Science.gov

Sample records for arrestin content studied

  1. GPCR Signaling: β-arrestins Kiss and Remember.

    PubMed

    Ranjan, Ravi; Gupta, Pragya; Shukla, Arun K

    2016-04-01

    β-arrestin-dependent activation of the ERK MAP kinase downstream of GPCRs typically originates from internalized receptor-β-arrestin-ERK complexes. A new study now reports that β-arrestin 2 is sufficiently 'primed' after 'kissing' the β1-adrenergic receptor to initiate ERK activation even in the absence of formation of the receptor-β-arrestin-ERK complex and receptor internalization.

  2. Different conformational dynamics of β-arrestin1 and β-arrestin2 analyzed by hydrogen/deuterium exchange mass spectrometry

    SciTech Connect

    Yun, Youngjoo; Kim, Dong Kyun; Seo, Min-Duk; Kim, Kyeong-Man; Chung, Ka Young

    2015-01-30

    Highlights: • The conformational dynamics of β-arrestin1 or β-arrestin2 were analyzed by HDX-MS. • β-Strands II through IV were more dynamic in β-arrestin2 than in β-arrestin1. • The middle loop was less dynamic in β-arrestin2 than in β-arrestin1. • Upon pre-activation by the R169E mutation, β-arrestins became more dynamic. • Pre-activation affected a wider region of β-arrestin1 compared to β-arrestin2. - Abstract: Arrestins have important roles in G protein-coupled receptor (GPCR) signaling including desensitization of GPCRs and G protein-independent signaling. There have been four arrestins identified: arrestin1, arrestin2 (e.g. β-arrestin1), arrestin3 (e.g. β-arrestin2), and arrestin4. β-Arrestin1 and β-arrestin2 are ubiquitously expressed and regulate a broad range of GPCRs, while arrestin1 and arrestin4 are expressed in the visual system. Although the functions of β-arrestin1 and β-arrestin2 widely overlap, β-arrestin2 has broader receptor selectivity, and a few studies have suggested that β-arrestin1 and β-arrestin2 have distinct cellular functions. Here, we compared the conformational dynamics of β-arrestin1 and β-arrestin2 by hydrogen/deuterium exchange mass spectrometry (HDX-MS). We also used the R169E mutant as a pre-activation model system. HDX-MS data revealed that β-strands II through IV were more dynamic in β-arrestin2 in the basal state, while the middle loop was more dynamic in β-arrestin1. With pre-activation, both β-arrestin1 and β-arrestin2 became more flexible, but broader regions of β-arrestin1 became flexible compared to β-arrestin2. The conformational differences between β-arrestin1 and β-arrestin2 in both the basal and pre-activated states might determine their different receptor selectivities and different cellular functions.

  3. Arrestin Expression in E. coli and Purification

    PubMed Central

    Vishnivetskiy, Sergey A.; Zhan, Xuanzhi; Chen, Qiuyan; Iverson, Tina M.; Gurevich, Vsevolod V.

    2014-01-01

    Purified arrestin proteins are necessary for biochemical, biophysical, and crystallographic studies of these versatile regulators of cell signaling. Here we describe a basic protocol for expression in E. coli and purification of tag-free wild type and mutant arrestins. The method includes ammonium sulfate precipitation of arrestins from cell lysates, followed by heparin-Sepharose chromatography. Depending on the arrestin type and/or mutations, this step is followed by Q-Sepharose or SP-Sepharose chromatography. In many cases the non-binding column is used as a pre-filter to bind contaminants without retaining arrestin. In some cases both chromatographic steps need to be performed sequentially to achieve high purity. Purified arrestins can be concentrated up to 10 mg/ml, remain fully functional, and can withstand several cycles of freezing and thawing, provided that overall salt concentration is kept at or above physiological levels. PMID:25446290

  4. β-arrestins in the Immune System

    PubMed Central

    Xie, Ting; Liang, Jiurong

    2015-01-01

    Summary β-arrestins regulate G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) through receptor desensitization while also acting as signaling scaffolds to facilitate numerous effector pathways. Recent studies have provided evidence that β-arrestins play a key role in inflammatory responses. We here summarize these advances on the roles of β-arrestins in immune regulation and inflammatory responses under physiological and pathological conditions, with an emphasis on translational implications of β-arrestins on human diseases. PMID:23764061

  5. β-Arrestins promote podocyte injury by inhibition of autophagy in diabetic nephropathy

    PubMed Central

    Liu, J; Li, Q X; Wang, X J; Zhang, C; Duan, Y Q; Wang, Z Y; Zhang, Y; Yu, X; Li, N J; Sun, J P; Yi, F

    2016-01-01

    β-Arrestins are multifunctional proteins originally identified as negative adaptors of G protein-coupled receptors (GPCRs). Emerging evidence has also indicated that β-arrestins can activate signaling pathways independent of GPCR activation. This study was to elucidate the role of β-arrestins in diabetic nephropathy (DN) and hypothesized that β-arrestins contribute to diabetic renal injury by mediating podocyte autophagic process. We first found that both β-arrestin-1 and β-arrestin-2 were upregulated in the kidney from streptozotocin-induced diabetic mice, diabetic db/db mice and kidney biopsies from diabetic patients. We further revealed that either β-arrestin-1 or β-arrestin-2 deficiency (Arrb1−/− or Arrb2−/−) ameliorated renal injury in diabetic mice. In vitro, we observed that podocytes increased both β-arrestin-1 and β-arrestin-2 expression levels under hyperglycemia condition and further demonstrated that β-arrestin-1 and β-arrestin-2 shared common mechanisms to suppress podocyte autophagy by negative regulation of ATG12–ATG5 conjugation. Collectively, this study for the first time demonstrates that β-arrestin-1 and β-arrestin-2 mediate podocyte autophagic activity, indicating that β-arrestins are critical components of signal transduction pathways that link renal injury to reduce autophagy in DN. Modulation of these pathways may be an innovative therapeutic strategy for treating patients with DN. PMID:27054338

  6. The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

    PubMed

    Robinson, Kelly A; Ou, Wei-Lin; Guan, Xinyu; Sugamori, Kim S; Bandyopadhyay, Abhishek; Ernst, Oliver P; Mitchell, Jane

    2015-12-01

    Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation.

  7. The effect of phosphorylation on arrestin-rhodopsin interaction in the squid visual system.

    PubMed

    Robinson, Kelly A; Ou, Wei-Lin; Guan, Xinyu; Sugamori, Kim S; Bandyopadhyay, Abhishek; Ernst, Oliver P; Mitchell, Jane

    2015-12-01

    Invertebrate visual opsins are G protein-coupled receptors coupled to retinoid chromophores that isomerize reversibly between inactive rhodopsin and active metarhodopsin upon absorption of photons of light. The squid visual system has an arrestin protein that binds to metarhodopsin to block signaling to Gq and activation of phospholipase C. Squid rhodopsin kinase (SQRK) can phosphorylate both metarhodopsin and arrestin, a dual role that is unique among the G protein-coupled receptor kinases. The sites and role of arrestin phosphorylation by SQRK were investigated here using recombinant proteins. Arrestin was phosphorylated on serine 392 and serine 397 in the C-terminus. Unphosphorylated arrestin bound to metarhodopsin and phosphorylated metarhodopsin with similar high affinities (Kd 33 and 21 nM respectively), while phosphorylation of arrestin reduced the affinity 3- to 5-fold (Kd 104 nM). Phosphorylation of metarhodopsin slightly increased the dissociation of arrestin observed during a 1 hour incubation. Together these studies suggest a unique role for SQRK in phosphorylating both receptor and arrestin and inhibiting the binding of these two proteins in the squid visual system. Invertebrate visual systems are inactivated by arrestin binding to metarhodopsin that does not require receptor phosphorylation. Here we show that squid rhodopsin kinase phosphorylates arrestin on two serines (S392,S397) in the C-terminus and phosphorylation decreases the affinity of arrestin for squid metarhodopsin. Metarhodopsin phosphorylation has very little effect on arrestin binding but does increase arrestin dissociation. PMID:26375013

  8. Depletion of β-arrestin2 in hepatic stellate cells reduces cell proliferation via ERK pathway.

    PubMed

    Sun, Wu-Yi; Song, Yang; Hu, Shan-Shan; Wang, Qing-Tong; Wu, Hua-Xun; Chen, Jing-Yu; Wei, Wei

    2013-05-01

    β-Arrestins are multifunctional adaptor proteins. Recently, some new roles of β-arrestins in regulating intracellular signaling networks have been discovered, which regulate cell growth, proliferation, and apoptosis. Though, the role of β-arrestins expression in the pathology of hepatic fibrosis remains unclear. In this study, the possible relationship between the expression of β-arrestins with the experimental hepatic fibrosis and the proliferation of hepatic stellate cells (HSCs) were investigated. Porcine serum induced liver fibrosis was established in this study. At five time points, the dynamic expression of β-arrestin1, β-arrestin2, and α-smooth muscle actin (α-SMA) in rat liver tissues, was measured by immunohistochemical staining, double immunofluorescent staining, and Western blotting. This study showed that aggravation of hepatic fibrosis with gradually increasing expression of β-arrestin2 in the hepatic tissues, but not β-arrestin1. Further, as hepatic fibrosis worsens, β-arrestin2-expressing activated HSCs accounts for an increasingly larger percentage of all activated HSCs. And the expression of β-arrestin2 had a significant positive correlation with the expression of α-SMA, an activated HSCs marker. In vitro studies, the dynamic expression of β-arrestin1 and β-arrestin2 in platelet derived growth factor-BB (PDGF-BB) stimulated HSCs was assessed by Western blotting. The expression of β-arrestin2 was remarkably increased in PDGF-BB stimulated HSCs. Furthermore, the small interfering RNA (siRNA) technique was used to explore the effect of β-arrestins on the proliferation of HSCs and the activation of ERK1/2. Transfection of siRNA targeting β-arrestin2 mRNA (siβ-arrestin2) into HSCs led to a 68% and 70% reduction of β-arrestin2 mRNA and protein expression, respectively. siβ-arrestin2 abolished the effect of PDGF-BB on the proliferation of HSCs. In addition, siβ-arrestin2 exerted the inhibition of the activation of ERK1/2 in HSCs. The

  9. Down-regulation of β-arrestin2 promotes tumour invasion and indicates poor prognosis of hepatocellular carcinoma

    PubMed Central

    Sun, Wu-Yi; Hu, Shan-Shan; Wu, Jing-Jing; Huang, Qiong; Ma, Yang; Wang, Qing-Tong; Chen, Jing-Yu; Wei, Wei

    2016-01-01

    β-arrestins, including β-arrestin1 and β-arrestin2, are multifunctional adaptor proteins. β-arrestins have recently been found to play new roles in regulating intracellular signalling networks associated with malignant cell functions. Altered β-arrestin expression has been reported in many cancers, but its role in hepatocellular carcinoma (HCC) is not clear. We therefore examined the roles of β-arrestins in HCC using an animal model of progressive HCC, HCC patient samples and HCC cell lines with stepwise metastatic potential. We demonstrated that β-arrestin2 level, but not β-arrestin1 level, decreased in conjunction with liver tumourigenesis in a mouse diethylnitrosamine-induced liver tumour model. Furthermore, β-arrestin2 expression was reduced in HCC tissues compared with noncancerous tissues in HCC patients. β-arrestin2 down-regulation in HCC was significantly associated with poor patient prognoses and aggressive pathologic features. In addition, our in vitro study showed that β-arrestin2 overexpression significantly reduced cell migration and invasion in cultured HCC cells. Furthermore, β-arrestin2 overexpression up-regulated E-cadherin expression and inhibited vimentin expression and Akt activation. These results suggest that β-arrestin2 down-regulation increases HCC cell migration and invasion ability. Low β-arrestin2 expression may be indicative of a poor prognosis or early cancer recurrence in patients who have undergone surgery for HCC. PMID:27759077

  10. β-Arrestin-1 directly interacts with Gαs and regulates its function.

    PubMed

    Li, Bo; Wang, Congcong; Zhou, Zhaocai; Zhao, Jian; Pei, Gang

    2013-03-01

    β-Arrestins function to mediate G protein-coupled receptor (GPCR) desensitization and internalization and to initiate G protein independent signaling of GPCRs. Elucidating how β-arrestin and G protein signal pathways coordinate with each other is important to fully understand GPCR signaling. Here we report that β-arrestin-1 directly interacts with Gα(s). Purified β-arrestin-1 binds to Gα(s) in a rapid association and dissociation manner. β-Arrestin-1 promotes the binding and the release of GTPγS from Gα(s) in vitro. β-Arrestin-1 L33K mutant shows reduced interaction with Gα(s) and has no detectable effects on Gα(s) function. Our study thus reveals a direct crosstalk of β-arrestin-1 with Gα(s).

  11. Agonist-Specific Recruitment of Arrestin Isoforms Differentially Modify Delta Opioid Receptor Function

    PubMed Central

    Perroy, Julie; Walwyn, Wendy M.; Smith, Monique L.; Vicente-Sanchez, Ana; Segura, Laura; Bana, Alia; Kieffer, Brigitte L.; Evans, Christopher J.

    2016-01-01

    ) preferentially recruit arrestin 3 and, surprisingly, KO of arrestin 3 produces acute tolerance and impaired receptor resensitization to these agonists. Arrestin 3 is in pre-engaged complexes with the delta opioid receptor at the cell membrane and low-internalizing agonists promote this interaction. This study reveals a novel role for arrestin 3 as a facilitator of receptor resensitization. PMID:27013682

  12. The functional cycle of visual arrestins in photoreceptor cells

    PubMed Central

    Gurevich, Vsevolod V.; Hanson, Susan M.; Song, Xiufeng; Vishnivetskiy, Sergey A.; Gurevich, Eugenia V.

    2011-01-01

    Visual arrestin-1 plays a key role in the rapid and reproducible shutoff of rhodopsin signaling. Its highly selective binding to light-activated phosphorylated rhodopsin is an integral part of the functional perfection of rod photoreceptors. Structure-function studies revealed key elements of the sophisticated molecular mechanism ensuring arrestin-1 selectivity and paved the way to the targeted manipulation of the arrestin-1 molecule to design mutants that can compensate for congenital defects in rhodopsin phosphorylation. Arrestin-1 self-association and light-dependent translocation in photoreceptor cells work together to keep a constant supply of active rhodopsin-binding arrestin-1 monomer in the outer segment. Recent discoveries of arrestin-1 interaction with other signaling proteins suggest that it is a much more versatile signaling regulator than previously thought, affecting the function of the synaptic terminals and rod survival. Elucidation of the fine molecular mechanisms of arrestin-1 interactions with rhodopsin and other binding partners is necessary for the comprehensive understanding of rod function and for devising novel molecular tools and therapeutic approaches to the treatment of visual disorders. PMID:21824527

  13. Multifaceted role of β-arrestins in inflammation and disease

    PubMed Central

    Sharma, Deepika; Parameswaran, Narayanan

    2015-01-01

    Arrestins are intracellular scaffolding proteins known to regulate a range of biochemical processes including G-protein coupled receptor (GPCR) desensitization, signal attenuation, receptor turnover and downstream signaling cascades. Their roles in regulation of signaling network have lately been extended to receptors outside of the GPCR family, demonstrating their roles as important scaffolding proteins in various physiological processes including proliferation, differentiation, and apoptosis. Recent studies have demonstrated a critical role for arrestins in immunological processes including key functions in inflammatory signaling pathways. In this review, we provide a comprehensive analysis of the different functions of the arrestin family of proteins especially related to immunity and inflammatory diseases. PMID:26378652

  14. Functional characterization of an arrestin gene on insecticide resistance of Culex pipiens pallens

    PubMed Central

    2012-01-01

    Background Continuous and excessive application of insecticides has resulted in the rapid development of insecticide resistance in several mosquito species, including Culex pipiens pallens. Previous studies in our laboratory found that arrestin gene expression was higher in the deltamethrin-resistant (DR) strain than in the deltamethrin-susceptible (DS) strain of Cx. pipiens pallens. Similarly, other studies reported that arrestin was highly expressed in permethrin-resistant Cx. quinquefasciatus and in dichlorodiphenyltrichloroethane (DDT)-resistant Drosophila melanogaster. Methods Full-length cDNAs of an arrestin gene were cloned from Cx. pipiens pallens via polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE). The mRNA levels of the arrestin gene in the whole life cycle of DR and DS strains of Cx. pipiens pallens were investigated via quantitative real-time PCR. In addition, the relationship between arrestin and deltamethrin (DM) resistance were identified using genetic overexpression strategies and arrestin RNAi in mosquito cells. Cell viability was analyzed with cholecystokinin octapeptide after DM treatment. Moreover, the mRNA levels of cytochrome P450 6A1 (CYP6A1) and opsin in the transfected cells and controls were analyzed. Results Complete arrestin gene sequence was cloned and expressed throughout the life cycle of Cx. pipiens pallens. Moreover, arrestin was significantly upregulated in the DR strain, compared with that in the DS strain at the egg, pupae, and adult stages. Arrestin overexpression comparably increased the mosquito cell viability, whereas arrestin knockdown by siRNA decreased mosquito cell viability with deltamethrin (DM) treatment. Meanwhile, the mRNA levels of CYP6A1 and opsin were upregulated in mosquito cells transfected with arrestin and downregulated in mosquito cells with arrestin knockdown. Conclusion This study presented the first evidence that arrestin might be associated with insecticide resistance in Cx

  15. Structure of an Arrestin2-clathrin Complex Reveals a Novel Clathrin Binding Domain that Modulates Receptor Trafficking

    SciTech Connect

    Kang, D.; Kern, R; Puthenveedu, M; von Zastrow, M; Williams, J; Benovic, J

    2009-01-01

    Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin {beta}-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formed by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I){sub 2}GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.

  16. Mammalian α arrestins link activated seven transmembrane receptors to Nedd4 family e3 ubiquitin ligases and interact with β arrestins.

