions in the context of an evolving history of national concern over natural resources and in light of lengthy congressional deliberations based on concern for future national environmental and energy needs. The analysis suggests that SMCRA's land-capability requirements are well within the constitutional authority of the national government. The history of environmental protection legislation in the 1970's suggests that Federal action will increasingly impose regulatory restraints on the use of privately-owned natural resources. This trend is meeting strong resistance, including varied demands to reduce the impact and scope of Federal regulation. While the conclusion may be limited to SMCRA, its approach to examining constitutional challenges to Federal regulation protecting natural resources can be broadly applied. Natural resources are essential, finite, and irrevocably threatened by human activity. In the absence of effective state action, Federal protective action to provide national management of those resources is necessary and consistent with constitutional principles. 184 references.
Sasang constitutional medicine as a holistic tailored medicine.

Science.gov (United States)

Kim, Jong Yeol; Pham, Duong Duc

2009-09-01

Sasang constitutional medicine (SCM) is a unique traditional Korean therapeutic alternative form of medicine. Based on the Yin and Yang theory and on Confucianism, humans are classified into four constitutions. These differ in terms of (i) sensitivity to certain groups of herbs and medicines, (ii) equilibrium among internal organic functions, (iii) physical features and (iv) psychological characteristics. We propose that two main axes in the physiopathology of SCM (food intake/waste discharge and consuming/storing Qi and body fluids) are equivalent to the process of internal-external exchange and catabolism/anabolism in modern physiology, respectively. We then used this hypothesis to discuss the physiological and pathological principles of SCM. Constitution-based medicine is based on the theory that some medicinal herbs and remedies are only appropriate for certain constitutions and can cause adverse effects in others. The constitutional approach of SCM share the same vision as tailored medicine; an individualized therapy that can minimize the risk of adverse reaction while increasing the efficacy and an individualized self-regulation that can help prevent specific susceptible chronic disease and live healthily. There is still a long way to this goal for both SCM and tailored medicine, but we may benefit from systems approaches such as systems biology. We suggest that constitutional perspective of SCM and our hypothesis of two main processes may provide a novel insight for further studies.
Sasang Constitutional Medicine as a Holistic Tailored Medicine

Directory of Open Access Journals (Sweden)

Jong Yeol Kim

2009-01-01

Full Text Available Sasang constitutional medicine (SCM is a unique traditional Korean therapeutic alternative form of medicine. Based on the Yin and Yang theory and on Confucianism, humans are classified into four constitutions. These differ in terms of (i sensitivity to certain groups of herbs and medicines, (ii equilibrium among internal organic functions, (iii physical features and (iv psychological characteristics. We propose that two main axes in the physiopathology of SCM (food intake/waste discharge and consuming/storing Qi and body fluids are equivalent to the process of internal–external exchange and catabolism/anabolism in modern physiology, respectively. We then used this hypothesis to discuss the physiological and pathological principles of SCM. Constitution-based medicine is based on the theory that some medicinal herbs and remedies are only appropriate for certain constitutions and can cause adverse effects in others. The constitutional approach of SCM share the same vision as tailored medicine; an individualized therapy that can minimize the risk of adverse reaction while increasing the efficacy and an individualized self-regulation that can help prevent specific susceptible chronic disease and live healthily. There is still a long way to this goal for both SCM and tailored medicine, but we may benefit from systems approaches such as systems biology. We suggest that constitutional perspective of SCM and our hypothesis of two main processes may provide a novel insight for further studies.
THE CONSTITUTIONAL CONCEPTS OF THE REFORM TREATY (THE LISBON TREATY

Directory of Open Access Journals (Sweden)

Emilian Ciongaru

2017-12-01

Full Text Available The Lisbon Treaty also known as the Reform Treaty provides only an amendment of the treaties considered as fundamental, namely the Treaty on the European Union and the Treaty on the functioning of the European Union and is the result of the constitutional process triggered by the Laeken Declaration adopted by the European Council. The Lisbon Treaty is still built on the content of the European Constitution from which they eliminated the most controversial provisions, first of all the title of Constitution that might produce concern and panic among the European Union population through the symbolic power it contained, and for Romania this new treaty was the first it signed in quality of a Union member state. Even if does not bear the name of European Constitution, the Lisbon Treaty is a European Constitution for the following reasons: first it is a Constitution because it gathers together most of the fundamental elements of the Constitutional Treaty, even if it does not have the structure or the name thereof, and second the treaties after the Lisbon reform have become small constitutions from the operational viewpoint, they develop the functions of a constitution, limit power and organize the operation of the organization.
The spirit of the constitution. The institutionalized unsociability in Greece

Directory of Open Access Journals (Sweden)

Savvas MAVRIDIS

2017-08-01

Full Text Available With this article attempts a comparison of the Greek Constitution of 1975, the constitutions of Germany, USA, Switzerland, Austria, France, UK and Italy on the issue of property relations and general interest, in order to highlight those notified differences relating to these communities and resulted in specific social situations. While the constitutions of the other countries, which are among the most representative democracies of the Western world, are characterized by an increased institutionalization of sociability, the Greek constitution on the issue of general interest takes an antisocial and, at best, neutral attitude. The convergence of the Greek constitution on this issue with the other constitutions is considered a prerequisite for overcoming the crisis and, more generally, for social development. This little effort hopes to contribute to a more social future formulation of the Greek constitution and a corresponding behaviour of Greek society.
Constitutive equations for Zr1Nb. II

International Nuclear Information System (INIS)

Novak, J.

1986-01-01

Based on existing knowledge and constitutive equations for non-irradiated material, constitutive equations were written for Zr1Nb irradiated at 573 K at deformation in the direction of forming. Constitutive equations express the following material characteristics: dependence of shear strength on fast neutron fluence, superposition of deformation hardening and subsequent radiation hardening, the effect of stress on deformation rate, and for fluences above ca. 10 24 n.m -2 (E>1 MeV) the course of the deformation curve for various fluence levels. The values apply for temperatures and rates of deformation which are characteristic of transient processes during changes in the power output of fuel elements of pressurized water reactors. (J.B.)
European constitution and EURATOM treaty

International Nuclear Information System (INIS)

Heller, W.

2003-01-01

The European Council held in Laeken in December 2001 had decided to call a convention preparing the next conference of the heads of state and government which, among other topics, was to deliberate the question of a fully formulated European constitution. Under the presidency of Giscard d'Estaing, all delegates to the European Convention on July 10, 2003 signed the draft treaty for a European constitution. This final document is the basis of the conference of the heads of state and government to begin in October 2003. On this occasion, the draft of a separate chapter on energy could well come up again for examination. This chapter had been introduced only at the end of the deliberations of the convention and adds to the competences of the EU institutions. Also the Euratom Treaty was a topic of the convention preparing the constitution. As the presidency felt that no specific issues had been raised in the Laeken declaration, it is proposed to adapt the Euratom Treaty to the new provisions of the constitution by adding a protocol. This would mean that the European Atomic Energy Community, for the time being, would retain its independent legal status. The contents would have to be examined at some later date. Consequently, the real discussion of the Euratom Treaty is yet to come. Also, the speedy completion of the single market for electricity would make it desirable for the Community to adopt a uniform, positive stance in the use of nuclear power at the best possible safety standards so as to ensure a level playing field. Current events entailing power failures in the United States and the United Kingdom have alerted the public to the problem of the continuity of power supply. This could well be the beginning of a new, unbiased, balanced energy discussion in a bigger Europe. (orig.)
Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors.

Science.gov (United States)

Weston, Cathryn; Winfield, Ian; Harris, Matthew; Hodgson, Rose; Shah, Archna; Dowell, Simon J; Mobarec, Juan Carlos; Woodlock, David A; Reynolds, Christopher A; Poyner, David R; Watkins, Harriet A; Ladds, Graham

2016-10-14

The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gα s -mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gα s and Gα q but also identify a Gα i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gα s , Gα i , and Gα q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased skin barrier disruption by sodium lauryl sulfate in mice expressing a constitutively active STAT6 in T cells.

Science.gov (United States)

DaSilva, Sonia C; Sahu, Ravi P; Konger, Raymond L; Perkins, Susan M; Kaplan, Mark H; Travers, Jeffrey B

2012-01-01

Atopic dermatitis (AD) is a pruritic, chronic inflammatory skin disease that affects 10-20% of children and 1-3% of adults worldwide. Recent studies have indicated that the ability of Th2 cytokines, such as interleukin-4 (IL-4) to regulate skin barrier function may be a predisposing factor for AD development. The present studies examined the ability of increased Th2 activity to affect cutaneous barrier function in vivo and epidermal thickening. Mice that express a constitutively active Signal Transducer and Activator of Transcription 6 (STAT6VT) have increased Th2 cells and a predisposition to allergic inflammation were used in these studies, they demonstrate that topical treatment with the irritant sodium lauryl sulfate (SLS) caused increased transepidermal water loss and epidermal thickening in STAT6VT mice over similarly treated wild-type mice. The proliferation marker Ki-67 was increased in the epidermis of STAT6VT compared to the wild-type mice. However, these differences do not appear to be linked to the addition of an irritant as control-treated STAT6VT skin also exhibited elevated Ki-67 levels, suggesting that the increased epidermal thickness in SLS-treated STAT6VT mice is primarily driven by epidermal cell hypertrophy rather than an increase in cellular proliferation. Our results suggest that an environment with increased Th2 cytokines results in abnormal responses to topical irritants.
Mathematical modeling and the two-phase constitutive equations

International Nuclear Information System (INIS)

Boure, J.A.

1975-01-01

The problems raised by the mathematical modeling of two-phase flows are summarized. The models include several kinds of equations, which cannot be discussed independently, such as the balance equations and the constitutive equations. A review of the various two-phase one-dimensional models proposed to date, and of the constitutive equations they imply, is made. These models are either mixture models or two-fluid models. Due to their potentialities, the two-fluid models are discussed in more detail. To avoid contradictions, the form of the constitutive equations involved in two-fluid models must be sufficiently general. A special form of the two-fluid models, which has particular advantages, is proposed. It involves three mixture balance equations, three balance equations for slip and thermal non-equilibriums, and the necessary constitutive equations [fr
Extended constitutive laws for lamellar phases

Directory of Open Access Journals (Sweden)

Chi-Deuk Yoo

2013-10-01

Full Text Available Classically, stress and strain rate in linear viscoelastic materials are related by a constitutive relationship involving the viscoelastic modulus G(t. The same constitutive law, within Linear Response Theory, relates currents of conserved quantities and gradients of existing conjugate variables, and it involves the autocorrelation functions of the currents in equilibrium. We explore the consequences of the latter relationship in the case of a mesoscale model of a block copolymer, and derive the resulting relationship between viscous friction and order parameter diffusion that would result in a lamellar phase. We also explicitly consider in our derivation the fact that the dissipative part of the stress tensor must be consistent with the uniaxial symmetry of the phase. We then obtain a relationship between the stress and order parameter autocorrelation functions that can be interpreted as an extended constitutive law, one that offers a way to determine them from microscopic experiment or numerical simulation.
Resolution and Letter to Congress from the Constitutional Convention

Science.gov (United States)

Potter, Lee Ann

2005-01-01

This article explores the drafting of the United States Constitution. The United States Constitution is the longest-lasting written national constitution in the world. Its four parchment pages serve as the blueprint for a government under which more than 290 million Americans live. It holds great significance for the American people and for others…
The constitutive activation of the CEF-4/9E3 chemokine gene depends on C/EBPbeta in v-src transformed chicken embryo fibroblasts

DEFF Research Database (Denmark)