    PubMed

    Shea, Fortune F; Rowell, Jennie L; Li, Yechaowei; Chang, Tien-Hsien; Alvarez, Carlos E

    2012-01-01

    The complement of fungal cell surface proteins is widely regulated by ubiquitination of membrane proteins, which results in their endocytosis and vacuolar degradation. For diverse fungal transporters, the specificity of ubiquitination is conferred by alpha arrestin adaptors, which recruit the Nedd4 family E3 ubiquitin ligase Rsp5. A recent study showed that one mammalian alpha arrestin also mediates ubiquitination and lysosomal trafficking of an activated plasma membrane receptor. Here we first screen all five widely-expressed human alpha arrestins for subcellular localization in ligand-stimulated and -unstimulated cells overexpressing the seven transmembrane receptor vasopressin 2. We then characterize the effects of alpha arrestins ARRDC3 and ARRDC4 upon activation of the seven transmembrane receptors vasopressin 2 and beta adrenergic 2. Using biochemical and imaging approaches, we show that ligand-activated receptors interact with alpha arrestins, and this results in recruitment of Nedd4 family E3 ubiquitin ligases and receptor ubiquitination - which are known to result in lysosomal trafficking. Our time course studies show these effects occur in the first 1-5 minutes after ligand activation, the same time that beta arrestins are known to have roles in receptor endocytic trafficking and kinase signaling. We tested the possibility that alpha and beta arrestins function coordinately and found co-immunoprecipitation and colocalization evidence to support this. Others recently reported that Arrdc3 knockout mice are lean and resistant to obesity. In the course of breeding our own Arrdc3-deficient mice, we observed two novel phenotypes in homozygotes: skin abnormalities, and embryonic lethality on normal chow diet, but not on high fat diet. Our findings suggest that alpha and beta arrestins function coordinately to maintain the optimal complement and function of cell surface proteins according to cellular physiological context and external signals. We discuss the

  17. An expanded family of arrestins regulate metabolism.

    PubMed

    Patwari, Parth; Lee, Richard T

    2012-05-01

    The classical visual and β-arrestins belong to a larger family of proteins that likely share structural similarity. Humans have an additional six related proteins sometimes termed the α-arrestins, whose functions are now emerging. Surprisingly, several α-arrestins play prominent roles in the regulation of metabolism and obesity. One α-arrestin, thioredoxin-interacting protein (Txnip), has crucial functions in regulating glucose uptake and glycolytic flux through the mitochondria. Another α-arrestin, Arrdc3, is linked to obesity in men and was recently identified in mice as a regulator of body mass, adiposity, and energy expenditure. Here we discuss recent evidence suggesting potential common themes for all arrestins, including physiological roles for classical arrestins in metabolism and the functions of α-arrestins in receptor signaling and endocytosis.

  18. In silico study of the human rhodopsin and meta rhodopsin II/S-arrestin complexes: impact of single point mutations related to retina degenerative diseases.

    PubMed

    Mokarzel-Falcón, Leonardo; Padrón-García, Juan Alexander; Carrasco-Velar, Ramón; Berry, Colin; Montero-Cabrera, Luis A

    2008-03-01

    We propose two models of the human S-arrestin/rhodopsin complex in the inactive dark adapted rhodopsin and meta rhodopsin II form, obtained by homology modeling and knowledge based docking. First, a homology model for the human S-arrestin was built and validated by molecular dynamics, showing an average root mean square deviation difference from the pattern behavior of 0.76 A. Then, combining the human S-arrestin model and the modeled structure of the two human rhodopsin forms, we propose two models of interaction for the human S-arrestin/rhodopsin complex. The models involve two S-arrestin regions related to the N domain (residues 68-78; 170-182) and a third constituent of the C domain (248-253), with the rhodopsin C terminus (330-348). Of the 22 single point mutations related to retinitis pigmentosa and congenital night blindness located in the cytoplasmatic portion of rhodopsin or in S-arrestin, our models locate 16 in the interaction region and relate two others to possible dimer formation. Our calculations also predict that the light activated complex is more stable than the dark adapted rhodopsin and, therefore, of higher affinity to S-arrestin.

  19. Beta-Arrestin2 regulates RANKL and ephrins gene expression in response to bone remodeling in mice.

    PubMed

    Pierroz, Dominique D; Rufo, Anna; Bianchi, Estelle N; Glatt, Vaida; Capulli, Mattia; Rucci, Nadia; Cavat, Fanny; Rizzoli, René; Teti, Anna; Bouxsein, Mary L; Ferrari, Serge L

    2009-05-01

    PTH-stimulated intracellular signaling is regulated by the cytoplasmic adaptor molecule beta-arrestin. We reported that the response of cancellous bone to intermittent PTH is reduced in beta-arrestin2(-/-) mice and suggested that beta-arrestins could influence the bone mineral balance by controlling RANKL and osteoprotegerin (OPG) gene expression. Here, we study the role of beta-arrestin2 on the in vitro development and activity of bone marrow (BM) osteoclasts (OCs) and Ephrins ligand (Efn), and receptor (Eph) mRNA levels in bone in response to PTH and the changes of bone microarchitecture in wildtype (WT) and beta-arrestin2(-/-) mice in models of bone remodeling: a low calcium diet (LoCa) and ovariectomy (OVX). The number of PTH-stimulated OCs was higher in BM cultures from beta-arrestin2(-/-) compared with WT, because of a higher RANKL/OPG mRNA and protein ratio, without directly influencing osteoclast activity. In vivo, high PTH levels induced by LoCa led to greater changes in TRACP5b levels in beta-arrestin2(-/-) compared with WT. LoCa caused a loss of BMD and bone microarchitecture, which was most prominent in beta-arrestin2(-/-). PTH downregulated Efn and Eph genes in beta-arrestin2(-/-), but not WT. After OVX, vertebral trabecular bone volume fraction and trabecular number were lower in beta-arrestin2(-/-) compared with WT. Histomorphometry showed that OC number was higher in OVX-beta-arrestin2(-/-) compared with WT. These results indicate that beta-arrestin2 inhibits osteoclastogenesis in vitro, which resulted in decreased bone resorption in vivo by regulating RANKL/OPG production and ephrins mRNAs. As such, beta-arrestins should be considered an important mechanism for the control of bone remodeling in response to PTH and estrogen deprivation. PMID:19113915

  20. Dual mode of glucagon receptor internalization: role of PKCα, GRKs and β-arrestins.

    PubMed

    Krilov, Lada; Nguyen, Amy; Miyazaki, Teruo; Unson, Cecilia G; Williams, Russell; Lee, Norman H; Ceryak, Susan; Bouscarel, Bernard

    2011-12-10

    Glucagon levels are elevated in diabetes and some liver diseases. Increased glucagon secretion leads to abnormal stimulation of glucagon receptors (GRs) and consequent elevated glucose production in the liver. Blocking glucagon receptor signaling has been proposed as a potential treatment option for diabetes and other conditions associated with hyperglycemia. Elucidating mechanisms of GR desensitization and downregulation may help identify new drug targets besides GR itself. The present study explores the mechanisms of GR internalization and the role of PKCα, GPCR kinases (GRKs) and β-arrestins therein. We have reported previously that PKCα mediates GR phosphorylation and desensitization. While the PKC agonist, PMA, did not affect GR internalization when tested alone, it increased glucagon-mediated GR internalization by 25-40% in GR-expressing HEK-293 cells (HEK-GR cells). In both primary hepatocytes and HEK-GR cells, glucagon treatment recruited PKCα to the plasma membrane where it colocalized with GR. We also observed that overexpression of GRK2, GRK3, or GRK5 enhanced GR internalization. In addition, we found that GR utilizes both clathrin- and caveolin-mediated endocytosis in HEK-GR cells. Glucagon triggered translocation of both β-arrestin1 and β-arrestin2 from the cytosol to the perimembrane region, and overexpression of β-arrestin1 and β-arrestin2 increased GR internalization. Furthermore, both β-arrestin1 and β-arrestin2 colocalized with GR and with Cav-1, suggesting the possible involvement of these arrestins in GR internalization.

  1. Quantification of beta adrenergic receptor subtypes in beta-arrestin knockout mouse airways.

    PubMed

    Hegde, Akhil; Strachan, Ryan T; Walker, Julia K L

    2015-01-01

    In allergic asthma Beta 2 adrenergic receptors (β2ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β2AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β2AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β2AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β2ARs predominate over β1ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β2AR expression. These data provide further evidence that β2ARs are expressed in greater abundance than β1ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β2AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference

  2. β-arrestin-1 contributes to brown fat function and directly interacts with PPARα and PPARγ

    PubMed Central

    Wang, Congcong; Zeng, Xianglu; Zhou, Zhaocai; Zhao, Jian; Pei, Gang

    2016-01-01

    The peroxisome proliferator-activated receptor (PPAR) family plays central roles in brown adipose tissue (BAT) adipogenesis and contributes to body temperature maintenance. The transcriptional activity of PPAR family has been shown to be tightly controlled by cellular signal networks. β-arrestins function as major secondary messengers of G protein-coupled receptors (GPCR) signaling by functional interactions with diverse proteins. Here, we report that β-arrestin-1 knock-out mice show enhanced cold tolerance. We found that β-arrestin-1 directly interacts with PPARα and PPARγ through a LXXXLXXXL motif, while D371 in PPARα and L311/N312/D380 in PPARγ are required for their interactions with β-arrestin-1. Further mechanistic studies showed that β-arrestin-1 promotes PPARα- but represses PPARγ-mediated transcriptional activities, providing potential regulatory pathway for BAT function. PMID:27301785

  3. β-arrestin-1 contributes to brown fat function and directly interacts with PPARα and PPARγ.

    PubMed

    Wang, Congcong; Zeng, Xianglu; Zhou, Zhaocai; Zhao, Jian; Pei, Gang

    2016-01-01

    The peroxisome proliferator-activated receptor (PPAR) family plays central roles in brown adipose tissue (BAT) adipogenesis and contributes to body temperature maintenance. The transcriptional activity of PPAR family has been shown to be tightly controlled by cellular signal networks. β-arrestins function as major secondary messengers of G protein-coupled receptors (GPCR) signaling by functional interactions with diverse proteins. Here, we report that β-arrestin-1 knock-out mice show enhanced cold tolerance. We found that β-arrestin-1 directly interacts with PPARα and PPARγ through a LXXXLXXXL motif, while D371 in PPARα and L311/N312/D380 in PPARγ are required for their interactions with β-arrestin-1. Further mechanistic studies showed that β-arrestin-1 promotes PPARα- but represses PPARγ-mediated transcriptional activities, providing potential regulatory pathway for BAT function. PMID:27301785

  4. Arrestins: ubiquitous regulators of cellular signaling pathways.

    PubMed

    Gurevich, Eugenia V; Gurevich, Vsevolod V

    2006-01-01

    In vertebrates, the arrestins are a family of four proteins that regulate the signaling and trafficking of hundreds of different G-protein-coupled receptors (GPCRs). Arrestin homologs are also found in insects, protochordates and nematodes. Fungi and protists have related proteins but do not have true arrestins. Structural information is available only for free (unbound) vertebrate arrestins, and shows that the conserved overall fold is elongated and composed of two domains, with the core of each domain consisting of a seven-stranded beta-sandwich. Two main intramolecular interactions keep the two domains in the correct relative orientation, but both of these interactions are destabilized in the process of receptor binding, suggesting that the conformation of bound arrestin is quite different. As well as binding to hundreds of GPCR subtypes, arrestins interact with other classes of membrane receptors and more than 20 surprisingly diverse types of soluble signaling protein. Arrestins thus serve as ubiquitous signaling regulators in the cytoplasm and nucleus.

  5. Ubiquitin-Related Roles of β-Arrestins in Endocytic Trafficking and Signal Transduction.

    PubMed

    Jean-Charles, Pierre-Yves; Rajiv, Vishwaesh; Shenoy, Sudha K

    2016-10-01

    The non-visual arrestins, β-arrestin1, and β-arrestin2 were originally identified as proteins that bind to seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors, and non-receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non-receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of β-arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of β-arrestins in protein ubiquitination is evolutionarily conserved. β-arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of β-arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on β-arrestin plays a critical role in stabilizing β-arrestin-7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin-dependent regulation of 7TMRs as well as β-arrestins and the potential role of reversible ubiquitination as a "biological switch" in signal transduction. J. Cell. Physiol. 231: 2071-2080, 2016. © 2016 Wiley Periodicals, Inc.

  6. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction.

    PubMed

    Wagener, Brant M; Marjon, Nicole A; Prossnitz, Eric R

    2016-01-01

    Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

  7. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction

    PubMed Central

    Wagener, Brant M.; Marjon, Nicole A.; Prossnitz, Eric R.

    2016-01-01

    Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking. PMID:26788723

  8. Arrestins in the cardiovascular system.

    PubMed

    Lymperopoulos, Anastasios; Bathgate, Ashley

    2013-01-01

    Of the four mammalian arrestins, only the β-arrestins (βarrs; Arrestin2 and -3) are expressed throughout the cardiovascular system, where they regulate, as either desensitizers/internalizers or signal transducers, several G-protein-coupled receptors (GPCRs) critical for cardiovascular homeostasis. The cardiovascular roles of βarrs have been delineated at an accelerated pace via a variety of techniques and tools, such as knockout mice, siRNA knockdown, artificial or naturally occurring polymorphic GPCRs, and availability of new βarr "biased" GPCR ligands. This chapter summarizes the current knowledge of cardiovascular arrestin physiology and pharmacology, addressing the individual cardiovascular receptors affected by βarrs in vivo, as well as the individual cell types, tissues, and organs of the cardiovascular system in which βarr effects are exerted; for example, cardiac myocyte or fibroblast, vascular smooth muscle, adrenal gland and platelet. In the broader scope of cardiovascular βarr pharmacology, a discussion of the βarr "bias" of certain cardiovascular GPCR ligands is also included.

  9. Roles of Dopamine D₂ Receptor Subregions in Interactions with β-Arrestin2.

    PubMed

    Zhang, Xiaohan; Choi, Bo-Gil; Kim, Kyeong-Man

    2016-09-01

    β-Arrestins are one of the protein families that interact with G protein-coupled receptors (GPCRs). The roles of β-arrestins are multifaceted, as they mediate different processes including receptor desensitization, endocytosis, and G protein-independent signaling. Thus, determining the GPCR regions involved in the interactions with β-arrestins would be a preliminary step in understanding the molecular mechanisms involved in the selective direction of each function. In the current study, we determined the roles of the N-terminus, intracellular loops, and C-terminal tail of a representative GPCR in the interaction with β-arrestin2. For this, we employed dopamine D₂ and D₃ receptors (D₂R and D₃R, respectively), since they display distinct agonist-induced interactions with β-arrestins. Our results showed that the second and third intracellular loops of D₂R are involved in the agonist-induced translocation of β-arrestins toward plasma membranes. In contrast, the N- and C-termini of D₂R exerted negative effects on the basal interaction with β-arrestins.

  10. Roles of Dopamine D2 Receptor Subregions in Interactions with β-Arrestin2

    PubMed Central

    Zhang, Xiaohan; Choi, Bo-Gil; Kim, Kyeong-Man

    2016-01-01

    β-Arrestins are one of the protein families that interact with G protein-coupled receptors (GPCRs). The roles of β-arrestins are multifaceted, as they mediate different processes including receptor desensitization, endocytosis, and G protein-independent signaling. Thus, determining the GPCR regions involved in the interactions with β-arrestins would be a preliminary step in understanding the molecular mechanisms involved in the selective direction of each function. In the current study, we determined the roles of the N-terminus, intracellular loops, and C-terminal tail of a representative GPCR in the interaction with β-arrestin2. For this, we employed dopamine D2 and D3 receptors (D2R and D3R, respectively), since they display distinct agonist-induced interactions with β-arrestins. Our results showed that the second and third intracellular loops of D2R are involved in the agonist-induced translocation of β-arrestins toward plasma membranes. In contrast, the N- and C-termini of D2R exerted negative effects on the basal interaction with β-arrestins. PMID:27068263

  11. Mechanisms of Biased β-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor

    PubMed Central

    Delgado-Peraza, Francheska; Ahn, Kwang H.; Nogueras-Ortiz, Carlos; Mungrue, Imran N.; Mackie, Ken; Kendall, Debra A.

    2016-01-01

    Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling. PMID:27009233

  12. β-Arrestin Regulation of Myosin Light Chain Phosphorylation Promotes AT1aR-mediated Cell Contraction and Migration

    PubMed Central

    Simard, Elie; Kovacs, Jeffrey J.; Miller, William E.; Kim, Jihee; Grandbois, Michel; Lefkowitz, Robert J.

    2013-01-01

    Over the last decade, it has been established that G-protein-coupled receptors (GPCRs) signal not only through canonical G-protein-mediated mechanisms, but also through the ubiquitous cellular scaffolds β-arrestin-1 and β-arrestin-2. Previous studies have implicated β-arrestins as regulators of actin reorganization in response to GPCR stimulation while also being required for membrane protrusion events that accompany cellular motility. One of the most critical events in the active movement of cells is the cyclic phosphorylation and activation of myosin light chain (MLC), which is required for cellular contraction and movement. We have identified the myosin light chain phosphatase Targeting Subunit (MYPT-1) as a binding partner of the β-arrestins and found that β-arrestins play a role in regulating the turnover of phosphorylated myosin light chain. In response to stimulation of the angiotensin Type 1a Receptor (AT1aR), MLC phosphorylation is induced quickly and potently. We have found that β-arrestin-2 facilitates dephosphorylation of MLC, while, in a reciprocal fashion, β-arrestin 1 limits dephosphorylation of MLC. Intriguingly, loss of either β-arrestin-1 or 2 blocks phospho-MLC turnover and causes a decrease in the contraction of cells as monitored by atomic force microscopy (AFM). Furthermore, by employing the β-arrestin biased ligand [Sar1,Ile4,Ile8]-Ang, we demonstrate that AT1aR-mediated cellular motility involves a β-arrestin dependent component. This suggests that the reciprocal regulation of MLC phosphorylation status by β-arrestins-1 and 2 causes turnover in the phosphorylation status of MLC that is required for cell contractility and subsequent chemotaxic motility. PMID:24255721

  13. The chemokine receptor CCR1 is constitutively active, which leads to G protein-independent, β-arrestin-mediated internalization.