Gagliardi, M; Maynard, S; Bojovic, B

2001-01-01

The CEF-4/9E3 chemokine gene is expressed constitutively in chicken embryo fibroblasts (CEF) transformed by the Rous sarcoma virus (RSV). This aberrant induction is controlled at the transcriptional and post-transcriptional levels. Transcriptional activation depends on multiple elements of the CEF....../EBPbeta binds to a second element located in proximity of the TRE. A mutation of this distal CAAT box impaired the activation of the CEF-4 promoter by pp60(v-src) indicating that this element is also part of the SRU. Using the RCASBP retroviral vector, we expressed a dominant negative mutant of C....../EBPbeta (designated Delta184-C/EBPbeta) in RSV-transformed CEF. Delta184-C/EBPbeta decreased the accumulation of the CEF-4 mRNA and activation of the CEF-4 promoter by pp60(v-src). The induction of the Cox-2 gene (CEF-147) was also reduced by Delta184-C/EBPbeta. The effect of the dominant negative mutant was observed...
A multiscale constitutive model for intergranular stress corrosion cracking in type 304 austenitic stainless steel

International Nuclear Information System (INIS)

Siddiq, A; Rahimi, S

2013-01-01

Intergranular stress corrosion cracking (IGSCC) is a fracture mechanism in sensitised austenitic stainless steels exposed to critical environments where the intergranular cracks extends along the network of connected susceptible grain boundaries. A constitutive model is presented to estimate the maximum intergranular crack growth by taking into consideration the materials mechanical properties and microstructure characters distribution. This constitutive model is constructed based on the assumption that each grain is a two phase material comprising of grain interior and grain boundary zone. The inherent micro-mechanisms active in the grain interior during IGSCC is based on crystal plasticity theory, while the grain boundary zone has been modelled by proposing a phenomenological constitutive model motivated from cohesive zone modelling approach. Overall, response of the representative volume is calculated by volume averaging of individual grain behaviour. Model is assessed by performing rigorous parametric studies, followed by validation and verification of the proposed constitutive model using representative volume element based FE simulations reported in the literature. In the last section, model application is demonstrated using intergranular stress corrosion cracking experiments which shows a good agreement
Constitutional collisions of criminal law

Directory of Open Access Journals (Sweden)

Sergey M. Inshakov

2016-12-01

Full Text Available Objective to identify and resolve conflicts between the norms of constitutional and criminal law which regulate the issue of legal liability of senior officials of the state. Methods formallogical systematic comparativelegal. Results the article analyzes the embodiment of the principle of citizensrsquo equality under the law regarding the criminal responsibility of the President of the Russian Federation as one of the segments of the elite right other criminal and legal conflicts are considered associated with the creation of conditions for derogation from the principle of equality. Basing on this analysis the means of overcoming collisions between the norms of constitutional and criminal law are formulated. Scientific novelty in the article for the first time it has been shown that in the Russian criminal law there are exceptions to the principle of citizensrsquo equality under the law relating to the President of the Russian Federation the conflicts are identified between the norms of constitutional and criminal law regulating the issue of legal liability of senior officials of the state ways of overcoming conflicts are suggested. Practical significance the main provisions and conclusions of the article can be used in research and teaching in the consideration of issues of senior state officialsrsquo criminal liability.
Governmental control of public expenditure in the constitutional State: thoughts regarding General Comptroller sanctioning powers approval on functional administrative responsibility

Directory of Open Access Journals (Sweden)

Erika García Cobián Castro

2013-12-01

Full Text Available Constitutional doctrine has little addressed on the General Comptroller of the Republic and its controlling function of public resources execution and management. Efforts have been made to explain this situation in other realities, regarding high budgetary, accounting and auditing content needed to carry out auditing activities constitutionally assigned to this kind of entities. Nevertheless, identifying competences of the General Comptroller of the Republic and its nature of autonomous constitutional body shapes an important expression of Constitutional State and the principle of powers division. This entity controls State’s Public Budget execution contributing to constitutional property protection, legality of budget implementation and the appropriate management of public resources, also the «efficiency» of social needs, proper functioning of public administration and prevention of corruption, among others duties. This article analyzes the constitutional mission of the General Comptroller in a Constitutional State promoting the process of given a constitutional status to the legal system in its area of influence, also optimizing constitutional properties protected. For such purpose, the 29622 recent law («Law that modifies the 27785 Organic Law of National Control System and the General Comptroller of the Republic extending powers in order to sanction in accordance of functional administrative responsibility» shall be used as object of constitutional analysis.
Constitutive Theories of Self-Knowledge and the Regress Problem ...

African Journals Online (AJOL)

... on the other hand, hold that self-knowledge is constitutive of intentional states. That is, self-ascription is a necessary condition for being in a particular mental state. Akeel Bilgrami is a defender of the constitutive model. I argue that the constitutive model gives rise to a regress problem. This paper will focus on that problem ...
Organic Law Of Judicial Guarantees And Constitutional Control

Directory of Open Access Journals (Sweden)

Ernesto López Freire

2013-01-01

Full Text Available This paper demonstrates the various unconstitutional and fallacies of the Organic Law of Judicial guarantees and Constitutional Control. For that, there will be a comprehensive collation between the mentioned law and the Constitution of the Republic of Ecuador and force. Through this analysis shows a lack of knowledge of Ecuadorian law or legal science by their authors. This study elucidated, inter alia, the inconsistencies in matters relating to the interpretation of constitutional provisions, full compensation, material and integral; challenge administrative acts, judicial unit.
Development of full sweet, umami, and bitter taste responsiveness requires Regulator of G protein Signaling-21 (RGS21).