    PubMed

    Gilliland, C Taylor; Salanga, Catherina L; Kawamura, Tetsuya; Trejo, JoAnn; Handel, Tracy M

    2013-11-01

    Activation of G protein-coupled receptors by their associated ligands has been extensively studied, and increasing structural information about the molecular mechanisms underlying ligand-dependent receptor activation is beginning to emerge with the recent expansion in GPCR crystal structures. However, some GPCRs are also able to adopt active conformations in the absence of agonist binding that result in the initiation of signal transduction and receptor down-modulation. In this report, we show that the CC-type chemokine receptor 1 (CCR1) exhibits significant constitutive activity leading to a variety of cellular responses. CCR1 expression is sufficient to induce inhibition of cAMP formation, increased F-actin content, and basal migration of human and murine leukocytes. The constitutive activity leads to basal phosphorylation of the receptor, recruitment of β-arrestin-2, and subsequent receptor internalization. CCR1 concurrently engages Gαi and β-arrestin-2 in a multiprotein complex, which may be accommodated by homo-oligomerization or receptor clustering. The data suggest the presence of two functional states for CCR1; whereas receptor coupled to Gαi functions as a canonical GPCR, albeit with high constitutive activity, the CCR1·β-arrestin-2 complex is required for G protein-independent constitutive receptor internalization. The pertussis toxin-insensitive uptake of chemokine by the receptor suggests that the CCR1·β-arrestin-2 complex may be related to a potential scavenging function of the receptor, which may be important for maintenance of chemokine gradients and receptor responsiveness in complex fields of chemokines during inflammation.

  14. Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

    PubMed Central

    Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2011-01-01

    Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

  15. Quantitative analysis of neuropeptide Y receptor association with β-arrestin2 measured by bimolecular fluorescence complementation

    PubMed Central

    Kilpatrick, LE; Briddon, SJ; Hill, SJ; Holliday, ND

    2010-01-01

    Background and purpose: β-Arrestins are critical scaffold proteins that shape spatiotemporal signalling from seven transmembrane domain receptors (7TMRs). Here, we study the association between neuropeptide Y (NPY) receptors and β-arrestin2, using bimolecular fluorescence complementation (BiFC) to directly report underlying protein–protein interactions. Experimental approach: Y1 receptors were tagged with a C-terminal fragment, Yc, of yellow fluorescent protein (YFP), and β-arrestin2 fused with the complementary N-terminal fragment, Yn. After Y receptor–β-arrestin association, YFP fragment refolding to regenerate fluorescence (BiFC) was examined by confocal microscopy in transfected HEK293 cells. Y receptor/β-arrestin2 BiFC responses were also quantified by automated imaging and granularity analysis. Key results: NPY stimulation promoted association between Y1–Yc and β-arrestin2–Yn, and the specific development of BiFC in intracellular compartments, eliminated when using non-interacting receptor and arrestin mutants. Responses developed irreversibly and were slower than for downstream Y1 receptor–YFP internalization, a consequence of delayed maturation and stability of complemented YFP. However, β-arrestin2 BiFC measurements delivered appropriate ligand pharmacology for both Y1 and Y2 receptors, and demonstrated higher affinity of Y1 compared to Y2 receptors for β-arrestin2. Receptor mutagenesis combined with β-arrestin2 BiFC revealed that alternative arrangements of Ser/Thr residues in the Y1 receptor C tail could support β-arrestin2 association, and that Y2 receptor–β-arrestin2 interaction was enhanced by the intracellular loop mutation H155P. Conclusions and implications: The BiFC approach quantifies Y receptor ligand pharmacology focused on the β-arrestin2 pathway, and provides insight into mechanisms of β-arrestin2 recruitment by activated and phosphorylated 7TMRs, at the level of protein–protein interaction. PMID:20438572

  16. Expression Analysis of Visual Arrestin gene during Ocular Development of Olive Flounder (Paralichthys olivaceus)

    PubMed Central

    Yang, Hyun; Lee, Young Mee; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Hwang, In Joon; Kim, Sung Yeon; Lee, Jeong-Ho

    2013-01-01

    Olive flounder (Paralichthys olivaceus) is one of the commercial important flatfish species in Korea. The ocular signal transduction pathway is important in newly hatched flounders because it is closely involved in the initial feeding phase thus essential for survival during the juvenile period. However, the study of gene expression during ocular development is incomplete in olive flounder. Therefore we examined the expression analysis of specifically induced genes during the development of the visual system in newly hatched flounders. We searched ocular development-involved gene in the database of expressed sequence tags (ESTs) from olive flounder eye and this gene similar to arrestin with a partial sequence homology. Microscopic observation of retinal formation corresponded with the time of expression of the arrestin gene in the developmental stage. These results suggest that arrestin plays a vital role in the visual signal transduction pathway of the retina during ocular development. The expression of arrestin was strong in the ocular system during the entirety of the development stages. Our findings regarding arrestin have important implications with respect to its biological role and evolution of G-protein coupled receptor (GPCR) signaling in olive flounder. Further studies are required on the GPCR-mediated signaling pathway and to decipher the functional role of arrestin. PMID:25949138

  17. ERK5 activation by Gq-coupled muscarinic receptors is independent of receptor internalization and β-arrestin recruitment.

    PubMed

    Sánchez-Fernández, Guzmán; Cabezudo, Sofía; García-Hoz, Carlota; Tobin, Andrew B; Mayor, Federico; Ribas, Catalina

    2013-01-01

    G-protein-coupled receptors (GPCRs) are known to activate both G protein- and β-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK) pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII) failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.

  18. Role of β-arrestins and arrestin domain-containing proteins in G protein-coupled receptor trafficking.

    PubMed

    Kang, Dong Soo; Tian, Xufan; Benovic, Jeffrey L

    2014-04-01

    The arrestin clan can now be broadly divided into three structurally similar subgroups: the originally identified arrestins (visual and β-arrestins), the α-arrestins and a group of Vps26-related proteins. The visual and β-arrestins selectively bind to agonist-occupied phosphorylated G protein-coupled receptors (GPCRs) and inhibit GPCR coupling to heterotrimeric G proteins while the β-arrestins also function as adaptor proteins to regulate GPCR trafficking and G protein-independent signaling. The α-arrestins have also recently been implicated in regulating GPCR trafficking while Vps26 regulates retrograde trafficking. In this review, we provide an overview of the α-arrestins and β-arrestins with a focus on our current understanding of how these adaptor proteins regulate GPCR trafficking.

  19. β-Arrestins Negatively Regulate the Toll Pathway in Shrimp by Preventing Dorsal Translocation and Inhibiting Dorsal Transcriptional Activity.

    PubMed

    Sun, Jie-Jie; Lan, Jiang-Feng; Shi, Xiu-Zhen; Yang, Ming-Chong; Niu, Guo-Juan; Ding, Ding; Zhao, Xiao-Fan; Yu, Xiao-Qiang; Wang, Jin-Xing

    2016-04-01

    The Toll signaling pathway plays an important role in the innate immunity ofDrosophila melanogasterand mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense againstStaphylococcus aureusby regulating expression of antimicrobial peptides in shrimp. We then found that β-arrestins negatively regulate Toll signaling in two different ways. β-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of β-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. β-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser(276)that impairs Dorsal transcriptional activity. Our study suggests that β-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity. PMID:26846853

  20. β-Arrestin signal complex plays a critical role in adipose differentiation.

    PubMed

    Santos-Zas, Icía; Lodeiro, María; Gurriarán-Rodríguez, Uxía; Bouzo-Lorenzo, Mónica; Mosteiro, Carlos S; Casanueva, Felipe F; Casabiell, Xesús; Pazos, Yolanda; Camiña, Jesús P

    2013-07-01

    β-Arrestins were identified as scaffold-proteins that have the capacity to desensitize G protein-coupled receptors. However, it has been found that β-arrestins activate signaling pathways independent of G protein activation. The diversity of these signaling pathways has also been recognized for receptor tyrosine kinase. The aim of the present study was to validate the β-arrestin-dependent signaling mechanism(s) responsible for regulation of adipogenesis. Two signal models were selected, ghrelin and insulin, based on its β-arrestin-associated Akt activity. Herein, we found that β-arrestin 1 and 2 were essential molecules for adipocyte differentiation. More specifically, the role of these scaffolding proteins was demonstrated by depletion of β-arrestin 1 and 2 during ghrelin-induced adipogenesis in 3T3-L1 cells, which decreased the adipocyte differentiation and the expression levels of master regulators of early, the CCAAT/enhancer-binding protein β (C/EBPβ) and the CCAAT/enhancer-binding protein δ (C/EBPδ), and terminal, the peroxisome proliferator-activated receptor (PPARγ) and the CCAAT/enhancer-binding protein α (C/EBPα), adipogenesis. Accordingly ghrelin-induced Akt activity and its downstream targets, the mammalian target of rapamycin complex 1 (mTORC1) and the ribosomal protein S6 kinase beta-1 (S6K1), were inhibited by β-arrestin 1 and 2 siRNAs. By contrast, assays performed during insulin-activated adipogenesis showed an intensifying effect on the adipocyte differentiation as well as on the expression of C/EBPβ, C/EBPδ, PPARγ and C/EBPα. The increase in insulin-induced adipogenesis by β-arrestin knock-down was concomitant to a decrease in the insulin receptor susbtrate-1 (IRS-1) serine phosphorylation, proving the loss of the negative feedback loop on IRS-1/phosphoinositide 3-kinase (PI3K)/Akt. Therefore, β-arrestins control the extent and intensity of the lipogenic and adipogenic factors associated to Akt signaling, although the

  1. Triphenylmethane Dye Activation of Beta-Arrestin

    PubMed Central

    2013-01-01

    β-Arrestins regulate G protein-coupled receptor signaling as competitive inhibitors and protein adaptors. Low molecular weight biased ligands that bind receptors and discriminate between the G protein dependent arm and β-arrestin, clathrin-associated arm of receptor signaling are considered therapeutically valuable as a result of this distinctive pharmacological behavior. Other than receptor agonists, compounds that activate β-arrestins are not available. We show that within minutes of exposure to the cationic triphenylmethane dyes malachite green and brilliant green, tissue culture cells recruit β-arrestins to clathrin scaffolds in a receptor-activation independent manner. In the presence of these compounds, G protein signaling is inhibited, ERK and GSK3β signaling are preserved, and the recruitment of the beta2-adaptin, AP2 adaptor complex to clathrin as well as transferrin internalization is reduced. Moreover, malachite green binds β-arrestin2-GFP coated immunotrap beads relative to GFP only coated beads. Triphenylmethane dyes are FDA approved for topical use on newborns as components of triple-dye preparations and are not approved but used effectively as aqueous antibiotics in fish husbandry. As possible carcinogens, their chronic ingestion in food preparations, particularly through farmed fish, is discouraged in the U.S. and Europe. Our results indicate triphenylmethane dyes as a result of novel pharmacology may have additional roles as β-arrestin/clathrin pathway signaling modulators in both pharmacology research and clinical therapy. PMID:23865508

  2. Structure and function of the third intracellular loop of the 5-hydroxytryptamine2A receptor: the third intracellular loop is alpha-helical and binds purified arrestins.

    PubMed

    Gelber, E I; Kroeze, W K; Willins, D L; Gray, J A; Sinar, C A; Hyde, E G; Gurevich, V; Benovic, J; Roth, B L

    1999-05-01

    Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5-hydroxytryptamine2A (5-HT2A) receptor, a model G(alpha)q-coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT2A receptor, a region previously implicated in G-protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with alpha-helical and beta-loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known arrestins (beta-arrestin, arrestin-3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT(2A) receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (beta-arrestin or arrestin-3) and 5-HT2A receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT2A receptor represents a structurally ordered domain composed of alpha-helical and beta-loop, -turn, and -sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5-HT2A receptors in vivo.

  3. β-Arrestin 1 and 2 and G protein-coupled receptor kinase 2 expression in pituitary adenomas: role in the regulation of response to somatostatin analogue treatment in patients with acromegaly.

    PubMed

    Gatto, Federico; Feelders, Richard; van der Pas, Rob; Kros, Johan M; Dogan, Fadime; van Koetsveld, Peter M; van der Lelij, Aart-Jan; Neggers, Sebastian J C M M; Minuto, Francesco; de Herder, Wouter; Lamberts, Steven W J; Ferone, Diego; Hofland, Leo J

    2013-12-01

    Recent in vitro studies highlighted G protein-coupled receptor kinase (GRK)2 and β-arrestins as important players in driving somatostatin receptor (SSTR) desensitization and trafficking. Our aim was to characterize GRK2 and β-arrestins expression in different pituitary adenomas and to investigate their potential role in the response to somatostatin analog (SSA) treatment in GH-secreting adenomas (GHomas). We evaluated mRNA expression of multiple SSTRs, GRK2, β-arrestin 1, and β-arrestin 2 in 41 pituitary adenomas (31 GHomas, 6 nonfunctioning [NFPAs], and 4 prolactinomas [PRLomas]). Within the GHomas group, mRNA data were correlated with the in vivo response to an acute octreotide test and with the GH-lowering effect of SSA in cultured primary cells. β-Arrestin 1 expression was low in all 3 adenoma histotypes. However, its expression was significantly lower in GHomas and PRLomas, compared with NFPAs (P < .01). GRK2 expression was higher in PRLomas and NFPAs compared with GHomas (P < .05). In the GHoma group, GRK2 expression was inversely correlated to β-arrestin 1 (P < .05) and positively correlated to β-arrestin 2 (P < .0001). SSA treatment did not affect GRK2 and β-arrestin expression in GHomas or in cultured rat pituitary tumor GH3 cells. Noteworthy, β-arrestin 1 was significantly lower (P < .05) in tumors responsive to octreotide treatment in vitro, whereas GRK2 and SSTR subtype 2 were significantly higher (P < .05). Likewise, β-arrestin 1 levels were inversely correlated with the in vivo response to acute octreotide test (P = .001), whereas GRK2 and SSTR subtype 2 expression were positively correlated (P < .05). In conclusion, for the first time, we characterized GRK2, β-arrestin 1, and β-arrestin 2 expression in a representative number of pituitary adenomas. β-Arrestin 1 and GRK2 seem to have a role in modulating GH secretion during SSA treatment.

  4. Alpha-arrestins Aly1 and Aly2 regulate intracellular trafficking in response to nutrient signaling.

    PubMed

    O'Donnell, Allyson F; Apffel, Alex; Gardner, Richard G; Cyert, Martha S

    2010-10-15

    Extracellular signals regulate trafficking events to reorganize proteins at the plasma membrane (PM); however, few effectors of this regulation have been identified. β-Arrestins relay signaling cues to the trafficking machinery by controlling agonist-stimulated endocytosis of G-protein-coupled receptors. In contrast, we show that yeast α-arrestins, Aly1 and Aly2, control intracellular sorting of Gap1, the general amino acid permease, in response to nutrients. These studies are the first to demonstrate association of α-arrestins with clathrin and clathrin adaptor proteins (AP) and show that Aly1 and Aly2 interact directly with the γ-subunit of AP-1, Apl4. Aly2-dependent trafficking of Gap1 requires AP-1, which mediates endosome-to-Golgi transport, and the nutrient-regulated kinase, Npr1, which phosphorylates Aly2. During nitrogen starvation, Npr1 phosphorylation of Aly2 may stimulate Gap1 incorporation into AP-1/clathrin-coated vesicles to promote Gap1 trafficking from endosomes to the trans-Golgi network. Ultimately, increased Aly1-/Aly2-mediated recycling of Gap1 from endosomes results in higher Gap1 levels within cells and at the PM by diverting Gap away from trafficking pathways that lead to vacuolar degradation. This work defines a new role for arrestins in membrane trafficking and offers insight into how α-arrestins coordinate signaling events with protein trafficking.

  5. Arrestins in host-pathogen interactions

    PubMed Central

    Marullo, Stefano; Coureuil, Mathieu

    2014-01-01

    In the context of host-pathogen interaction, host cell receptors and signaling pathways are essential for both invading pathogens, which exploit them at their own profit, and the defending organism, which activates early mechanism of defense, known as innate immunity, to block the aggression. Because of their central role as scaffolding proteins downstream of activated receptors, β-arrestins are involved in multiple signaling pathways activated in host cells by pathogens. Some of these pathways participate to the innate immunity and the inflammatory response. Other β-arrestin-dependent pathways are actually hijacked by microbes and toxins to penetrate into host cells and spread in the organism. PMID:24292839

  6. Structure and function of the visual arrestin oligomer

    PubMed Central

    Hanson, Susan M; Van Eps, Ned; Francis, Derek J; Altenbach, Christian; Vishnivetskiy, Sergey A; Arshavsky, Vadim Y; Klug, Candice S; Hubbell, Wayne L; Gurevich, Vsevolod V

    2007-01-01

    A distinguishing feature of rod arrestin is its ability to form oligomers at physiological concentrations. Using visible light scattering, we show that rod arrestin forms tetramers in a cooperative manner in solution. To investigate the structure of the tetramer, a nitroxide side chain (R1) was introduced at 18 different positions. The effects of R1 on oligomer formation, EPR spectra, and inter-spin distance measurements all show that the structures of the solution and crystal tetramers are different. Inter-subunit distance measurements revealed that only arrestin monomer binds to light-activated phosphorhodopsin, whereas both monomer and tetramer bind microtubules, which may serve as a default arrestin partner in dark-adapted photoreceptors. Thus, the tetramer likely serves as a ‘storage' form of arrestin, increasing the arrestin-binding capacity of microtubules while readily dissociating to supply active monomer when it is needed to quench rhodopsin signaling. PMID:17332750

  7. Helix formation in arrestin accompanies recognition of photoactivated rhodopsin.

    PubMed

    Feuerstein, Sophie E; Pulvermüller, Alexander; Hartmann, Rudolf; Granzin, Joachim; Stoldt, Matthias; Henklein, Peter; Ernst, Oliver P; Heck, Martin; Willbold, Dieter; Koenig, Bernd W

    2009-11-17

    Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification of visual signals in photoreceptor cells. Currently, there is no crystal structure of a rhodopsin-arrestin complex available, although structures of unbound rhodopsin and arrestin have been determined. High-affinity receptor binding is dependent on distinct arrestin sites responsible for recognition of rhodopsin activation and phosphorylation. The loop connecting beta-strands V and VI in rod arrestin has been implicated in the recognition of active rhodopsin. We report the structure of receptor-bound arrestin peptide Arr(67-77) mimicking this loop based on solution NMR data. The peptide binds photoactivated rhodopsin in the unphosphorylated and phosphorylated form with similar affinities and stabilizes the metarhodopsin II photointermediate. A largely alpha-helical conformation of the receptor-bound peptide is observed. PMID:19835414

  8. Arrestin 1 and Cone Arrestin 4 Have Unique Roles in Visual Function in an All-Cone Mouse Retina

    PubMed Central

    Deming, Janise D.; Pak, Joseph S.; Shin, Jung-a; Brown, Bruce M.; Kim, Moon K.; Aung, Moe H.; Lee, Eun-Jin; Pardue, Machelle T.; Craft, Cheryl Mae