Science.gov (United States)

Schroer, Adam B; Gross, Joshua D; Kaski, Shane W; Wix, Kim; Siderovski, David P; Vandenbeuch, Aurelie; Setola, Vincent

2018-04-26

The mammalian tastes of sweet, umami, and bitter are initiated by activation of G protein-coupled receptors (GPCRs) of the T1R and T2R families on taste receptor cells. GPCRs signal via nucleotide exchange and hydrolysis, the latter hastened by GTPase-accelerating proteins (GAPs) that include the Regulators of G protein Signaling (RGS) protein family. We previously reported that RGS21, uniquely expressed in Type II taste receptor cells, decreases the potency of bitter-stimulated T2R signaling in cultured cells, consistent with its in vitro GAP activity. However, the role of RGS21 in organismal responses to GPCR-mediated tastants was not established. Here, we characterized mice lacking the Rgs21 fifth exon. Eliminating Rgs21 expression had no effect on body mass accumulation (a measure of alimentation), fungiform papillae number and morphology, circumvallate papillae morphology, and taste bud number. Two-bottle preference tests, however, revealed that Rgs21-null mice have blunted aversion to quinine and denatonium, and blunted preference for monosodium glutamate, the sweeteners sucrose and SC45647, and (surprisingly) NaCl. Observed reductions in GPCR-mediated tastant responses upon Rgs21 loss are opposite to original expectations, given that loss of RGS21 -- a GPCR signaling negative regulator -- should lead to increased responsiveness to tastant-mediated GPCR signaling (all else being equal). Yet, reduced organismal tastant responses are consistent with observations of reduced chorda tympani nerve recordings in Rgs21-null mice. Reduced tastant-mediated responses and behaviors exhibited by adult mice lacking Rgs21 expression since birth have thus revealed an underappreciated requirement for a GPCR GAP to establish the full character of tastant signaling.
Constitutive Modelling of Resins in the Stiffness Domain

Science.gov (United States)

Klasztorny, M.

2004-09-01

An analytic method for inverting the constitutive compliance equations of viscoelasticity for resins is developed. These equations describe the HWKK/H rheological model, which makes it possible to simulate, with a good accuracy, short-, medium- and long-term viscoelastic processes in epoxy and polyester resins. These processes are of first-rank reversible isothermal type. The time histories of deviatoric stresses are simulated with three independent strain history functions of fractional and normal exponential types. The stiffness equations are described by two elastic and six viscoelastic constants having a clear physic meaning (three long-term relaxation coefficients and three relaxation times). The time histories of axiatoric stresses are simulated as perfectly elastic. The inversion method utilizes approximate constitutive stiffness equations of viscoelasticity for the HWKK/H model. The constitutive compliance equations for the model are a basis for determining the exact complex shear stiffness, whereas the approximate constitutive stiffness equations are used for determining the approximate complex shear stiffness. The viscoelastic constants in the stiffness domain are derived by equating the exact and approximate complex shear stiffnesses. The viscoelastic constants are obtained for Epidian 53 epoxy and Polimal 109 polyester resins. The accuracy of the approximate constitutive stiffness equations are assessed by comparing the approximate and exact complex shear stiffnesses. The constitutive stiffness equations for the HWKK/H model are presented in uncoupled (shear/bulk) and coupled forms. Formulae for converting the constants of shear viscoelasticity into the constants of coupled viscoelasticity are given as well.
Gospel and constitutional imperatives: the right to life

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S.P. Giles

2011-06-01

Full Text Available Gospel and constitutional imperatives: the right to life In the Republic of South Africa, Christians are called to live out gospel imperatives within the legal framework of the Constitution. Ethical issues about the right to life are considered from the perspectives of selected gospel and constitutional imperatives. Gospel imperatives impose themselves as a consequence of Christian discipleship. These are many and diverse, both explicit and implicit. Christian vocation, discipleship, witness and perseverance, are foundational and integral to the praxis of Christian daily living. These facets of Christian life are illustrated by the selected gospel imperatives, “Follow me” (Matt. 4:19 and synoptic parallels, “Love God, and your neighbour as yourself” (Matt. 22:34-40 and synoptic parallels, and “Take up your cross” (Matt. 10:38 and synoptic parallels. The central theoretical argument of this article is driven from a reformed ethical perspective. Gospel imperatives have priority over constitutional imperatives since gospel imperatives are of divine origin and constitutional imperatives of human origin. Acknowledgement of these priorities informs the Christian ethical perspective on the right to life and on abortion.
Limits of verification by the Federal Constitutional Court

International Nuclear Information System (INIS)

Anon.

1985-01-01

The court of administration of Baden-Wuerttemberg passed a resolution on October 27,1983 - 10 S 1102/83 - dismissing the application to revoke the immediate implementation of the first partial construction permit for the joint nuclear power plant Neckar II. As the plaintiff thereupon lodged a complaint with the Federal Constitutional Court the resolution did not become effective immediately. However, the Federal Constitutional Court on October 1, 1984 decreed unanimously in accordance with the section 93a subsection 3 of the law on the Federal Constitutional Court - 1 BvR 231/84 -: ''The constitutional complaint lodged is not taken up for decision because its chances of success are nil.'' Thus the original decision of the court of administration of Baden-Wuerttemberg passed on October 27, 1983 was confirmed by the endorsement of its immediate implementation. (orig./HSCH) [de
ARF1 recruits RAC1 to leading edge in neutrophil chemotaxis.

Science.gov (United States)

Mazaki, Yuichi; Onodera, Yasuhito; Higashi, Tsunehito; Horinouchi, Takahiro; Oikawa, Tsukasa; Sabe, Hisataka

2017-10-02

The small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the Gβγ-PAK1-αPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which αPIX activates RAC1/CDC42, which then employs PAK1. However, it has remained unclear as to why GIT2 is included in this complex. We investigated the association between ARF1 and RAC1/CDC42 during the fMLP-stimulated chemotaxis of HL60 cells. We found that the silencing of GBF1 significantly impaired the recruitment of RAC1 to the leading edges, but not PAK1, αPIX, RAC2, or CDC42. A significant population of RAC1 colocalized with ARF1 at the leading edges in stimulated cells, whereas fMLP activated both ARF1 and ARF5. Consistently, the silencing of ARF1, but not ARF5, impaired the recruitment of RAC1, whereas the silencing of RAC1 did not affect the recruitment of ARF1 to the leading edges. Our results indicated that the activation of ARF1 triggers the plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling. Thus, membrane remodeling at the leading edges appears to precede actin remodeling in chemotaxis. Together with the fact that GIT2, which inactivates ARF1, is an integral component of the machinery activating RAC1, we proposed a model in which the ARF1-RAC1 linkage enables the regulation of ARF1 by repetitive on/off cycles during GPCR-mediated neutrophil chemotaxis.
Public health and the Australian Constitution.