    2015-01-01

    Purpose Previous studies discovered cone phototransduction shutoff occurs normally for Arr1−/− and Arr4−/−; however, it is defective when both visual arrestins are simultaneously not expressed (Arr1−/−Arr4−/−). We investigated the roles of visual arrestins in an all-cone retina (Nrl−/−) since each arrestin has differential effects on visual function, including ARR1 for normal light adaptation, and ARR4 for normal contrast sensitivity and visual acuity. Methods We examined Nrl−/−, Nrl−/−Arr1−/−, Nrl−/−Arr4−/−, and Nrl−/−Arr1−/−Arr4−/− mice with photopic electroretinography (ERG) to assess light adaptation and retinal responses, immunoblot and immunohistochemical localization analysis to measure retinal expression levels of M- and S-opsin, and optokinetic tracking (OKT) to measure the visual acuity and contrast sensitivity. Results Study results indicated that Nrl−/− and Nrl−/−Arr4−/− mice light adapted normally, while Nrl−/−Arr1−/− and Nrl−/−Arr1−/−Arr4−/− mice did not. Photopic ERG a-wave, b-wave, and flicker amplitudes followed a general pattern in which Nrl−/−Arr4−/− amplitudes were higher than the amplitudes of Nrl−/−, while the amplitudes of Nrl−/−Arr1−/− and Nrl−/−Arr1−/−Arr4−/− were lower. All three visual arrestin knockouts had faster implicit times than Nrl−/− mice. M-opsin expression is lower when ARR1 is not expressed, while S-opsin expression is lower when ARR4 is not expressed. Although M-opsin expression is mislocalized throughout the photoreceptor cells, S-opsin is confined to the outer segments in all genotypes. Contrast sensitivity is decreased when ARR4 is not expressed, while visual acuity was normal except in Nrl−/−Arr1−/−Arr4−/−. Conclusions Based on the opposite visual phenotypes in an all-cone retina in the Nrl−/−Arr1−/− and Nrl−/−Arr4−/− mice, we conclude that ARR1 and ARR4 perform unique

  9. Arrestin development: emerging roles for beta-arrestins in developmental signaling pathways.

    PubMed

    Kovacs, Jeffrey J; Hara, Makoto R; Davenport, Chandra L; Kim, Jihee; Lefkowitz, Robert J

    2009-10-01

    Arrestins were identified as mediators of G protein-coupled receptor (GPCR) desensitization and endocytosis. However, it is now clear that they scaffold many intracellular signaling networks to modulate the strength and duration of signaling by diverse types of receptors--including those relevant to the Hedgehog, Wnt, Notch, and TGFbeta pathways--and downstream kinases such as the MAPK and Akt/PI3K cascades. The involvement of arrestins in many discrete developmental signaling events suggests an indispensable role for these multifaceted molecular scaffolds. PMID:19853559

  10. Engineering visual arrestin-1 with special functional characteristics.

    PubMed

    Vishnivetskiy, Sergey A; Chen, Qiuyan; Palazzo, Maria C; Brooks, Evan K; Altenbach, Christian; Iverson, Tina M; Hubbell, Wayne L; Gurevich, Vsevolod V

    2013-02-01

    Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations. PMID:23250748

  11. β-arrestin regulates estradiol membrane-initiated signaling in hypothalamic neurons.

    PubMed

    Wong, Angela M; Abrams, Matthew C; Micevych, Paul E

    2015-01-01

    Estradiol (E2) action in the nervous system is the result of both direct nuclear and membrane-initiated signaling (EMS). E2 regulates membrane estrogen receptor-α (ERα) levels through opposing mechanisms of EMS-mediated trafficking and internalization. While ß-arrestin-mediated mERα internalization has been described in the cortex, a role of ß-arrestin in EMS, which underlies multiple physiological processes, remains undefined. In the arcuate nucleus of the hypothalamus (ARH), membrane-initiated E2 signaling modulates lordosis behavior, a measure of female sexually receptivity. To better understand EMS and regulation of ERα membrane levels, we examined the role of ß-arrestin, a molecule associated with internalization following agonist stimulation. In the present study, we used an immortalized neuronal cell line derived from embryonic hypothalamic neurons, the N-38 line, to examine whether ß-arrestins mediate internalization of mERα. β-arrestin-1 (Arrb1) was found in the ARH and in N-38 neurons. In vitro, E2 increased trafficking and internalization of full-length ERα and ERαΔ4, an alternatively spliced isoform of ERα, which predominates in the membrane. Treatment with E2 also increased phosphorylation of extracellular-signal regulated kinases 1/2 (ERK1/2) in N-38 neurons. Arrb1 siRNA knockdown prevented E2-induced ERαΔ4 internalization and ERK1/2 phosphorylation. In vivo, microinfusions of Arrb1 antisense oligodeoxynucleotides (ODN) into female rat ARH knocked down Arrb1 and prevented estradiol benzoate-induced lordosis behavior compared with nonsense scrambled ODN (lordosis quotient: 3 ± 2.1 vs. 85.0 ± 6.0; p < 0.0001). These results indicate a role for Arrb1 in both EMS and internalization of mERα, which are required for the E2-induction of female sexual receptivity.

  12. β-Arrestin 2 mediates the anti-inflammatory effects of fluoxetine in lipopolysaccharide-stimulated microglial cells.

    PubMed

    Du, Ren-Wei; Du, Ren-Hong; Bu, Wen-Guang

    2014-09-01

    Recent evidence has suggested that microglial activation plays an important role in the pathogenesis of depression. Activated microglia can secrete various pro-inflammatory cytokines, which may contribute to the development and maintenance of depression. Thus, inhibition of microglial activation may have a therapeutic benefit in the treatment of depression. In the present study, we found that fluoxetine significantly inhibited lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α), interleukin- 6 (IL-6) and nitric oxide (NO) and reduced the phosphorylation of transforming growth factor-beta-activated kinase 1 (TAK1) and nuclear factor-kappa B (NF-κB) p65 nuclear translocation in microglia. We further found that fluoxetine increased the expression of β-arrestin 2 and enhanced the association of β-arrestin 2 with TAK1-binding protein 1 (TAB1) and disrupted TAK1-TAB1 interaction. Moreover, β-arrestin 2 knock-down abolished the anti-inflammatory effects of fluoxetine in lipopolysaccharide-stimulated microglial cells. Collectively, our findings suggest that β-arrestin 2 is necessary for the anti-inflammatory effects of fluoxetine and offers novel drug targets in the convergent fluoxetine/β-arrestin 2 and inflammatory pathways for treating microglial inflammatory neuropathologies like depression.

  13. Differential regulation of somatostatin receptor dephosphorylation by β-arrestin1 and β-arrestin2.

    PubMed

    Kliewer, Andrea; Schulz, Stefan

    2014-03-01

    Signaling of G protein-coupled receptors (GPCRs) is tightly regulated by coordinated phosphorylation of intracellular serine and threonine residues. Although the mechanisms of agonist-induced phosphorylation have been deciphered for many GPCRs, the regulation of their dephosphorylation remains poorly understood. Using a combination of siRNA knockdown screening and phosphosite-specific antibodies, we have recently identified the catalytic subunit β of protein phosphatase 1 (PP1β) as major constituent of the GPCR phosphatase responsible for dephosphorylation of the sst2 somatostatin receptor. However, PP1-targeting subunits specifically required for GPCR dephosphorylation have not been identified so far. Here, we show that siRNA knockdown of β-arrestin1 strongly inhibits sst2 receptor dephosphorylation. Co-immunoprecipitation experiments demonstrate that β-arrestin1 and PP1β exist as constitutive complex that mediates rapid dephosphorylation of sst2 receptors at or near the plasma membrane. By contrast, β-arrestin2 is not essential for rapid sst2 receptor dephosphorylation. Together, these findings reveal a novel scaffolding function of β-arrestin1 that facilitates efficient targeting of PP1β to phosphorylated GPCRs.

  14. Differential regulation of class IA phosphoinositide 3-kinase catalytic subunits p110 alpha and beta by protease-activated receptor 2 and beta-arrestins.

    PubMed

    Wang, Ping; Kumar, Puneet; Wang, Chang; Defea, Kathryn A

    2007-12-01

    PAR-2 (protease-activated receptor 2) is a GPCR (G-protein-coupled receptor) that can elicit both G-protein-dependent and -independent signals. We have shown previously that PAR-2 simultaneously promotes Galphaq/Ca2+-dependent activation and beta-arrestin-1-dependent inhibition of class IA PI3K (phosphoinositide 3-kinase), and we sought to characterize further the role of beta-arrestins in the regulation of PI3K activity. Whereas the ability of beta-arrestin-1 to inhibit p110alpha (PI3K catalytic subunit alpha) has been demonstrated, the role of beta-arrestin-2 in PI3K regulation and possible differences in the regulation of the two catalytic subunits (p110alpha and p110beta) associated with p85alpha (PI3K regulatory subunit) have not been examined. In the present study we have demonstrated that: (i) PAR-2 increases p110alpha- and p110beta-associated lipid kinase activities, and both p110alpha and p110beta are inhibited by over-expression of either beta-arrestin-1 or -2; (ii) both beta-arrestin-1 and -2 directly inhibit the p110alpha catalytic subunit in vitro, whereas only beta-arrestin-2 directly inhibited p110beta; (iii) examination of upstream pathways revealed that PAR-2-induced PI3K activity required the small GTPase Cdc (cell-division cycle)42, but not tyrosine phosphorylation of p85; and (iv) beta-arrestins inhibit PAR-2-induced Cdc42 activation. Taken together, these results indicated that beta-arrestins could inhibit PAR-2-stimulated PI3K activity, both directly and through interference with upstream pathways, and that the two beta-arrestins differ in their ability to inhibit the p110alpha and p110beta catalytic subunits. These results are particularly important in light of the growing interest in PAR-2 as a pharmacological target, as commonly used biochemical assays that monitor G-protein coupling would not screen for beta-arrestin-dependent signalling events.

  15. Arrestin-mediated endocytosis of yeast plasma membrane transporters.

    PubMed

    Nikko, Elina; Pelham, Hugh R B

    2009-12-01

    Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters.

  16. Targeting β-arrestin2 in the treatment of l-DOPA–induced dyskinesia in Parkinson’s disease

    PubMed Central

    Urs, Nikhil M.; Bido, Simone; Peterson, Sean M.; Daigle, Tanya L.; Bass, Caroline E.; Gainetdinov, Raul R.; Bezard, Erwan; Caron, Marc G.

    2015-01-01

    Parkinson’s disease (PD) is characterized by severe locomotor deficits and is commonly treated with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (l-DOPA), but its prolonged use causes dyskinesias referred to as l-DOPA–induced dyskinesias (LIDs). Recent studies in animal models of PD have suggested that dyskinesias are associated with the overactivation of G protein-mediated signaling through DA receptors. β-Arrestins desensitize G protein signaling at DA receptors (D1R and D2R) in addition to activating their own G protein-independent signaling events, which have been shown to mediate locomotion. Therefore, targeting β-arrestins in PD l-DOPA therapy might prove to be a desirable approach. Here we show in a bilateral DA-depletion mouse model of Parkinson’s symptoms that genetic deletion of β-arrestin2 significantly limits the beneficial locomotor effects while markedly enhancing the dyskinesia-like effects of acute or chronic l-DOPA treatment. Viral rescue or overexpression of β-arrestin2 in knockout or control mice either reverses or protects against LIDs and its key biochemical markers. In other more conventional animal models of DA neuron loss and PD, such as 6-hydroxydopamine–treated mice or rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated nonhuman primates, β-arrestin2 overexpression significantly reduced dyskinesias while maintaining the therapeutic effect of l-DOPA. Considerable efforts are being spent in the pharmaceutical industry to identify therapeutic approaches to block LIDs in patients with PD. Our results point to a potential therapeutic approach, whereby development of either a genetic or pharmacological intervention to enhance β-arrestin2- or limit G protein-dependent D1/D2R signaling could represent a more mechanistically informed strategy. PMID:25918399

  17. Regulation of GPR54 signaling by GRK2 and {beta}-arrestin.

    PubMed

    Pampillo, Macarena; Camuso, Natasha; Taylor, Jay E; Szereszewski, Jacob M; Ahow, Maryse R; Zajac, Mateusz; Millar, Robert P; Bhattacharya, Moshmi; Babwah, Andy V

    2009-12-01

    Kisspeptin and its receptor, GPR54, are major regulators of the hypothalamic-pituitary-gonadal axis as well as regulators of human placentation and tumor metastases. GPR54 is a G(q/11)-coupled G protein-coupled receptor (GPCR), and activation by kisspeptin stimulates phosphatidy linositol 4, 5-biphosphate hydrolysis, Ca(2+) mobilization, arachidonic acid release, and ERK1/2 MAPK phosphorylation. Physiological evidence suggests that GPR54 undergoes agonist-dependent desensitization, but underlying molecular mechanisms are unknown. Furthermore, very little has been reported on the early events that regulate GPR54 signaling. The lack of information in these important areas led to this study. Here we report for the first time on the role of GPCR serine/threonine kinase (GRK)2 and beta-arrestin in regulating GPR54 signaling in human embryonic kidney (HEK) 293 cells, a model cell system for studying the molecular regulation of GPCRs, and genetically modified MDA MB-231 cells, an invasive breast cancer cell line expressing about 75% less beta-arrestin-2 than the control cell line. Our study reveals that in HEK 293 cells, GPR54 is expressed both at the plasma membrane and intracellularly and also that plasma membrane expression is regulated by cytoplasmic tail sequences. We also demonstrate that GPR54 exhibits constitutive activity, internalization, and association with GRK2 and beta- arrestins-1 and 2 through sequences in the second intracellular loop and cytoplasmic tail of the receptor. We also show that GRK2 stimulates the desensitization of GPR54 in HEK 293 cells and that beta-arrestin-2 mediates GPR54 activation of ERK1/2 in MDA-MB-231 cells. The significance of these findings in developing molecular-based therapies for treating certain endocrine-related disorders is discussed.

  18. Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

    SciTech Connect

    Calabrese, G.; Sallese, M.; Stornaiuolo, A.

    1994-09-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.

  19. Α-arrestin 1 (ARRDC1) and β-arrestins cooperate to mediate Notch degradation in mammals.

    PubMed

    Puca, Loredana; Chastagner, Patricia; Meas-Yedid, Vannary; Israël, Alain; Brou, Christel

    2013-10-01

    Notch signaling is a conserved signaling pathway implicated in embryogenesis and adult tissue maintenance. Notch signaling strength is strictly regulated, notably by maintaining a controlled pool of functional receptor at the cell surface. Mammalian non-activated Notch receptor is internalized, ubiquitylated by the Itch E3 ubiquitin ligase and degraded in the lysosomes. Here, we show that β-arrestins are necessary for Itch-Notch interaction and for Itch-driven ubiquitylation and degradation of Notch. Interestingly, β-arrestins do not directly bind Itch but heterodimerize with a member of another subfamily of arrestins called ARRDC1 or α-arrestin 1, which harbors PPxY motifs that allow direct interaction with Itch. Cells transfected with ARRDC1 mutated in PPxY motifs show reduced Itch-mediated Notch ubiquitylation and impaired lysosomal degradation of Notch, as observed in β-arrestin(-/-) or Itch(-/-) cells. Our data show for the first time that ARRDC1 and β-arrestins heterodimerize and cooperate in the same complex to promote non-activated Notch receptor degradation, thus acting as negative regulators of Notch signaling.

  20. α-Arrestins participate in cargo selection for both clathrin-independent and clathrin-mediated endocytosis.

    PubMed

    Prosser, Derek C; Pannunzio, Anthony E; Brodsky, Jeffrey L; Thorner, Jeremy; Wendland, Beverly; O'Donnell, Allyson F

    2015-11-15

    Clathrin-mediated endocytosis (CME) is a well-studied mechanism to internalize plasma membrane proteins; however, to endocytose such cargo, most eukaryotic cells also use alternative clathrin-independent endocytic (CIE) pathways, which are less well characterized. The budding yeast Saccharomyces cerevisiae, a widely used model for studying CME, was recently shown to have a CIE pathway that requires the GTPase Rho1, the formin Bni1, and their regulators. Nevertheless, in both yeast and mammalian cells, the mechanisms underlying cargo selection in CME and CIE are only beginning to be understood. For CME in yeast, particular α-arrestins contribute to recognition of specific cargos and promote their ubiquitylation by recruiting the E3 ubiquitin protein ligase Rsp5. Here, we show that the same α-arrestin-cargo pairs promote internalization through the CIE pathway by interacting with CIE components. Notably, neither expression of Rsp5 nor its binding to α-arrestins is required for CIE. Thus, α-arrestins are important for cargo selection in both the CME and CIE pathways, but function by distinct mechanisms in each pathway.

  1. GPR54 regulates ERK1/2 activity and hypothalamic gene expression in a Gα(q/11) and β-arrestin-dependent manner.

    PubMed

    Szereszewski, Jacob M; Pampillo, Macarena; Ahow, Maryse R; Offermanns, Stefan; Bhattacharya, Moshmi; Babwah, Andy V

    2010-01-01

    G protein-coupled receptor 54 (GPR54) is a G(q/11)-coupled 7 transmembrane-spanning receptor (7TMR). Activation of GPR54 by kisspeptin (Kp) stimulates PIP(2) hydrolysis, Ca(2+) mobilization and ERK1/2 MAPK phosphorylation. Kp and GPR54 are established regulators of the hypothalamic-pituitary-gonadal (HPG) axis and loss-of-function mutations in GPR54 are associated with an absence of puberty and hypogonadotropic hypogonadism, thus defining an important role of the Kp/GPR54 signaling system in reproductive function. Given the tremendous physiological and clinical importance of the Kp/GPR54 signaling system, we explored the contributions of the GPR54-coupled G(q/11) and β-arrestin pathways on the activation of a major downstream signaling molecule, ERK, using G(q/11) and β-arrestin knockout mouse embryonic fibroblasts. Our study revealed that GPR54 employs the G(q/11) and β-arrestin-2 pathways in a co-dependent and temporally overlapping manner to positively regulate ERK activity and pERK nuclear localization. We also show that while β-arrestin-2 potentiates GPR54 signaling to ERK, β-arrestin-1 inhibits it. Our data also revealed that diminished β-arrestin-1 and -2 expression in the GT1-7 GnRH hypothalamic neuronal cell line triggered distinct patterns of gene expression following Kp-10 treatment. Thus, β-arrestin-1 and -2 also regulate distinct downstream responses in gene expression. Finally, we showed that GPR54, when uncoupled from the G(q/11) pathway, as is the case for several naturally occurring GPR54 mutants associated with hypogonadotropic hypogonadism, continues to regulate gene expression in a G protein-independent manner. These new and exciting findings add significantly to our mechanistic understanding of how this important receptor signals intracellularly in response to kisspeptin stimulation.