Science.gov (United States)

Reynolds, C

1995-06-01

The powers vested in the Commonwealth Government by the Constitution contain the basis of much public health law in Australia. Yet this is not often recognised; public health law is generally, and historically, seen as the states' responsibility. This article surveys the broad range of constitutional powers that the Commonwealth Government can rely upon to make public health laws. It considers areas of power specified in the Constitution, such as those with respect to external affairs and corporations. Decisions of the High Court have interpreted the various heads of power very broadly and have significantly enhanced the potential of the Commonwealth to pass detailed and far-reaching public health law. To this fact must be added the taxation arrangements in Australia and, with these, the very extensive control that the Commonwealth can exercise through its monopoly of major taxation sources. Its power to make financial arrangements can tie dependent states into specific policies (including public health policies) as a condition of the grants made to them. However, these broad powers may be limited in some important respects: the High Court is increasingly identifying rights and freedoms in the Constitution that may increasingly bring both state and Commonwealth public health law under challenge. Despite this possibility, the Commonwealth may prove to be our most significant source of public health law, and public health policy makers should recognise the full potential of its power to make such laws.
Book review: Advanced Introduction to Comparative Constitutional Law.

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Ainhoa Martinez

2016-12-01

Full Text Available Tushnet presents a thoughtful introduction to the field of comparative constitutional law through a review of recent literature and an analysis of the key contemporary issues in constitutional design and structure. In the following lines a review of his book is presented.Book review of Mark Tushnet. Advanced Introduction to Comparative Constitutional Law. Cheltenham; Northampton: Edward Elgar Publishing, 2014. ISBN (Hb 978 1 78100 731 0 £58.50, ISBN (Pb 978 1 78347 351 9 £12.76.DOWNLOAD THIS PAPER FROM SSRN: https://ssrn.com/abstract=2887014
Constitutive phosphorylation of Shc proteins in human tumors

DEFF Research Database (Denmark)

Pelicci, G; Lanfrancone, L; Salcini, A E

1995-01-01

The Shc gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are ubiquitously expressed and are downstream targets and effectors of activated tyrosine kinases (TK). We investigated tyrosine-phosphorylation of Shc proteins in normal and transformed...... of the Shc-associated phosphoproteins (EGFR, PDGFR, erbB-2, Met, bcr-abl, H4-ret) bound both the Shc- and Grb2-SH2 domains in vitro; others (p175; p70-p80) only the Shc-SH2 domain and yet others (p140) only the Grb2-SH3 domains. These results indicate that Shc proteins are common substrates of constitutively...
15 years of constitutional review in the Supreme Court of Estonia : systematized extracts of constitutional review judgments and rulings of the Supreme Court en banc and the Constitutional Review Chamber in 1993-2008 / Riigikohus ; toim. ja eess.: Ge

Index Scriptorium Estoniae

2009-01-01

Sisaldab ka: The outset of judicial constitutional review / Rait Maruste. Judicial constitutional review at the turn of the century / Uno Lõhmus. Judicial constitutional review through the eyes of Chancellor of Justice / Eerik-Juhan Truuväli. The courts and the Supreme Court in concrete norm control / Madis Ernits
A phenomenological constitutive model for low density polyurethane foams

International Nuclear Information System (INIS)

Neilsen, M.K.; Morgan, H.S.; Krieg, R.D.

1987-04-01

Results from a series of hydrostatic and triaxial compression tests which were performed on polyurethane foams are presented in this report. These tests indicate that the volumetric and deviatoric parts of the foam behavior are strongly coupled. This coupling behavior could not be captured with any of several commonly used plasticity models. Thus, a new constitutive model was developed. This new model was based on a decomposition of the foam response into two parts: (1) response of the polymer skeleton, and (2) response of the air inside the cells. The air contribution was completely volumetric. The new constitutive model was implemented in two finite element codes, SANCHO and PRONTO. Results from a series of analyses completed with these codes indicated that the new constitutive model captured all of the foam behaviors that had been observed in the experiments. Finally, a typical dynamic problem was analyzed using the new constitutive model and other constitutive models to demonstrate differences between the models. Results from this series of analyses indicated that the new constitutive model generated displacement and acceleration predictions that were between predictions obtained using the other models. This result was expected. 9 refs., 45 figs., 4 tabs

Latency-Associated Expression of Human Cytomegalovirus US28 Attenuates Cell Signaling Pathways To Maintain Latent Infection

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Benjamin A. Krishna

2017-12-01

Full Text Available Reactivation of human cytomegalovirus (HCMV latent infection from early myeloid lineage cells constitutes a threat to immunocompromised or immune-suppressed individuals. Consequently, understanding the control of latency and reactivation to allow targeting and killing of latently infected cells could have far-reaching clinical benefits. US28 is one of the few viral genes that is expressed during latency and encodes a cell surface G protein-coupled receptor (GPCR, which, during lytic infection, is a constitutive cell-signaling activator. Here we now show that in monocytes, which are recognized sites of HCMV latency in vivo, US28 attenuates multiple cell signaling pathways, including mitogen-activated protein (MAP kinase and NF-κB, and that this is required to establish a latent infection; viruses deleted for US28 initiate a lytic infection in infected monocytes. We also show that these monocytes then become potent targets for the HCMV-specific host immune response and that latently infected cells treated with an inverse agonist of US28 also reactivate lytic infection and similarly become immune targets. Consequently, we suggest that the use of inhibitors of US28 could be a novel immunotherapeutic strategy to reactivate the latent viral reservoir, allowing it to be targeted by preexisting HCMV-specific T cells.
Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium

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Hayoz Sébastien

2012-05-01

Full Text Available Abstract Background ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. Results Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αβMeATP and Bz-ATP and a G protein-coupled P2Y receptor agonist (UTP. Calcium imaging of P2X2-transfected HEK293 “biosensor” cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. Conclusions The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP
“People”, constitutions and politics in Antioquia, 1810-1877

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Renzo Ramírez Bacca

2014-07-01

Full Text Available This article analyzes the term people which is used in the provincial constitutions of Antioquia and Colombia. The interest lies on presenting their representations and usage in different phases of the Colombian political and constitutional history. The intention is to understand its correlation with the electoral and participative citizenship practice with respect to the State, and also to point out contradictions between different discourses (the constitutional and the political party ones around the people and its actors: women, artisans and farmers. This is a hermeneutic understanding with a historicist focus which, in turn, develops a diachronic analysis based on constitutionals texts and primary sources.
Constitutive equations for discrete electromagnetic problems over polyhedral grids

International Nuclear Information System (INIS)

Codecasa, Lorenzo; Trevisan, Francesco

2007-01-01

In this paper a novel approach is proposed for constructing discrete counterparts of constitutive equations over polyhedral grids which ensure both consistency and stability of the algebraic equations discretizing an electromagnetic field problem. The idea is to construct discrete constitutive equations preserving the thermodynamic relations for constitutive equations. In this way, consistency and stability of the discrete equations are ensured. At the base, a purely geometric condition between the primal and the dual grids has to be satisfied for a given primal polyhedral grid, by properly choosing the dual grid. Numerical experiments demonstrate that the proposed discrete constitutive equations lead to accurate approximations of the electromagnetic field
A constitutional economics perspective on soft paternalism

OpenAIRE

Schnellenbach, Jan

2015-01-01

Using a framework that distinguishes short-term consumer preferences, individual reflective preferences and political preferences, we discuss from a constitutional economics perspective whether individuals find it in their common constitutional interest to endow representatives and bureaucrats with the competence to impose soft paternalist policies. The focus is specifically on soft paternalist policies, because these often work with non-transparent 'nudges' that are considered as manipulativ...
The flow behavior and constitutive equation in isothermal compression of FGH4096-GH4133B dual alloy

International Nuclear Information System (INIS)

Liu, Yanhui; Yao, Zekun; Ning, Yongquan; Nan, Yang; Guo, Hongzhen; Qin, Chun; Shi, Zhifeng

2014-01-01

Highlights: • Hot compression behaviors of the FGH4096-GH4133B dual alloy were investigated. • Constitutive equation also represented deformation behavior of a dual alloy. • The effects of deformation activation energy on the microstructures were discussed. • Constitutive equation represented an accurate and precise estimate of flow stress. - Abstract: The electron beam welding of superalloy FGH4096 and GH4133B was conducted, and the cylindrical compression specimens were machined from the central part of the electron beam weldments. Isothermal compression tests were carried out on electron beam weldments FGH4096-GH4133B alloy at the temperatures of 1020–11140 °C (the nominal γ′-transus temperature is about 1080 °C) and the strain rates of 0.001–1.0 s −1 with the height reduction of 50%. True stress–true strain curves are sensitive to the deformation temperature and strain rate, and the flow stress decreases with the increasing deformation temperature and the decreasing strain rate. The true stress–true strain curves can indicate the intrinsic relationship between the flow stress and the thermal-dynamic behavior. The apparent activation energy of deformation at the strain of 0.6 was calculated to be 550 kJ/mol, and the apparent activation energy has a great effect on the microstructure. The constitutive equation that describes the flow stress as a function of strain rate and deformation temperature was proposed for modeling the hot deformation process of FGH4096-GH4133B electron beam weldments. The constitutive equation at the strain of 0.6 was established using the hyperbolic law. The relationship between the strain and the values of parameters was studied, and the cubic functions were built. The constitutive equation during the whole process can be obtained based on the parameters under different strains. Comparing the experimental flow stress and the calculated flow stress, the constitutive equation obtained in this paper can be very good
Virally delivered, constitutively active NFκB improves survival of injured retinal ganglion cells.

Science.gov (United States)

Dvoriantchikova, Galina; Pappas, Steve; Luo, Xueting; Ribeiro, Marcio; Danek, Dagmara; Pelaez, Daniel; Park, Kevin K; Ivanov, Dmitry

2016-12-01

As axon damage and retinal ganglion cell (RGC) loss lead to blindness, therapies that increase RGC survival and axon regrowth have direct clinical relevance. Given that NFκB signaling is critical for neuronal survival and may regulate neurite growth, we investigated the therapeutic potential of NFκB signaling in RGC survival and axon regeneration. Although both NFκB subunits (p65 and p50) are present in RGCs, p65 exists in an inactive (unphosphorylated) state when RGCs are subjected to neurotoxic conditions. In this study, we used a phosphomimetic approach to generate DNA coding for an activated (phosphorylated) p65 (p65mut), then employed an adeno-associated virus serotype 2 (AAV2) to deliver the DNA into RGCs. We tested whether constitutive p65mut expression prevents death and facilitates neurite outgrowth in RGCs subjected to transient retinal ischemia or optic nerve crush (ONC), two models of neurotoxicity. Our data indicate that RGCs treated with AAV2-p65mut displayed a significant increase in survival compared to controls in ONC model (77 ± 7% vs. 25 ± 3%, P-value = 0.0001). We also found protective effect of modified p65 in RGCs of ischemic retinas (55 ± 12% vs. 35 ± 6%), but not to a statistically significant degree (P-value = 0.14). We did not detect a difference in axon regeneration between experimental and control animals after ONC. These findings suggest that increased NFκB signaling in RGCs attenuates retinal damage in animal models of neurodegeneration, but insignificantly impacts axon regeneration. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.
Constitutive activation of BMP signalling abrogates experimental metastasis of OVCA429 cells via reduced cell adhesion