  2. BRET-based β-arrestin2 recruitment to the histamine H1 receptor for investigating antihistamine binding kinetics.

    PubMed

    Bosma, Reggie; Moritani, Ryo; Leurs, Rob; Vischer, Henry F

    2016-09-01

    Ligand residence time is thought to be a critical parameter for optimizing the in vivo efficacy of drug candidates. For the histamine H1 receptor (H1R) and other G protein-coupled receptors, the kinetics of ligand binding are typically measured by low throughput radioligand binding experiments using homogenized cell membranes expressing the target receptor. In this study, a real-time proximity assay between H1R and β-arrestin2 in living cells was established to investigate the dynamics of antihistamine binding to the H1R. No receptor reserve was found for the histamine-induced recruitment of β-arrestin2 to the H1R and the transiently recruited β-arrestin2 therefore reflected occupancy of the receptor by histamine. Antihistamines displayed similar kinetic signatures on antagonizing histamine-induced β-arrestin2 recruitment as compared to displacing radioligand binding from the H1R. This homogeneous functional method unambiguously determined the fifty-fold difference in the dissociation rate constant between mepyramine and the long residence time antihistamines levocetirizine and desloratadine. PMID:27468652

  3. Α-arrestins - new players in Notch and GPCR signaling pathways in mammals.

    PubMed

    Puca, Loredana; Brou, Christel

    2014-04-01

    For many years, β-arrestins have been known to be involved in G-protein-coupled receptor (GPCR) desensitization. However, β-arrestins belong to a family of proteins that act as multifunctional scaffolding proteins, in particular during trafficking of transmembrane receptors. The arrestin family comprises visual arrestins, β-arrestins and α-arrestins. In mammals, the functions of the α-arrestins are beginning to be elucidated, and they are described as versatile adaptors that link GPCRs or the Notch receptor to E3 ubiquitin ligases and endocytic factors. These α-arrestins can act in sequence, complementarily or cooperatively with β-arrestins in trafficking and ubiquitylation events. This Commentary will summarize the recent advances in our understanding of the functions and properties of these α-arrestin proteins in comparison to β-arrestins, and will highlight a new hypothesis linking their functional complementarity to their physical interactions. α- and β-arrestins could form transient and versatile heterodimers that form a bridge between cargo and E3 ubiquitin ligases, thus allowing trafficking to proceed.

  4. The conformational signature of arrestin3 predicts its trafficking and signaling functions

    PubMed Central

    Lee, Mi-Hye; Appleton, Kathryn M.; Strungs, Erik G.; Kwon, Joshua Y.; Morinelli, Thomas A.; Peterson, Yuri K.; Laporte, Stephane A.; Luttrell, Louis M.

    2016-01-01

    Arrestins are cytosolic proteins that regulate G protein-coupled receptor (GPCR) desensitization, internalization, trafficking, and signaling1,2. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets them for internalization via clathrin-coated pits3,4. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G protein effectors into GPCR-based ‘signalsomes’5,6. While the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify divergent arrestin functions is not understood. Using a panel of intramolecular FlAsH-BRET reporters7 to monitor conformational changes in arrestin3, we show here that GPCRs impose distinctive arrestin ‘conformational signatures’ that reflect the stability of the receptor-arrestin complex and role of arrestin3 in activating or dampening downstream signaling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in arrestin3 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average arrestin3 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands8,9 and in identifying factors that dictate arrestin conformation and function. PMID:27007854

  5. Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-01-01

    Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies. PMID:25318610

  6. Biased signaling favoring gi over β-arrestin promoted by an apelin fragment lacking the C-terminal phenylalanine.

    PubMed

    Ceraudo, Emilie; Galanth, Cécile; Carpentier, Eric; Banegas-Font, Inmaculada; Schonegge, Anne-Marie; Alvear-Perez, Rodrigo; Iturrioz, Xavier; Bouvier, Michel; Llorens-Cortes, Catherine

    2014-08-29

    Apelin plays a prominent role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or Gi protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in BP decrease may be as a Gi-independent but β-arrestin-dependent signaling pathway that could involve MAPKs. For this purpose, we have used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates Gi and promotes β-arrestin recruitment to the receptor, K16P had a much reduced ability to promote β-arrestin recruitment while maintaining its Gi activating property, revealing the biased agonist character of K16P. We further show that both β-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity, whereas the K16P-promoted ERK1/2 activity is entirely Gi-dependent. In addition to providing new insights on the structural basis underlying the functional selectivity of apelin peptides, our study indicates that the β-arrestin-dependent ERK1/2 activation and not the Gi-dependent signaling may participate in K17F-induced BP decrease.

  7. Beta-arrestin-2 negatively modulates inflammation response in mouse chondrocytes induced by 4-mer hyaluronan oligosaccharide.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-01-01

    Beta-arrestin-2 is an adaptor protein that terminates G protein activation and seems to be involved in the modulation of the inflammatory response. Small hyaluronan (HA) fragments, such as 4-mer HA oligosaccharides, are known to interact with the toll-like receptor-4 (TLR-4) with consequent activation of the nuclear factor kappaB (NF-kB) that in turn stimulates the inflammation response. NF-kB activation is mediated by different pathways, in particular by the transforming growth factor-activated kinase-1 (TAK-1). Conversely, increased levels of protein kinase A (PKA), induced by cyclic adenosine monophosphate (cAMP), seem to inhibit NF-kB activation. We studied the involvement and role of beta-arrestin-2 in mouse chondrocytes stimulated with 4-mer HA fragments. The exposure of chondrocytes to 4-mer HA produced a significant up-regulation in TLR-4, cAMP, beta-arrestin-2, TAK-1, protein 38 mitogen-activated protein kinase (p38MAPK), and PKA, both in terms of mRNA expression and of the related protein levels. NF-kB was significantly activated, thereby producing the transcription of pro-inflammatory mediators, including tumor necrosis factor alpha, interleukin-6, and interleukin-17. The treatment of 4-mer HA-stimulated chondrocytes with antibodies against beta-arrestin-2 and/or a specific PKA inhibitor, significantly increased the inflammatory response, while the treatment with a specific p38MAPK inhibitor significantly reduced the inflammatory response. Interestingly, the anti-inflammatory action exerted by beta-arrestin-2 appeared to be mediated in part through the direct inhibition of p38MAPK, preventing NF-kB activation, and in part through cAMP and PKA activation primed by G protein signaling, which exerted an inhibitory effect on NF-kB. Taken together, these results could be useful for future anti-inflammatory strategies.

  8. Arrestin-3 is essential for the activation of Fyn by the luteinizing hormone receptor (LHR) in MA-10 cells*

    PubMed Central

    Galet, Colette; Ascoli, Mario

    2008-01-01

    Recent studies showed that Fyn is a mediator of the LHR-induced activation of the ERK1/2 cascade in MA-10 cells. Since the LHR is a G protein-coupled receptor and the Src family of kinases can be activated by some Gα subunits and by the non-visual arrestins we investigated the role of these signaling molecules in the LHR-provoked activation of Fyn. Small interfering RNAs (siRNAs) that target two Gα subunits that participate in LHR signaling (Gαs and Gα11) and one that targets arrestin-3 were co-transfected with the hLHR in MA-10 cells. We then determined the effects of these siRNAs on the LHR-provoked activation of Fyn, the phosphorylation of FAK (a prominent Fyn substrate) and the release of EGF-like growth factors (a Fyn-mediated process). Expression of the siRNA against Gαs decreased the level of Gαs and LHR-stimulated cAMP production by ~50% but did not affect LHR-stimulated Fyn activation or FAK phosphorylation. Likewise, expression of the siRNA against Gα11 decreased the level of Gα11 and LHR-stimulated inositol phosphate production by ~50% but did not affect LHR-stimulated Fyn activation or FAK phosphorylation. Expression of the siRNA against arrestin-3 decreased the level of arrestin-3 and the rate of internalization of hCG by ~50% and it also inhibited the LHR-provoked stimulation of Fyn, the phosphorylation of FAK and the release of EGF-like growth factors. These results show that, in MA-10 cells, the hLHR activates Fyn through and arrestin-3-dependent pathway and that this pathway is a mediator of the hLHR-provoked release of EGF-like growth factors. PMID:18647647

  9. Pharmacological Profile of Nociceptin/Orphanin FQ Receptors Interacting with G-Proteins and β-Arrestins 2

    PubMed Central

    Malfacini, D.; Ambrosio, C.; Gro’, M. C.; Sbraccia, M.; Trapella, C.; Guerrini, R.; Bonora, M.; Pinton, P.; Costa, T.; Calo’, G.

    2015-01-01

    Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified. PMID:26248189

  10. β-Arrestin-2 knockout prevents development of cellular μ-opioid receptor tolerance but does not affect opioid-withdrawal-related adaptations in single PAG neurons

    PubMed Central

    Connor, M; Bagley, E E; Chieng, B C; Christie, M J

    2015-01-01

    BACKGROUND AND PURPOSE Tolerance to the behavioural effects of morphine is blunted in β-arrestin-2 knockout mice, but opioid withdrawal is largely unaffected. The cellular mechanisms of tolerance have been studied in some neurons from β-arrestin-2 knockouts, but tolerance and withdrawal mechanisms have not been examined at the cellular level in periaqueductal grey (PAG) neurons, which are crucial for central tolerance and withdrawal phenomena. EXPERIMENTAL APPROACH μ-Opioid receptor (MOPr) inhibition of voltage-gated calcium channel currents (ICa) was examined by patch-clamp recordings from acutely dissociated PAG neurons from wild-type and β-arrestin-2 knockout mice treated chronically with morphine (CMT) or vehicle. Opioid withdrawal-induced activation of GABA transporter type 1 (GAT-1) currents was determined using perforated patch recordings from PAG neurons in brain slices. KEY RESULTS MOPr inhibition of ICa in PAG neurons was unaffected by β-arrestin-2 deletion. CMT impaired coupling of MOPrs to ICa in PAG neurons from wild-type mice, but this cellular tolerance was not observed in neurons from CMT β-arrestin-2 knockouts. However, β-arrestin-2 knockouts displayed similar opioid-withdrawal-induced activation of GAT-1 currents as wild-type PAG neurons. CONCLUSIONS AND IMPLICATIONS In β-arrestin-2 knockout mice, the central neurons involved in the anti-nociceptive actions of opioids also fail to develop cellular tolerance to opioids following chronic morphine. The results also provide the first cellular physiological evidence that opioid withdrawal is not disrupted by β-arrestin-2 deletion. However, the unaffected basal sensitivity to opioids in PAG neurons provides further evidence that changes in basal MOPr sensitivity cannot account for the enhanced acute nociceptive response to morphine reported in β-arrestin-2 knockouts. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other

  11. Dexamethasone in the presence of desipramine enhances MAPK/ERK1/2 signaling possibly via its interference with β-arrestin.

    PubMed

    Lucki, Anat; Klein, Ehud; Karry, Rachel; Ben-Shachar, Dorit

    2014-01-01

    Antidepressant medication is the standard treatment for major depression disorder (MDD). However, the response to these treatments is often incomplete and many patients remain refractory. In the present study, we show that the glucocorticoid receptor (GR) agonist dexamethasone (DEX) increased MAPK/ERK1/2 signaling in the presence of the noradrenergic antidepressant, desipramine (DMI), while no such effect was induced by DEX or DMI alone in human neuroblastoma SH-SY5Y cells. This enhancement was dependent on the activation of both α(2) adrenergic receptors (AR) and GR. The timing of MAPK/ERK1/2 activation as well as DEX-induced reduction in membranous α(2) AR suggests the involvement of a β-arrestin-dependent mechanism. In line with the latter, DEX increased cytosolic and decreased membranous levels of β-arrestin. Concomitantly, DEX induced a time-dependent increase in cytosolic α(2) AR-β-arrestin interaction and a decrease in β-arrestin interaction with Mdm2 E3 ubiquitin ligase. All of these effects of DEX were prevented by the GR antagonist RU486. Our data suggest an additional intracellular role for DEX, in which activation of GR interferes with the trafficking and degradation of β-arrestin-α2c-AR complex. We suggest that such an interaction in the presence of DMI can enhance MAPK/ERK1/2 signaling, a key player in neural plasticity and neurogenesis processes, which is impaired in MDD, while stimulated by antidepressants.

  12. The conformational signature of β-arrestin2 predicts its trafficking and signalling functions.

    PubMed

    Lee, Mi-Hye; Appleton, Kathryn M; Strungs, Erik G; Kwon, Joshua Y; Morinelli, Thomas A; Peterson, Yuri K; Laporte, Stephane A; Luttrell, Louis M

    2016-03-31

    Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based 'signalsomes'. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in β-arrestin2, here we show that GPCRs impose distinctive arrestin 'conformational signatures' that reflect the stability of the receptor-arrestin complex and role of β-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in β-arrestin2 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average β-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function. PMID:27007854

  13. Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK*

    PubMed Central

    Khoury, Etienne; Nikolajev, Ljiljana; Simaan, May; Namkung, Yoon; Laporte, Stéphane A.

    2014-01-01

    β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET178P), but not rat β-arrestin-1 (PER177P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK178P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species. PMID:25016018

  14. Differential regulation of endosomal GPCR/β-arrestin complexes and trafficking by MAPK.

    PubMed

    Khoury, Etienne; Nikolajev, Ljiljana; Simaan, May; Namkung, Yoon; Laporte, Stéphane A

    2014-08-22

    β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET(178)P), but not rat β-arrestin-1 (PER(177)P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK(178)P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species.

  15. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

    PubMed

    Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    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.

  16. Co-purification of arrestin like proteins with alpha-enolase from bovine myocardial tissues and the possible role in heart diseases as an autoantigen

    SciTech Connect

    Mirshahi, M. Le Marchand, S.

    2015-05-08

    Aim: Previously, we reported that visual arrestin co-purified with glycolytic enzymes. The aim of this study was to analyze the co-purification of arrestin like proteins (ALP) in bovine cardiac tissues with enolases. Methods: The soluble extract of bovine myocardial tissues from different regions such as left and right atriums and ventricles of the bovine heart (n = 3) was analyzed by ACA-34 gel filtration, immuno-affinity column, SDS-PAGE, ELISA, western blot and a sandwich immune assay for quantification of ALP and sequence analysis. Results: We observed that; 1) The cardiac muscle contained a 50 kDa ALP at a concentration of 751 pg/mg of soluble protein extract, 2) ALP purified, by immunoaffinity, contained alpha-enolase of 48 kDa confirmed by protein sequence analysis; 3) Cardiomyocyte cells exposed to anti arrestin and anti enolase monoclonal antibodies showed decreased proliferation in vitro, 4) High level of autoantibodies were detected by ELISA (3.57% for arrestin and 9.12% for α-enolase) in serum of patients with infarcted heart disease. Conclusion: We suggest a possible interaction between ALP and alpha-enolases yielding a complex that may be involved in the induction of cardiac autoimmune diseases. - Highlights: • We examine a possible interaction between arrestin like protein and alpha-enolases in cardiomyocyte. • We demonstrated the effect of antibodies against arrestin and enolase on cardiomyocyte cell proliferation. • We suggest that this proteins complex may be involved in the induction of cardiac autoimmune diseases.

  17. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation.

    PubMed

    Eichel, K; Jullié, D; von Zastrow, M

    2016-03-01

    β-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase. Despite enormous recent progress towards understanding biophysical aspects of arrestin function, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR-β-arrestin complexes. We show here, using β1-adrenergic receptors, that β-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and β-arrestin-dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signalling from CCSs. We propose a β-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery.

  18. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation.

    PubMed

    Eichel, K; Jullié, D; von Zastrow, M

    2016-03-01

    β-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase. Despite enormous recent progress towards understanding biophysical aspects of arrestin function, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR-β-arrestin complexes. We show here, using β1-adrenergic receptors, that β-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and β-arrestin-dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signalling from CCSs. We propose a β-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

  19. KISS1R signals independently of Gαq/11 and triggers LH secretion via the β-arrestin pathway in the male mouse.

    PubMed

    Ahow, Maryse; Min, Le; Pampillo, Macarena; Nash, Connor; Wen, Junping; Soltis, Kathleen; Carroll, Rona S; Glidewell-Kenney, Christine A; Mellon, Pamela L; Bhattacharya, Moshmi; Tobet, Stuart A; Kaiser, Ursula B; Babwah, Andy V

    2014-11-01

    Hypothalamic GnRH is the master regulator of the neuroendocrine reproductive axis, and its secretion is regulated by many factors. Among these is kisspeptin (Kp), a potent trigger of GnRH secretion. Kp signals via the Kp receptor (KISS1R), a Gαq/11-coupled 7-transmembrane-spanning receptor. Until this study, it was understood that KISS1R mediates GnRH secretion via the Gαq/11-coupled pathway in an ERK1/2-dependent manner. We recently demonstrated that KISS1R also signals independently of Gαq/11 via β-arrestin and that this pathway also mediates ERK1/2 activation. Because GnRH secretion is ERK1/2-dependent, we hypothesized that KISS1R regulates GnRH secretion via both the Gαq/11- and β-arrestin-coupled pathways. To test this hypothesis, we measured LH secretion, a surrogate marker of GnRH secretion, in mice lacking either β-arrestin-1 or β-arrestin-2. Results revealed that Kp-dependent LH secretion was significantly diminished relative to wild-type mice (P < .001), thus supporting that β-arrestin mediates Kp-induced GnRH secretion. Based on this, we hypothesized that Gαq/11-uncoupled KISS1R mutants, like L148S, will display Gαq/11-independent signaling. To test this hypothesis, L148S was expressed in HEK 293 cells. and results confirmed that, although strongly uncoupled from Gαq/11, L148S retained the ability to trigger significant Kp-dependent ERK1/2 phosphorylation (P < .05). Furthermore, using mouse embryonic fibroblasts lacking β-arrestin-1 and -2, we demonstrated that L148S-mediated ERK1/2 phosphorylation is β-arrestin-dependent. Overall, we conclude that KISS1R signals via Gαq/11 and β-arrestin to regulate GnRH secretion. This novel and important finding could explain why patients bearing some types of Gαq/11-uncoupled KISS1R mutants display partial gonadotropic deficiency and even a reversal of the condition, idiopathic hypogonadotropic hypogonadism.