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Shepherd Trevor G

2010-02-01

Full Text Available Abstract Background Activation of bone morphogenetic protein (BMP4 signalling in human ovarian cancer cells induces a number of phenotypic changes in vitro, including altered cell morphology, adhesion, motility and invasion, relative to normal human ovarian surface epithelial cells. From these in vitro analyses, we had hypothesized that active BMP signalling promotes the metastatic potential of ovarian cancer. Methods To test this directly, we engineered OVCA429 human ovarian cancer cells possessing doxycycline-inducible expression of a constitutively-active mutant BMP receptor, ALK3QD, and administered these cells to immunocompromised mice. Further characterization was performed in vitro to address the role of activated BMP signalling on the EOC phenotype, with particular emphasis on epithelial-mesenchymal transition (EMT and cell adhesion. Results Unexpectedly, doxycycline-induced ALK3QD expression in OVCA429 cells reduced tumour implantation on peritoneal surfaces and ascites formation when xenografted into immunocompromised mice by intraperitoneal injection. To determine the potential mechanisms controlling this in vivo observation, we followed with several cell culture experiments. Doxycycline-induced ALK3QD expression enhanced the refractile, spindle-shaped morphology of cultured OVCA429 cells eliciting an EMT-like response. Using in vitro wound healing assays, we observed that ALK3QD-expressing cells migrated with long, cytoplasmic projections extending into the wound space. The phenotypic alterations of ALK3QD-expressing cells correlated with changes in specific gene expression patterns of EMT, including increased Snail and Slug and reduced E-cadherin mRNA expression. In addition, ALK3QD signalling reduced β1- and β3-integrin expression, critical molecules involved in ovarian cancer cell adhesion. The combination of reduced E-cadherin and β-integrin expression correlates directly with the reduced EOC cell cohesion in spheroids and
Initiating the judicial review in the European model of constitutional justice

OpenAIRE

Stojanović Dragan

2014-01-01

Judicial review is the core competence of the constitutional judicature in Europe, which is largely shaped by the Austrian and German models of constitutional justice. In that context, the issue of initiating the constitutional review of legislation is extremely important. Depending on the subject who is authorized to initiate this proceeding, the constitutional review may be twofold: the abstract control and the incidental control. The former type of constitutional review is generally initia...
Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+

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Nigou Jérôme

2007-08-01

Full Text Available Abstract Background The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase. Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear. Results We determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own. Conclusion The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity.
Constitutionalism and conflict in Ternate, North Maluku, Indonesia

NARCIS (Netherlands)

Hermkens, A.K.

2010-01-01

Allegedly the oldest in Indonesia, and to some even beyond, the constitution treasured in the Kedaton (traditional palace) of the Sultan of Ternate (North Maluku, Indonesia) constitutes a dividing line between North and South Ternate in terms of government, ethnicity, and, spirituality. Moreover, it
Gender and the Constitution

Science.gov (United States)

Ginsburg, Ruth Bader

1975-01-01

In discussing the constitutional aspects of the sex-role debate in the U.S. the author traces the tradition, compares the present criterion of equal protection to the equal rights argument, and analyzes the equality principle with reference to affirmative action and to childbearing and childrearing, supporting the proposed equal rights amendment.…
GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine

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David B. Finlay

2016-03-01

Full Text Available The orphan receptor GPR18 has become a research target following the discovery of a putative endogenous agonist, N-arachidonoyl glycine (NAGly. Chemical similarity between NAGly and the endocannabinoid anandamide suggested the hypothesis that GPR18 is a third cannabinoid receptor. GPR18-mediated cellular signalling through inhibition of cyclic adenosine monophosphate (cAMP and phosphorylation of extracellular signal-regulated kinase (ERK, in addition to physiological consequences such as regulation of cellular migration and proliferation/apoptosis have been described in response to both NAGly and anandamide. However, discordant findings have also been reported. Here we sought to describe the functional consequences of GPR18 activation in heterologously-expressing HEK cells. GPR18 expression was predominantly intracellular in stably transfected cell lines, but moderate cell surface expression could be achieved in transiently transfected cells which also had higher overall expression. Assays were employed to characterise the ability of NAGly or anandamide to inhibit cAMP or induce ERK phosphorylation through GPR18, or induce receptor trafficking. Positive control experiments, which utilised cells expressing hCB1 receptors (hCB1R, were performed to validate assay design and performance. While these functional pathways in GPR18-expressing cells were not modified on treatment with a panel of putative GPR18 ligands, a constitutive phenotype was discovered for this receptor. Our data reveal that GPR18 undergoes rapid constitutive receptor membrane trafficking—several-fold faster than hCB1R, a highly constitutively active receptor. To enhance the likelihood of detecting agonist-mediated receptor signalling responses, we increased GPR18 protein expression (by tagging with a preprolactin signal sequence and generated a putative constitutively inactive receptor by mutating the hGPR18 gene at amino acid site 108 (alanine to asparagine. This A108N mutant
Perspective of the Human Body in Sasang Constitutional Medicine

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Junhee Lee

2009-01-01

Full Text Available The Sasang constitutional medicine (SCM, a medical tradition originating from Korea, is distinguished from the traditional Chinese medicine in its philosophical background, theoretical development and especially, the fundamental rationale that analyzes the structure and function of the human body within a quadrifocal scheme. In SCM, the structure of the body is comprehended within the Sasang quadrifocal scheme, and the function of the body is understood within the context of the energy-fluid metabolism and the water-food metabolism controlled by the four main organs (lung, spleen, liver and kidney. Also, the concept of Seong-Jeong is used to explain the structural and functional variations between different constitutional types that arise from the constitutional variations in organ system scheme, which are in turn caused by deviations in the constitutional Seong-Jeong. Therefore, understanding the SCM perspective of the human body is essential in order to fully appreciate the advantages of the constitutional typological system (which focuses on individual idiosyncrasies found in SCM.
Constitutional reform processes and political parties: principles for practice

OpenAIRE

Vliet, van, M.; Wahiu, W.; Magolowondo, A.

2012-01-01

This publication provides a set of guiding principles for constitutional reform on the basis of practical experiences of constitutional reform processes in selected countries: Bolivia, Ghana, Indonesia, Iraq, Kenya, Malawi, Zimbabwe and South Africa. The focus is on the role of political parties in constitution-building processes. The publication shows that although country-specific reform processes may be unique in terms of objectives, context, popular involvement, and achievements, they go ...
Social pacts and Constitutional Justice: A comparative approach between Korea and Colombia.