  20. Fluorescence correlation spectroscopy, combined with bimolecular fluorescence complementation, reveals the effects of β-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors

    PubMed Central

    Kilpatrick, Laura E.; Briddon, Stephen J.; Holliday, Nicholas D.

    2012-01-01

    Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis are powerful ways to study mobility and stoichiometry of G protein coupled receptor complexes, within microdomains of single living cells. However, relating these properties to molecular mechanisms can be challenging. We investigated the influence of β-arrestin adaptors and endocytosis mechanisms on plasma membrane diffusion and particle brightness of GFP-tagged neuropeptide Y (NPY) receptors. A novel GFP-based bimolecular fluorescence complementation (BiFC) system also identified Y1 receptor-β-arrestin complexes. Diffusion co-efficients (D) for Y1 and Y2-GFP receptors in HEK293 cell plasma membranes were 2.22 and 2.15 × 10− 9 cm2 s− 1 respectively. At a concentration which promoted only Y1 receptor endocytosis, NPY treatment reduced Y1-GFP motility (D 1.48 × 10− 9 cm2 s− 1), but did not alter diffusion characteristics of the Y2-GFP receptor. Agonist induced changes in Y1 receptor motility were inhibited by mutations (6A) which prevented β-arrestin recruitment and internalisation; conversely they became apparent in a Y2 receptor mutant with increased β-arrestin affinity. NPY treatment also increased Y1 receptor-GFP particle brightness, changes which indicated receptor clustering, and which were abolished by the 6A mutation. The importance of β-arrestin recruitment for these effects was illustrated by reduced lateral mobility (D 1.20–1.33 × 10− 9 cm2 s− 1) of Y1 receptor-β-arrestin BiFC complexes. Thus NPY-induced changes in Y receptor motility and brightness reflect early events surrounding arrestin dependent endocytosis at the plasma membrane, results supported by a novel combined BiFC/FCS approach to detect the underlying receptor-β-arrestin signalling complex. PMID:22487268

  1. Characterization of methadone as a β-arrestin-biased μ-opioid receptor agonist

    PubMed Central

    Doi, Seira; Mori, Tomohisa; Uzawa, Naoki; Arima, Takamichi; Takahashi, Tomoyuki; Uchida, Masashi; Yawata, Ayaka; Narita, Michiko; Uezono, Yasuhito; Suzuki, Tsutomu

    2016-01-01

    Background Methadone is a unique µ-opioid receptor agonist. Although several researchers have insisted that the pharmacological effects of methadone are mediated through the blockade of NMDA receptor, the underlying mechanism by which methadone exerts its distinct pharmacological effects compared to those of other µ-opioid receptor agonists is still controversial. In the present study, we further investigated the pharmacological profile of methadone compared to those of fentanyl and morphine as measured mainly by the discriminative stimulus effect and in vitro assays for NMDA receptor binding, µ-opioid receptor-internalization, and µ-opioid receptor-mediated β-arrestin recruitment. Results We found that fentanyl substituted for the discriminative stimulus effects of methadone, whereas a relatively high dose of morphine was required to substitute for the discriminative stimulus effects of methadone in rats. Under these conditions, the non-competitive NMDA receptor antagonist MK-801 did not substitute for the discriminative stimulus effects of methadone. In association with its discriminative stimulus effect, methadone failed to displace the receptor binding of MK801 using mouse brain membrane. Methadone and fentanyl, but not morphine, induced potent µ-opioid receptor internalization accompanied by the strong recruitment of β-arrestin-2 in µ-opioid receptor-overexpressing cells. Conclusions These results suggest that methadone may, at least partly, produce its pharmacological effect as a β-arrestin-biased µ-opioid receptor agonist, similar to fentanyl, and NMDA receptor blockade is not the main contributor to the pharmacological profile of methadone. PMID:27317580

  2. Visual arrestins in olfactory pathways of Drosophila and the malaria vector mosquito Anopheles gambiae

    PubMed Central

    Merrill, C. E.; Riesgo-Escovar, J.; Pitts, R. J.; Kafatos, F. C.; Carlson, J. R.; Zwiebel, L. J.

    2002-01-01

    Arrestins are important components for desensitization of G protein-coupled receptor cascades that mediate neurotransmission as well as olfactory and visual sensory reception. We have isolated AgArr1, an arrestin-encoding cDNA from the malaria vector mosquito, Anopheles gambiae, where olfaction is critical for vectorial capacity. Analysis of AgArr1 expression revealed an overlap between chemosensory and photoreceptor neurons. Furthermore, an examination of previously identified arrestins from Drosophila melanogaster exposed similar bimodal expression, and Drosophila arrestin mutants demonstrate impaired electrophysiological responses to olfactory stimuli. Thus, we show that arrestins in Drosophila are required for normal olfactory physiology in addition to their previously described role in visual signaling. These findings suggest that individual arrestins function in both olfactory and visual pathways in Dipteran insects; these genes may prove useful in the design of control strategies that target olfactory-dependent behaviors of insect disease vectors. PMID:11792843

  3. Caspase-cleaved arrestin-2 and BID cooperatively facilitate cytochrome C release and cell death.

    PubMed

    Kook, S; Zhan, X; Cleghorn, W M; Benovic, J L; Gurevich, V V; Gurevich, E V

    2014-01-01

    Apoptosis is programmed cell death triggered by activation of death receptors or cellular stress. Activation of caspases is the hallmark of apoptosis. Arrestins are best known for their role in homologous desensitization of G protein-coupled receptors (GPCRs). Arrestins quench G protein activation by binding to activated phosphorylated GPCRs. Recently, arrestins have been shown to regulate multiple signalling pathways in G protein-independent manner via scaffolding signalling proteins. Here we demonstrate that arrestin-2 isoform is cleaved by caspases during apoptosis induced via death receptor activation or by DNA damage at evolutionarily conserved sites in the C-terminus. Caspase-generated arrestin-2-(1-380) fragment translocates to mitochondria increasing cytochrome C release, which is the key checkpoint in cell death. Cells lacking arrestin-2 are significantly more resistant to apoptosis. The expression of wild-type arrestin-2 or its cleavage product arrestin-2-(1-380), but not of its caspase-resistant mutant, restores cell sensitivity to apoptotic stimuli. Arrestin-2-(1-380) action depends on tBID: at physiological concentrations, arrestin-2-(1-380) directly binds tBID and doubles tBID-induced cytochrome C release from isolated mitochondria. Arrestin-2-(1-380) does not facilitate apoptosis in BID knockout cells, whereas its ability to increase caspase-3 activity and facilitate cytochrome C release is rescued when BID expression is restored. Thus, arrestin-2-(1-380) cooperates with another product of caspase activity, tBID, and their concerted action significantly contributes to cell death.

  4. Assignment of the {beta}-arrestin 1 gene (ARRB1) to human chromosome 11q13

    SciTech Connect

    Calabrese, G.; Morizio, E.; Palka, G.

    1994-11-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor, and its functional cofactor, {beta}-arrestin. {beta}ARK is a member of a multigene family, consisting of six known subtypes, which have also been named G-protein-coupled receptor kinases (GRK 1 to 6) due to the apparently unique functional association of such kinases with this receptor family. The gene for {beta}ARK1 has been localized to human chromosome 11q13. The four members of the arrestin/{beta}-arrestin gene family identified so far are arrestin, X-arrestin, {beta}-arrestin 1, and {beta}-arrestin 2. Here the authors report the chromosome mapping of the human gene for {beta}-arrestin 1 (ARRB1) to chromosome 11q13 by fluorescence in situ hybridization (FISH). Two-color FISH confirmed that the two genes coding for the functionally related proteins {beta}ARK1 and {beta}arrestin 1 both map to 11q13. 16 refs., 1 fig., 1 tab.

  5. β-arrestin drives MAP kinase signaling from clathrin-coated structures after GPCR dissociation

    PubMed Central

    Eichel, K.; Jullié, D.

    2016-01-01

    β-arrestins critically regulate G protein-coupled receptor (GPCR) signaling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G protein-independent signal via MAP kinase1–3. Despite enormous recent progress toward understanding biophysical aspects of arrestin function4,5, its cell biology remains relatively poorly understood. Two key tenets underlie the present dogma: (1) β-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and (2) MAP kinase activation requires endocytosis of formed GPCR - β-arrestin complexes6–9. We show here, using β1-adrenergic receptors, that β-arrestin-2 (Arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin and β-arrestin -dependent MAP kinase signal. These results demonstrate β-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signaling from CCSs. We propose a β-arrestin signaling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery. PMID:26829388

  6. Proximal tubule NHE3 activity is inhibited by beta-arrestin-biased angiotensin II type 1 receptor signaling.

    PubMed

    Carneiro de Morais, Carla P; Polidoro, Juliano Z; Ralph, Donna L; Pessoa, Thaissa D; Oliveira-Souza, Maria; Barauna, Valério G; Rebouças, Nancy A; Malnic, Gerhard; McDonough, Alicia A; Girardi, Adriana C C

    2015-10-15

    Physiological concentrations of angiotensin II (ANG II) upregulate the activity of Na(+)/H(+) exchanger isoform 3 (NHE3) in the renal proximal tubule through activation of the ANG II type I (AT1) receptor/G protein-coupled signaling. This effect is key for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the beta-arrestin-biased AT1 receptor signaling pathway induces diuresis and natriuresis independent of G protein-mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G-protein coupling, and stimulates beta-arrestin signaling on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na(+)-dependent intracellular pH recovery. We found that 10(-7) M TRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro. Additionally, stimulation of NHE3 by ANG II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. These findings indicate that biased signaling of the beta-arrestin pathway through the AT1 receptor inhibits NHE3 activity in the proximal tubule at least in part due to changes in NHE3 subcellular localization. PMID:26246427

  7. Gamma-aminobutyric acid induces tumor cells apoptosis via GABABR1·β-arrestins·JNKs signaling module.

    PubMed

    Tian, Hui; Wu, Jin-Xia; Shan, Feng-Xiao; Zhang, Shang-Nuan; Cheng, Qian; Zheng, Jun-Nian; Pei, Dong-Sheng

    2015-03-01

    Gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter in central nervous system, has yet been found to widely exist in tumor tissues to regulate tumor cells growth. However, the function of GABA on inducing tumor cells apoptosis and the potential mechanism are still unclear. In order to detect whether GABA via GABAB receptor GABABR1 would activate c-Jun N-terminal kinases (JNKs) to promote tumor cells apoptosis, co-immunoprecipitation assay was used to investigate the association of β-arrestins with GABABR1 and JNKs in the different four cancer cell lines. Our observation demonstrated that β-arrestins, in addition to their role in G protein-coupled receptors desensitization, had an additional function as adapter proteins to recruit JNKs to GABABR1, thereby conferring distinct enzymatic activities upon the receptor, which may trigger JNKs signal pathway involved in the regulation of cellular growth. Activated JNKs subsequently phosphorylated downstream c-Jun to transcribe a wide variety of pro-apoptotic genes. Additionally, GABA up-regulated the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2, and thus facilitated caspase-3 cleavage, leading to tumor cells apoptosis in a mitochondrial-dependent pathway. In contrast, GABABR antagonist CGP35348 reversed GABA-induced JNKs phosphorylation and its downstream proteins activation, which consequently restrained tumor cells apoptosis. Taken together, our study suggested that GABA via its receptor GABABR1 recruited β-arrestins to facilitate the activation of JNKs cascade, resulting in tumor cells growth inhibition.

  8. The antidepressant desipramine is an arrestin-biased ligand at the α(2A)-adrenergic receptor driving receptor down-regulation in vitro and in vivo.

    PubMed

    Cottingham, Christopher; Chen, Yunjia; Jiao, Kai; Wang, Qin

    2011-10-14

    The neurobiological mechanisms of action underlying antidepressant drugs remain poorly understood. Desipramine (DMI) is an antidepressant classically characterized as an inhibitor of norepinephrine reuptake. Available evidence, however, suggests a mechanism more complex than simple reuptake inhibition. In the present study, we have characterized the direct interaction between DMI and the α(2A)-adrenergic receptor (α(2A)AR), a key regulator of noradrenergic neurotransmission with altered expression and function in depression. DMI alone was found to be sufficient to drive receptor internalization acutely and a robust down-regulation of α(2A)AR expression and signaling following prolonged stimulation in vitro. These effects are achieved through arrestin-biased regulation of the receptor, as DMI selectively induces recruitment of arrestin but not activation of heterotrimeric G proteins. Meanwhile, a physiologically relevant concentration of endogenous agonist (norepinephrine) was unable to sustain a down-regulation response. Prolonged in vivo administration of DMI resulted in significant down-regulation of synaptic α(2A)AR expression, a response that was lost in arrestin3-null animals. We contend that direct DMI-driven arrestin-mediated α(2A)AR down-regulation accounts for the therapeutically desirable but mechanistically unexplained adaptive alterations in receptor expression associated with this antidepressant. Our results provide novel insight into both the pharmacology of this antidepressant drug and the targeting of the α(2A)AR in depression.

  9. Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge.

    PubMed

    Vishnivetskiy, Sergey A; Hirsch, Joel A; Velez, Maria-Gabriela; Gurevich, Yulia V; Gurevich, Vsevolod V

    2002-11-15

    Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.

  10. Comparative analyses of downstream signal transduction targets modulated after activation of the AT1 receptor by two β-arrestin-biased agonists.

    PubMed

    Santos, Geisa A; Duarte, Diego A; Parreiras-E-Silva, Lucas T; Teixeira, Felipe R; Silva-Rocha, Rafael; Oliveira, Eduardo B; Bouvier, Michel; Costa-Neto, Claudio M

    2015-01-01

    G protein-coupled receptors (GPCRs) are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with β-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or β-arrestin pathways. The angiotensin II (AngII) AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known β-arrestin-biased agonists, such as [Sar(1), Ile(4), Ile(8)]-AngII (SII), and [Sar(1), D-Ala(8)]-AngII (TRV027). The aim of this study was to comparatively analyze the two above mentioned β-arrestin-biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between "G protein-dependent" and "β-arrestin-dependent" signaling. We observed clusters of activation profiles common to AngII, SII, and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of β-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization "G protein-dependent" vs. "β-arrestin-dependent" signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with

  11. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

    PubMed

    Kang, Yanyong; Zhou, X Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Ke, Jiyuan; Tan, M H Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M; Pascal, Bruce D; Van Eps, Ned; Caro, Lydia N; Vishnivetskiy, Sergey A; Lee, Regina J; Suino-Powell, Kelly M; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P; Katritch, Vsevolod; Gurevich, Vsevolod V; Griffin, Patrick R; Hubbell, Wayne L; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

    2015-07-30

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

  12. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

    SciTech Connect

    Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Ke, Jiyuan; Tan, M. H. Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M.; Pascal, Bruce D.; Van Eps, Ned; Caro, Lydia N.; Vishnivetskiy, Sergey A.; Lee, Regina J.; Suino-Powell, Kelly M.; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J.; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A.; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E.; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S.; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P.; Katritch, Vsevolod; Gurevich, Vsevolod V.; Griffin, Patrick R.; Hubbell, Wayne L.; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

    2015-07-22

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. In conclusion, this structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

  13. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

    DOE PAGES

    Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; et al

    2015-07-22

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotationmore » between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. In conclusion, this structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.« less

  14. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

    PubMed Central

    Kang, Yanyong; Zhou, X. Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Ke, Jiyuan; Eileen Tan, M. H.; Zhang, Chenghai; Moeller, Arne; West, Graham M.; Pascal, Bruce; Van Eps, Ned; Caro, Lydia N.; Vishnivetskiy, Sergey A.; Lee, Regina J.; Suino-Powell, Kelly M.; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J.; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A.; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N.; Spence, John C. H.; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P.; Katritch, Vsevolod; Gurevich, Vsevolod V.; Griffin, Patrick R.; Hubbell, Wayne L.; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

    2015-01-01

    G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the N- and C- domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. PMID:26200343

  15. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

    PubMed

    Kang, Yanyong; Zhou, X Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Ke, Jiyuan; Tan, M H Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M; Pascal, Bruce D; Van Eps, Ned; Caro, Lydia N; Vishnivetskiy, Sergey A; Lee, Regina J; Suino-Powell, Kelly M; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P; Katritch, Vsevolod; Gurevich, Vsevolod V; Griffin, Patrick R; Hubbell, Wayne L; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

    2015-07-30

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. PMID:26200343

  16. β-Arrestin-mediated Angiotensin II Signaling Controls the Activation of ARF6 Protein and Endocytosis in Migration of Vascular Smooth Muscle Cells.

    PubMed

    Charles, Ricardo; Namkung, Yoon; Cotton, Mathieu; Laporte, Stéphane A; Claing, Audrey

    2016-02-19

    Angiotensin II (Ang II) is a vasopressive hormone but is also a potent activator of cellular migration. We have previously shown that it can promote the activation of the GTPase ARF6 in a heterologous overexpressing system. The molecular mechanisms by which receptors control the activation of this small G protein remain, however, largely unknown. Furthermore, how ARF6 coordinates the activation of complex cellular responses needs to be further elucidated. In this study, we demonstrate that Ang II receptors engage β-arrestin, but not Gq, to mediate ARF6 activation in HEK 293 cells. To further confirm the key role of β-arrestin proteins, we overexpressed β-arrestin2-(1-320), a dominant negative mutant known to block receptor endocytosis. We show that expression of this truncated construct does not support the activation of the GTPase nor cell migration. Interestingly, β-arrestin2 can interact with the ARF guanine nucleotide exchange factor ARNO, although the C-terminally lacking mutant does not. We finally examined whether receptor endocytosis controlled ARF6 activation and cell migration. Although the clathrin inhibitor PitStop2 did not impact the ability of Ang II to activate ARF6, cell migration was markedly impaired. To further show that ARF activation regulates key signaling events leading to migration, we also examined MAPK activation. We demonstrate that this signaling axis is relevant in smooth muscle cells of the vasculature. Altogether, our findings show for the first time that Ang II receptor signaling to β-arrestin regulates ARF6 activation. These proteins together control receptor endocytosis and ultimately cell migration.

  17. The type III transforming growth factor-beta receptor negatively regulates nuclear factor kappa B signaling through its interaction with beta-arrestin2.

    PubMed

    You, Hye Jin; How, Tam; Blobe, Gerard C

    2009-08-01

    Transforming growth factor-beta (TGF-beta) increases or decreases nuclear factor kappa B (NFkappaB) signaling in a context-dependent manner through mechanisms that remain to be defined. The type III transforming growth factor-beta receptor (TbetaRIII) is a TGF-beta superfamily co-receptor with emerging roles in both mediating and regulating TGF-beta superfamily signaling. We have previously reported a novel interaction of TbetaRIII with the scaffolding protein, beta-arrestin2, which results in TbetaRIII internalization and downregulation of TGF-beta signaling. beta-arrestin2 also scaffolds interacting receptors with the mitogen-activated protein kinase and NFkappaB-signaling pathways. Here, we demonstrate that TbetaRIII, through its interaction with beta-arrestin2, negatively regulates NFkappaB signaling in MCF10A breast epithelial and MDA-MB-231 breast cancer cells. Increasing TbetaRIII expression reduced NFkappaB-mediated transcriptional activation and IkappaBalpha degradation, whereas a TbetaRIII mutant unable to interact with beta-arrestin2, TbetaRIII-T841A, had no effect. In a reciprocal manner, short hairpin RNA-mediated silencing of either TbetaRIII expression or beta-arrestin2 expression increased NFkappaB-mediated transcriptional activation and IkappaBalpha degradation. Functionally, TbetaRIII-mediated repression of NFkappaB signaling is important for TbetaRIII-mediated inhibition of breast cancer cell migration. These studies define a mechanism through which TbetaRIII regulates NFkappaB signaling and expand the roles of this TGF-beta superfamily co-receptor in regulating epithelial cell homeostasis.

  18. The type III transforming growth factor-β receptor negatively regulates nuclear factor kappa B signaling through its interaction with β-arrestin2

    PubMed Central

    You, Hye Jin; How, Tam; Blobe, Gerard C.

    2009-01-01

    Transforming growth factor-β (TGF-β) increases or decreases nuclear factor kappa B (NFκB) signaling in a context-dependent manner through mechanisms that remain to be defined. The type III transforming growth factor-β receptor (TβRIII) is a TGF-β superfamily co-receptor with emerging roles in both mediating and regulating TGF-β superfamily signaling. We have previously reported a novel interaction of TβRIII with the scaffolding protein, β-arrestin2, which results in TβRIII internalization and downregulation of TGF-β signaling. β-arrestin2 also scaffolds interacting receptors with the mitogen-activated protein kinase and NFκB-signaling pathways. Here, we demonstrate that TβRIII, through its interaction with β-arrestin2, negatively regulates NFκB signaling in MCF10A breast epithelial and MDA-MB-231 breast cancer cells. Increasing TβRIII expression reduced NFκB-mediated transcriptional activation and IκBα degradation, whereas a TβRIII mutant unable to interact with β-arrestin2, TβRIII-T841A, had no effect. In a reciprocal manner, short hairpin RNA-mediated silencing of either TβRIII expression or β-arrestin2 expression increased NFκB-mediated transcriptional activation and IκBα degradation. Functionally, TβRIII-mediated repression of NFκB signaling is important for TβRIII-mediated inhibition of breast cancer cell migration. These studies define a mechanism through which TβRIII regulates NFκB signaling and expand the roles of this TGF-β superfamily co-receptor in regulating epithelial cell homeostasis. PMID:19325136

  19. β-Arrestin-Dependent Dopaminergic Regulation of Calcium Channel Activity in the Axon Initial Segment.

    PubMed

    Yang, Sungchil; Ben-Shalom, Roy; Ahn, Misol; Liptak, Alayna T; van Rijn, Richard M; Whistler, Jennifer L; Bender, Kevin J

    2016-08-01

    G-protein-coupled receptors (GPCRs) initiate a variety of signaling cascades, depending on effector coupling. β-arrestins, which were initially characterized by their ability to "arrest" GPCR signaling by uncoupling receptor and G protein, have recently emerged as important signaling effectors for GPCRs. β-arrestins engage signaling pathways that are distinct from those mediated by G protein. As such, arrestin-dependent signaling can play a unique role in regulating cell function, but whether neuromodulatory GPCRs utilize β-arrestin-dependent signaling to regulate neuronal excitability remains unclear. Here, we find that D3 dopamine receptors (D3R) regulate axon initial segment (AIS) excitability through β-arrestin-dependent signaling, modifying CaV3 voltage dependence to suppress high-frequency action potential generation. This non-canonical D3R signaling thereby gates AIS excitability via pathways distinct from classical GPCR signaling pathways.

  20. Functional map of arrestin binding to phosphorylated opsin, with and without agonist

    PubMed Central

    Peterhans, Christian; Lally, Ciara C. M.; Ostermaier, Martin K.; Sommer, Martha E.; Standfuss, Jörg

    2016-01-01

    Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct. PMID:27350090

  1. Structural evidence for the role of polar core residue Arg175 in arrestin activation

    PubMed Central

    Granzin, Joachim; Stadler, Andreas; Cousin, Anneliese; Schlesinger, Ramona; Batra-Safferling, Renu

    2015-01-01

    Binding mechanism of arrestin requires photoactivation and phosphorylation of the receptor protein rhodopsin, where the receptor bound phosphate groups cause displacement of the long C-tail ‘activating’ arrestin. Mutation of arginine 175 to glutamic acid (R175E), a central residue in the polar core and previously predicted as the ‘phosphosensor’ leads to a pre-active arrestin that is able to terminate phototransduction by binding to non-phosphorylated, light-activated rhodopsin. Here, we report the first crystal structure of a R175E mutant arrestin at 2.7 Å resolution that reveals significant differences compared to the basal state reported in full-length arrestin structures. These differences comprise disruption of hydrogen bond network in the polar core, and three-element interaction including disordering of several residues in the receptor-binding finger loop and the C-terminus (residues 361–404). Additionally, R175E structure shows a 7.5° rotation of the amino and carboxy-terminal domains relative to each other. Consistent to the biochemical data, our structure suggests an important role of R29 in the initial activation step of C-tail release. Comparison of the crystal structures of basal arrestin and R175E mutant provide insights into the mechanism of arrestin activation, where binding of the receptor likely induces structural changes mimicked as in R175E. PMID:26510463

  2. β-Arrestin-kinase scaffolds: turn them on or turn them off?

    PubMed

    Lin, Alice; DeFea, Kathryn A

    2013-01-01

    G-protein-coupled receptors (GPCRs) can signal through heterotrimeric G-proteins or through β-arrestins to elicit responses to a plethora of extracellular stimuli. While the mechanisms underlying G-protein signaling is relatively well understood, the mechanisms by which β-arrestins regulate the diverse set of proteins with which they associate remain unclear. Multi-protein complexes are a common feature of β-arrestin-dependent signaling. The first two such complexes discovered were the mitogen-activated kinases modules associated with extracellular regulated kinases (ERK1/2) and Jnk3. Subsequently a number of other kinases have been shown to undergo β-arrestin-dependent regulation, including Akt, phosphatidylinositol-3kinase (PI3K), Lim-domain-containing kinase (LIMK), calcium calmodulin kinase II (CAMKII), and calcium calmodulin kinase kinase β (CAMKKβ). Some are positively and some negatively regulated by β-arrestin association. One of the missing links to understanding these pathways is the molecular mechanisms by which the activity of these kinases is regulated. Do β-arrestins merely serve as scaffolds to bring enzyme and substrate together or do they have a direct effect on the enzymatic activities of target kinases? Recent evidence suggests that both mechanisms are involved and that the mechanisms by which β-arrestins regulate kinase activity varies with the target kinase. This review discusses recent advances in the field focusing on 5 kinases for which considerable mechanistic detail and specific sites of interaction have been elucidated.

  3. South Dakota Social Studies Content Standards.

    ERIC Educational Resources Information Center

    South Dakota State Dept. of Education and Cultural Affairs, Pierre.

    This document presents the South Dakota Content Standards for K-12 Social Studies. The document outlines the four major areas of social studies: history, geography, civics, and economics. Standards are provided for each major area according to grade level, separately for grades K-8 and collectively for grades 9-12. Grade level standards represent…

  4. Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site.

    PubMed Central

    Neuschäfer-Rube, Frank; Hermosilla, Ricardo; Rehwald, Mathias; Rönnstrand, Lars; Schülein, Ralf; Wernstedt, Christer; Püschel, Gerhard Paul

    2004-01-01

    hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary. PMID:14709160

  5. The beta-arrestin pathway-selective type 1A angiotensin receptor (AT1A) agonist [Sar1,Ile4,Ile8]angiotensin II regulates a robust G protein-independent signaling network.

    PubMed

    Kendall, Ryan T; Strungs, Erik G; Rachidi, Saleh M; Lee, Mi-Hye; El-Shewy, Hesham M; Luttrell, Deirdre K; Janech, Michael G; Luttrell, Louis M

    2011-06-01

    The angiotensin II peptide analog [Sar(1),Ile(4),Ile(8)]AngII (SII) is a biased AT(1A) receptor agonist that stimulates receptor phosphorylation, β-arrestin recruitment, receptor internalization, and β-arrestin-dependent ERK1/2 activation without activating heterotrimeric G-proteins. To determine the scope of G-protein-independent AT(1A) receptor signaling, we performed a gel-based phosphoproteomic analysis of AngII and SII-induced signaling in HEK cells stably expressing AT(1A) receptors. A total of 34 differentially phosphorylated proteins were detected, of which 16 were unique to SII and eight to AngII stimulation. MALDI-TOF/TOF mass fingerprinting was employed to identify 24 SII-sensitive phosphoprotein spots, of which three (two peptide inhibitors of protein phosphatase 2A (I1PP2A and I2PP2A) and prostaglandin E synthase 3 (PGES3)) were selected for validation and further study. We found that phosphorylation of I2PP2A was associated with rapid and transient inhibition of a β-arrestin 2-associated pool of protein phosphatase 2A, leading to activation of Akt and increased phosphorylation of glycogen synthase kinase 3β in an arrestin signalsome complex. SII-stimulated PGES3 phosphorylation coincided with an increase in β-arrestin 1-associated PGES3 and an arrestin-dependent increase in cyclooxygenase 1-dependent prostaglandin E(2) synthesis. These findings suggest that AT(1A) receptors regulate a robust G protein-independent signaling network that affects protein phosphorylation and autocrine/paracrine prostaglandin production and that these pathways can be selectively modulated by biased ligands that antagonize G protein activation.

  6. Visual Cone Arrestin 4 Contributes to Visual Function and Cone Health

    PubMed Central

    Deming, Janise D.; Pak, Joseph S.; Brown, Bruce M.; Kim, Moon K.; Aung, Moe H.; Eom, Yun Sung; Shin, Jung-a; Lee, Eun-Jin; Pardue, Machelle T.; Craft, Cheryl Mae

    2015-01-01

    Purpose Visual arrestins (ARR) play a critical role in shutoff of rod and cone phototransduction. When electrophysiological responses are measured for a single mouse cone photoreceptor, ARR1 expression can substitute for ARR4 in cone pigment desensitization; however, each arrestin may also contribute its own, unique role to modulate other cellular functions. Methods A combination of ERG, optokinetic tracking, immunohistochemistry, and immunoblot analysis was used to investigate the retinal phenotypes of Arr4 null mice (Arr4−/−) compared with age-matched control, wild-type mice. Results When 2-month-old Arr4−/− mice were compared with wild-type mice, they had diminished visual acuity and contrast sensitivity, yet enhanced ERG flicker response and higher photopic ERG b-wave amplitudes. In contrast, in older Arr4−/− mice, all ERG amplitudes were significantly reduced in magnitude compared with age-matched controls. Furthermore, in older Arr4−/− mice, the total cone numbers decreased and cone opsin protein immunoreactive expression levels were significantly reduced, while overall photoreceptor outer nuclear layer thickness was unchanged. Conclusions Our study demonstrates that Arr4−/− mice display distinct phenotypic differences when compared to controls, suggesting that ARR4 modulates essential functions in high acuity vision and downstream cellular signaling pathways that are not fulfilled or substituted by the coexpression of ARR1, despite its high expression levels in all mouse cones. Without normal ARR4 expression levels, cones slowly degenerate with increasing age, making this a new model to study age-related cone dystrophy. PMID:26284544

  7. β-Arrestin1 inhibits chemotherapy-induced intestinal stem cell apoptosis and mucositis

    PubMed Central

    Zhan, Y; Xu, C; Liu, Z; Yang, Y; Tan, S; Yang, Y; Jiang, J; Liu, H; Chen, J; Wu, B

    2016-01-01

    The mechanism of chemotherapy-induced gastrointestinal (GI) syndrome (CIGIS) is still controversial, and it is unclear whether chemotherapy induces intestinal stem cell (ISC) apoptosis. β-Arrestins are regulators and mediators of G protein-coupled receptor signaling in cell apoptosis, division and growth. In this study, we aimed to investigate whether chemotherapy induces ISC apoptosis to contribute to mucositis in CIGIS and whether β-arrestin1 (β-arr1) is involved in this apoptosis. Different chemotherapeutic agents were used to generate a CIGIS model. Lgr5-EGFP-IRES-creERT2+/− knock-in mice were used as a CIGIS model to investigate ISC apoptosis. β-arr1 knockout mice were used to determine whether β-arr1 is involved in the apoptosis in CIGIS. Intestinal histology was performed, the ISC apoptosis was analyzed and the mucosal barrier was examined. The effects of β-arr1 in apoptosis were investigated in the samples from humans and mice as well as in cell lines. Here, we demonstrate that chemotherapy induced intestinal mucositis by promoting crypt cell apoptosis, especially in Lgr5+ stem cells and Paneth cells but not in goblet cells, epithelial cells or vascular endothelial cells. Furthermore, β-arr1 deficiency exacerbated the Lgr5+ stem cell apoptosis, but not Paneth cell apoptosis, in CIGIS. In addition, the data showed that β-arr1 reduced the chemotherapy-induced Lgr5+ stem cell apoptosis by inhibiting endoplasmic reticulum stress-mediated mitochondrial apoptotic signaling. Our study indicates that β-arr1 inhibits chemotherapy-induced ISC apoptosis to alleviate intestinal mucositis in CIGIS. PMID:27195676

  8. Identifying bias in CCR1 antagonists using radiolabelled binding, receptor internalization, β-arrestin translocation and chemotaxis assays

    PubMed Central

    Gilchrist, A; Gauntner, T D; Fazzini, A; Alley, K M; Pyen, D S; Ahn, J; Ha, S J; Willett, A; Sansom, S E; Yarfi, J L; Bachovchin, K A; Mazzoni, M R; Merritt, J R

    2014-01-01

    Background and Purpose Investigators have suggested that the chemokine receptor CCR1 plays a role in multiple myeloma. Studies using antisense and neutralizing antibodies to CCR1 showed that down-regulation of the receptor altered disease progression in a mouse model. More recently, experiments utilizing scid mice injected with human myeloma cells demonstrated that the CCR1 antagonist BX471 reduced osteolytic lesions, while the CCR1 antagonist MLN-3897 prevented myeloma cell adhesion to osteoclasts. However, information is limited regarding the pharmacology of CCR1 antagonists in myeloma cells. Experimental Approach We compared several well-studied CCR1 antagonists including AZD4818, BX471, CCX354, CP-481715, MLN-3897 and PS899877 for their ability to inhibit binding of [125I]-CCL3 in vitro using membranes prepared from RPMI 8226 cells, a human multiple myeloma cell line that endogenously expresses CCR1. In addition, antagonists were assessed for their ability to modulate CCL3-mediated internalization of CCR1 and CCL3-mediated cell migration using RPMI 8226 cells. As many GPCRs signal through β–arrestin-dependent pathways that are separate and distinct from those driven by G-proteins, we also evaluated the compounds for their ability to alter β-arrestin translocation. Key Results There were clear differences between the CCR1 antagonists in their ability to inhibit CCL3 binding to myeloma cells, as well as in their ability to inhibit G–protein-dependent and -independent functional responses. Conclusions and Implications Our studies demonstrate that tissue phenotype seems to be relevant with regards to CCR1. Moreover, it appears that for CCR1 antagonists, inhibition of β-arrestin translocation is not necessarily linked to chemotaxis or receptor internalization. PMID:24990525

  9. Connecting Literacy with Social Studies Content.

    ERIC Educational Resources Information Center

    Johnson, Margaret J.; Janisch, Carole

    1998-01-01

    Explains how teachers at Ramirez Elementary School in Lubbock (Texas) use thematic teaching to make connections across subject areas and to provide students with rich literacy instruction. Describes how these teachers implement social studies content to enhance their students' reading, writing, and thinking skills. Illustrates the power of…

  10. Inhibitory effects of omega-3 fatty acids on early brain injury after subarachnoid hemorrhage in rats: Possible involvement of G protein-coupled receptor 120/β-arrestin2/TGF-β activated kinase-1 binding protein-1 signaling pathway.

    PubMed

    Yin, Jia; Li, Haiying; Meng, Chengjie; Chen, Dongdong; Chen, Zhouqing; Wang, Yibin; Wang, Zhong; Chen, Gang

    2016-06-01

    Omega-3 fatty acids have been reported to improve neuron functions during aging and in patients affected by mild cognitive impairment, and mediate potent anti-inflammatory via G protein-coupled receptor 120 (GPR120) signal pathway. Neuron dysfunction and inflammatory response also contributed to the progression of subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was to examine the effects of omega-3 fatty acids on SAH-induced EBI. Two weeks before SAH, 30% Omega-3 fatty acids was administered by oral gavage at 1g/kg body weight once every 24h. Specific siRNA for GPR120 was exploited. Terminal deoxynucleotidyl transferase dUTP nick end labeling, fluoro-Jade B staining, and neurobehavioral scores and brain water content test showed that omega-3 fatty acids effectively suppressed SAH-induced brain cell apoptosis and neuronal degradation, behavioral impairment, and brain edema. Western blot, immunoprecipitation, and electrophoretic mobility shift assays results showed that omega-3 fatty acids effectively suppressed SAH-induced elevation of inflammatory factors, including cyclooxygenase-2, monocyte chemoattractant protein-1, and inducible nitric oxide synthase. In addition, omega-3 fatty acids could inhibit phosphorylation of transforming growth factor β activated kinase-1 (TAK1), MEK4, c-Jun N-terminal kinase, and IkappaB kinase as well as activation of nuclear factor kappa B through regulating GPR120/β-arrestin2/TAK1 binding protein-1 pathway. Furthermore, siRNA-induced GPR120 silencing blocked the protective effects of omega-3 fatty acids. Here, we show that stimulation of GPR120 with omega-3 fatty acids pretreatment causes anti-apoptosis and anti-inflammatory effects via β-arrestin2/TAK1 binding protein-1/TAK1 pathway in the brains of SAH rats. Fish omega-3 fatty acids as part of a daily diet may reduce EBI in an experimental rat model of SAH.