Directory of Open Access Journals (Sweden)

Rodrigo González Quintero

2011-12-01

Full Text Available During the last quarter of the twentieth century, new constitutions were adopted in South Korea and Colombia and these documents established constitutional courts. Thus, under specific circumstances of institutional crisis, two societies and their leaders embraced the task of overcoming said crisis through the enactment of a new constitution. The establishment of constitutional courts entailed the validity towards the future of the reached constitutional consensus,as the successful way to surpass the prior crisis. The new constitutions at our countries are perceived as the reformulation of the social compact, and the constitutional courts play a constant and noteworthy function as they actualize said democratic-constitutional compact.
Pathophysiological consequences of receptor mistraffic: Tales from the platelet P2Y12 receptor.

Science.gov (United States)

Cunningham, Margaret R; Aungraheeta, Riyaad; Mundell, Stuart J

2017-07-05

Genetic variations in G protein-coupled receptor (GPCR) genes can disrupt receptor function in a wide variety of human genetic diseases, including platelet bleeding disorders. Platelets are critical for haemostasis with inappropriate platelet activation leading to the development of arterial thrombosis, which can result in heart attack and stroke whilst decreased platelet activity is associated with an increased risk of bleeding. GPCRs expressed on the surface of platelets play key roles in regulating platelet activity and therefore function. Receptors include purinergic receptors (P2Y 1 and P2Y 12 ), proteinase-activated receptor (PAR1 and PAR4) and thromboxane receptors (TPα), among others. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis. With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms (SNPs) and insertion or deletions that have the potential to alter GPCR expression or function. A number of defects in platelet GPCRs that disrupt receptor function have now been characterized in patients with mild bleeding disorders. This review will focus on rare, function-disrupting variants of platelet GPCRs with particular emphasis upon mutations in the P2Y 12 receptor gene that affect receptor traffic to modulate platelet function. Further this review will outline how the identification and characterization of function-disrupting GPCR mutations provides an essential link in translating our detailed understanding of receptor traffic and function in cell line studies into relevant human biological systems. Copyright © 2017. Published by Elsevier B.V.
Vestigialization of an Allosteric Switch: Genetic and Structural Mechanisms for the Evolution of Constitutive Activity in a Steroid Hormone Receptor

Science.gov (United States)

Bridgham, Jamie T.; Keay, June; Ortlund, Eric A.; Thornton, Joseph W.

2014-01-01

An important goal in molecular evolution is to understand the genetic and physical mechanisms by which protein functions evolve and, in turn, to characterize how a protein's physical architecture influences its evolution. Here we dissect the mechanisms for an evolutionary shift in function in the mollusk ortholog of the steroid hormone receptors (SRs), a family of biologically essential transcription factors. In vertebrates, the activity of SRs allosterically depends on binding a hormonal ligand; in mollusks, however, the SR ortholog (called ER, because of high sequence similarity to vertebrate estrogen receptors) activates transcription in the absence of ligand and does not respond to steroid hormones. To understand how this shift in regulation evolved, we combined evolutionary, structural, and functional analyses. We first determined the X-ray crystal structure of the ER of the Pacific oyster Crassostrea gigas (CgER), and found that its ligand pocket is filled with bulky residues that prevent ligand occupancy. To understand the genetic basis for the evolution of mollusk ERs' unique functions, we resurrected an ancient SR progenitor and characterized the effect of historical amino acid replacements on its functions. We found that reintroducing just two ancient replacements from the lineage leading to mollusk ERs recapitulates the evolution of full constitutive activity and the loss of ligand activation. These substitutions stabilize interactions among key helices, causing the allosteric switch to become “stuck” in the active conformation and making activation independent of ligand binding. Subsequent changes filled the ligand pocket without further affecting activity; by degrading the allosteric switch, these substitutions vestigialized elements of the protein's architecture required for ligand regulation and made reversal to the ancestral function more complex. These findings show how the physical architecture of allostery enabled a few large-effect mutations
Vestigialization of an allosteric switch: genetic and structural mechanisms for the evolution of constitutive activity in a steroid hormone receptor.

Directory of Open Access Journals (Sweden)

Jamie T Bridgham

2014-01-01

Full Text Available An important goal in molecular evolution is to understand the genetic and physical mechanisms by which protein functions evolve and, in turn, to characterize how a protein's physical architecture influences its evolution. Here we dissect the mechanisms for an evolutionary shift in function in the mollusk ortholog of the steroid hormone receptors (SRs, a family of biologically essential transcription factors. In vertebrates, the activity of SRs allosterically depends on binding a hormonal ligand; in mollusks, however, the SR ortholog (called ER, because of high sequence similarity to vertebrate estrogen receptors activates transcription in the absence of ligand and does not respond to steroid hormones. To understand how this shift in regulation evolved, we combined evolutionary, structural, and functional analyses. We first determined the X-ray crystal structure of the ER of the Pacific oyster Crassostrea gigas (CgER, and found that its ligand pocket is filled with bulky residues that prevent ligand occupancy. To understand the genetic basis for the evolution of mollusk ERs' unique functions, we resurrected an ancient SR progenitor and characterized the effect of historical amino acid replacements on its functions. We found that reintroducing just two ancient replacements from the lineage leading to mollusk ERs recapitulates the evolution of full constitutive activity and the loss of ligand activation. These substitutions stabilize interactions among key helices, causing the allosteric switch to become "stuck" in the active conformation and making activation independent of ligand binding. Subsequent changes filled the ligand pocket without further affecting activity; by degrading the allosteric switch, these substitutions vestigialized elements of the protein's architecture required for ligand regulation and made reversal to the ancestral function more complex. These findings show how the physical architecture of allostery enabled a few large
The application of section 8(3) of the constitution in the development of customary law values in South Africa's new constitutional dispensation

OpenAIRE

Ntlama, Nomthandazo

2012-01-01

The constitutional recognition of customary law alongside common law in the Constitution of the Republic of South Africa, 1996 is highly commendable. It also raises the question of whether or not the recognition was undertaken out of genuine respect for customary law or merely forgotten in section 8(3) of the Constitution. It is argued that the exclusion of customary law from the provision of the section is nothing more than the advancement of the dominant status enjoyed by common law, as was...

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