  11. Inhibitory effects of omega-3 fatty acids on early brain injury after subarachnoid hemorrhage in rats: Possible involvement of G protein-coupled receptor 120/β-arrestin2/TGF-β activated kinase-1 binding protein-1 signaling pathway.

    PubMed

    Yin, Jia; Li, Haiying; Meng, Chengjie; Chen, Dongdong; Chen, Zhouqing; Wang, Yibin; Wang, Zhong; Chen, Gang

    2016-06-01

    Omega-3 fatty acids have been reported to improve neuron functions during aging and in patients affected by mild cognitive impairment, and mediate potent anti-inflammatory via G protein-coupled receptor 120 (GPR120) signal pathway. Neuron dysfunction and inflammatory response also contributed to the progression of subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was to examine the effects of omega-3 fatty acids on SAH-induced EBI. Two weeks before SAH, 30% Omega-3 fatty acids was administered by oral gavage at 1g/kg body weight once every 24h. Specific siRNA for GPR120 was exploited. Terminal deoxynucleotidyl transferase dUTP nick end labeling, fluoro-Jade B staining, and neurobehavioral scores and brain water content test showed that omega-3 fatty acids effectively suppressed SAH-induced brain cell apoptosis and neuronal degradation, behavioral impairment, and brain edema. Western blot, immunoprecipitation, and electrophoretic mobility shift assays results showed that omega-3 fatty acids effectively suppressed SAH-induced elevation of inflammatory factors, including cyclooxygenase-2, monocyte chemoattractant protein-1, and inducible nitric oxide synthase. In addition, omega-3 fatty acids could inhibit phosphorylation of transforming growth factor β activated kinase-1 (TAK1), MEK4, c-Jun N-terminal kinase, and IkappaB kinase as well as activation of nuclear factor kappa B through regulating GPR120/β-arrestin2/TAK1 binding protein-1 pathway. Furthermore, siRNA-induced GPR120 silencing blocked the protective effects of omega-3 fatty acids. Here, we show that stimulation of GPR120 with omega-3 fatty acids pretreatment causes anti-apoptosis and anti-inflammatory effects via β-arrestin2/TAK1 binding protein-1/TAK1 pathway in the brains of SAH rats. Fish omega-3 fatty acids as part of a daily diet may reduce EBI in an experimental rat model of SAH. PMID:27000704

  12. Fanpage metrics analysis. "Study on content engagement"

    NASA Astrophysics Data System (ADS)

    Rahman, Zoha; Suberamanian, Kumaran; Zanuddin, Hasmah Binti; Moghavvemi, Sedigheh; Nasir, Mohd Hairul Nizam Bin Md

    2016-08-01

    Social Media is now determined as an excellent communicative tool to connect directly with consumers. One of the most significant ways to connect with the consumers through these Social Networking Sites (SNS) is to create a facebook fanpage with brand contents and to place different posts periodically on these fanpages. In measuring social networking sites' effectiveness, corporate houses are now analyzing metrics in terms of calculating engagement rate, number of comments/share and likings in fanpages. So now, it is very important for the marketers to know the effectiveness of different contents or posts of fanpages in order to increase the fan responsiveness and engagement rate in the fan pages. In the study the authors have analyzed total 1834 brand posts from 17 international brands of Electronics companies. Data of 9 months (From December 2014 to August 2015) have been collected for analyses, which were available online in the Brand' fan pages. An econometrics analysis is conducted using Eviews 9, to determine the impact of different contents on fanpage engagement. The study picked the four most frequently posted content to determine their impact on PTA (people Talking About) metrics and Fanpage engagement activities.

  13. Regulation of mitochondrial oxidative stress by β-arrestins in cultured human cardiac fibroblasts.

    PubMed

    Philip, Jennifer L; Razzaque, Md Abdur; Han, Mei; Li, Jinju; Theccanat, Tiju; Xu, Xianyao; Akhter, Shahab A

    2015-12-01

    Oxidative stress in cardiac fibroblasts (CFs) promotes transformation to myofibroblasts and collagen synthesis leading to myocardial fibrosis, a precursor to heart failure (HF). NADPH oxidase 4 (Nox4) is a major source of cardiac reactive oxygen species (ROS); however, mechanisms of Nox4 regulation are unclear. β-arrestins are scaffold proteins that signal in G-protein-dependent and -independent pathways; for example, in ERK activation. We hypothesize that β-arrestins regulate oxidative stress in a Nox4-dependent manner and increase fibrosis in HF. CFs were isolated from normal and failing adult human left ventricles. Mitochondrial ROS/superoxide production was quantitated using MitoSox. β-arrestin and Nox4 expressions were manipulated using adenoviral overexpression or short interfering RNA (siRNA)-mediated knockdown. Mitochondrial oxidative stress and Nox4 expression in CFs were significantly increased in HF. Nox4 knockdown resulted in inhibition of mitochondrial superoxide production and decreased basal and TGF-β-stimulated collagen and α-SMA expression. CF β-arrestin expression was upregulated fourfold in HF. β-arrestin knockdown in failing CFs decreased ROS and Nox4 expression by 50%. β-arrestin overexpression in normal CFs increased mitochondrial superoxide production twofold. These effects were prevented by inhibition of either Nox or ERK. Upregulation of Nox4 seemed to be a primary mechanism for increased ROS production in failing CFs, which stimulates collagen deposition. β-arrestin expression was upregulated in HF and plays an important and newly identified role in regulating mitochondrial superoxide production via Nox4. The mechanism for this effect seems to be ERK-mediated. Targeted inhibition of β-arrestins in CFs might decrease oxidative stress as well as pathological cardiac fibrosis.

  14. Regulation of mitochondrial oxidative stress by β-arrestins in cultured human cardiac fibroblasts

    PubMed Central

    Philip, Jennifer L.; Razzaque, Md. Abdur; Han, Mei; Li, Jinju; Theccanat, Tiju; Xu, Xianyao; Akhter, Shahab A.

    2015-01-01

    ABSTRACT Oxidative stress in cardiac fibroblasts (CFs) promotes transformation to myofibroblasts and collagen synthesis leading to myocardial fibrosis, a precursor to heart failure (HF). NADPH oxidase 4 (Nox4) is a major source of cardiac reactive oxygen species (ROS); however, mechanisms of Nox4 regulation are unclear. β-arrestins are scaffold proteins that signal in G-protein-dependent and -independent pathways; for example, in ERK activation. We hypothesize that β-arrestins regulate oxidative stress in a Nox4-dependent manner and increase fibrosis in HF. CFs were isolated from normal and failing adult human left ventricles. Mitochondrial ROS/superoxide production was quantitated using MitoSox. β-arrestin and Nox4 expressions were manipulated using adenoviral overexpression or short interfering RNA (siRNA)-mediated knockdown. Mitochondrial oxidative stress and Nox4 expression in CFs were significantly increased in HF. Nox4 knockdown resulted in inhibition of mitochondrial superoxide production and decreased basal and TGF-β-stimulated collagen and α-SMA expression. CF β-arrestin expression was upregulated fourfold in HF. β-arrestin knockdown in failing CFs decreased ROS and Nox4 expression by 50%. β-arrestin overexpression in normal CFs increased mitochondrial superoxide production twofold. These effects were prevented by inhibition of either Nox or ERK. Upregulation of Nox4 seemed to be a primary mechanism for increased ROS production in failing CFs, which stimulates collagen deposition. β-arrestin expression was upregulated in HF and plays an important and newly identified role in regulating mitochondrial superoxide production via Nox4. The mechanism for this effect seems to be ERK-mediated. Targeted inhibition of β-arrestins in CFs might decrease oxidative stress as well as pathological cardiac fibrosis. PMID:26449263

  15. Evaluation of the arrestin gene in patients with retinitis pigmentosa or an allied disease

    SciTech Connect

    DeStefano, D.J.; Berson, E.L.; Dryja, T.P.

    1994-09-01

    Arrestin, also called 48K protein or S-antigen, plays a role in deactivating rhodopsin, the photosensitive, seven-helix, G-protein receptor found in rod photoreceptors. In Drosophila, null mutations in arrestin genes cause a light-dependent photoreceptor degeneration. It is possible that a comparable photoreceptor degeneration in humans is caused by defects in the rod arrestin gene. In order to evaluate this possibility, we are characterizing the human arrestin locus on chromosome 2q. We screened a genomic library (5 million plaques) using an arrestin cDNA clone. Sixty-eight hybridizing clones were identified; portions of 7 clones were sequenced to determine the intron sequence flanking the exons. We are using SSCP analysis and direct genomic sequencing to screen the entire coding region, splice donor and acceptor sites, and the promoter region of the arrestin gene in 188 patients with autosomal dominant and 104 patients with autosomal recessive retinitis pigmentosa. We have already obtained flanking intron sequences necessary for SSCP analysis for 13 of 16 exons. So far, we have identified 4 silent base changes at codons 67 (TGC-to-TGT), 107 (CTG-to-CTC), 163 (GCC-to-GCT), and 288 (CTG-to-TGT), all with allele frequencies at 1% or less. Several other variant bands detected by SSCP analysis are currently being sequenced.

  16. Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gβγ complex.

    PubMed

    Wehbi, Vanessa L; Stevenson, Hilary P; Feinstein, Timothy N; Calero, Guillermo; Romero, Guillermo; Vilardaga, Jean-Pierre

    2013-01-22

    G protein-coupled receptors (GPCRs) participate in ubiquitous transmembrane signal transduction processes by activating heterotrimeric G proteins. In the current "canonical" model of GPCR signaling, arrestins terminate receptor signaling by impairing receptor-G-protein coupling and promoting receptor internalization. However, parathyroid hormone receptor type 1 (PTHR), an essential GPCR involved in bone and mineral metabolism, does not follow this conventional desensitization paradigm. β-Arrestins prolong G protein (G(S))-mediated cAMP generation triggered by PTH, a process that correlates with the persistence of arrestin-PTHR complexes on endosomes and which is thought to be associated with prolonged physiological calcemic and phosphate responses. This presents an inescapable paradox for the current model of arrestin-mediated receptor-G-protein decoupling. Here we show that PTHR forms a ternary complex that includes arrestin and the Gβγ dimer in response to PTH stimulation, which in turn causes an accelerated rate of G(S) activation and increases the steady-state levels of activated G(S), leading to prolonged generation of cAMP. This work provides the mechanistic basis for an alternative model of GPCR signaling in which arrestins contribute to sustaining the effect of an agonist hormone on the receptor.

  17. Cloning of a member of the arrestin family from a human thyroid cDNA library.

    PubMed

    Rapoport, B; Kaufman, K D; Chazenbalk, G D

    1992-04-01

    We used the cDNA of human retinal arrestin as a probe to screen a human thyroid cDNA library. We isolated and plaque-purified one clone (hTHY-ARRX). The nucleotide sequence of the 1.8 kb cDNA insert had an open reading frame of 1227 bp coding for a protein of 409 amino acids. Northern blot analysis revealed a single transcript of 1.7 kb in human thyroid cells. There is significant homology between amino acid sequences of human thyroid arrestin and human retinal arrestin (63%) and bovine beta-arrestin (74%), respectively. The hTHY-ARRX cDNA was stably transfected into Chinese hamster ovary cells already expressing a functional human thyrotropin (TSH) receptor. The cAMP response to TSH stimulation was unaltered in these cells, and homologous desensitization to TSH stimulation was not restored. It is not presently known whether hTHY-ARRX is human beta-adrenergic arrestin or a new member of the arrestin family.

  18. Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR

    PubMed Central

    Yang, Fan; Yu, Xiao; Liu, Chuan; Qu, Chang-Xiu; Gong, Zheng; Liu, Hong-Da; Li, Fa-Hui; Wang, Hong-Mei; He, Dong-Fang; Yi, Fan; Song, Chen; Tian, Chang-Lin; Xiao, Kun-Hong; Wang, Jiang-Yun; Sun, Jin-Peng

    2015-01-01

    Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors. PMID:26347956

  19. The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

    PubMed Central

    de Munnik, Sabrina M.; Kooistra, Albert J.; van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, Chris; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi’s sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74. PMID:25894435

  20. The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines.

    PubMed

    de Munnik, Sabrina M; Kooistra, Albert J; van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, Chris; Smit, Martine J; Leurs, Rob; Vischer, Henry F

    2015-01-01

    Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi's sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

  1. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET.

    PubMed

    Namkung, Yoon; Le Gouill, Christian; Lukashova, Viktoria; Kobayashi, Hiroyuki; Hogue, Mireille; Khoury, Etienne; Song, Mideum; Bouvier, Michel; Laporte, Stéphane A

    2016-01-01

    Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes. PMID:27397672

  2. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET

    PubMed Central

    Namkung, Yoon; Le Gouill, Christian; Lukashova, Viktoria; Kobayashi, Hiroyuki; Hogue, Mireille; Khoury, Etienne; Song, Mideum; Bouvier, Michel; Laporte, Stéphane A.

    2016-01-01

    Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes. PMID:27397672

  3. β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1.

    PubMed

    Mancini, Arturo D; Bertrand, Gyslaine; Vivot, Kevin; Carpentier, Éric; Tremblay, Caroline; Ghislain, Julien; Bouvier, Michel; Poitout, Vincent

    2015-08-21

    FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses ("biased agonism"). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.

  4. β-arrestin-1 drives endothelin-1-mediated podocyte activation and sustains renal injury.

    PubMed

    Buelli, Simona; Rosanò, Laura; Gagliardini, Elena; Corna, Daniela; Longaretti, Lorena; Pezzotta, Anna; Perico, Luca; Conti, Sara; Rizzo, Paola; Novelli, Rubina; Morigi, Marina; Zoja, Carlamaria; Remuzzi, Giuseppe; Bagnato, Anna; Benigni, Ariela

    2014-03-01

    Activation of endothelin-A receptor (ET(A)R) by endothelin-1 (ET-1) drives epithelial-to-mesenchymal transition in ovarian tumor cells through β-arrestin signaling. Here, we investigated whether this pathogenetic pathway could affect podocyte phenotype in proliferative glomerular disorders. In cultured mouse podocytes, ET-1 caused loss of the podocyte differentiation marker synaptopodin and acquisition of the mesenchymal marker α-smooth muscle actin. ET-1 promoted podocyte migration via ET(A)R activation and increased β-arrestin-1 expression. Activated ET(A)R recruited β-arrestin-1 to form a trimeric complex with Src leading to epithelial growth factor receptor (EGFR) transactivation and β-catenin phosphorylation, which promoted gene transcription of Snail. Increased Snail expression fostered ET-1-induced migration as confirmed by Snail knockdown experiments. Silencing of β-arrestin-1 prevented podocyte phenotypic changes and motility and inhibited ET(A)R-driven signaling. In vitro findings were confirmed in doxorubicin (Adriamycin)-induced nephropathy. Mice receiving Adriamycin developed renal injury with loss of podocytes and hyperplastic lesion formation; β-arrestin-1 expression increased in visceral podocytes and in podocytes entrapped in pseudo-crescents. Administration of the selective ET(A)R antagonist sitaxsentan prevented podocyte loss, formation of the hyperplastic lesions, and normalized expression of glomerular β-arrestin-1 and Snail. Increased β-arrestin-1 levels in podocytes retrieved from crescents of patients with proliferative glomerulopathies confirmed the translational relevance of these findings and suggest the therapeutic potential of ET(A)R antagonism for a group of diseases still needing a specific treatment.

  5. β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1*

    PubMed Central

    Mancini, Arturo D.; Bertrand, Gyslaine; Vivot, Kevin; Carpentier, Éric; Tremblay, Caroline; Ghislain, Julien; Bouvier, Michel; Poitout, Vincent

    2015-01-01

    FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses (“biased agonism”). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes. PMID:26157145

  6. G Protein and β-Arrestin Signaling Bias at the Ghrelin Receptor*

    PubMed Central

    Evron, Tama; Peterson, Sean M.; Urs, Nikhil M.; Bai, Yushi; Rochelle, Lauren K.; Caron, Marc G.; Barak, Larry S.

    2014-01-01

    The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through Gq/11, Gi/o, and G12/13 as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca2+ mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events. PMID:25261469

  7. Fenoterol inhibits LPS-induced AMPK activation and inflammatory cytokine production through β-arrestin-2 in THP-1 cell line

    SciTech Connect

    Wang, Wei; Zhang, Yuan; Xu, Ming; Zhang, You-Yi; He, Bei

    2015-06-26

    The AMP-activated protein kinase (AMPK) pathway is involved in regulating inflammation in several cell lines. We reported that fenoterol, a β{sub 2}-adrenergic receptor (β{sub 2}-AR) agonist, had anti-inflammatory effects in THP-1 cells, a monocytic cell line. Whether the fenoterol anti-inflammatory effect involves the AMPK pathway is unknown. In this study, we explored the mechanism of β{sub 2}-AR stimulation with fenoterol in a lipopolysaccharide (LPS)-induced inflammatory cytokine secretion in THP-1 cells. We studied whether fenoterol and β-arrestin-2 or AMPKα1 subunit knockdown could affect LPS-induced AMPK activation, nuclear factor-kappa B (NF-κB) activation and inflammatory cytokine secretion. LPS-induced AMPK activation and interleukin 1β (IL-1β) release were reduced with fenoterol pretreatment of THP-1 cells. SiRNA knockdown of β-arrestin-2 abolished the fenoterol inhibition of LPS-induced AMPK activation and interleukin 1β (IL-1β) release, thus β-arrestin-2 mediated the anti-inflammatory effects of fenoterol on LPS-treated THP-1 cells. In addition, siRNA knockdown of AMPKα1 significantly attenuated the LPS-induced NF-κB activation and IL-1β release, so AMPKα1 was a key signaling molecule involved in LPS-induced inflammatory cytokine production. These results suggested the β{sub 2}-AR agonist fenoterol inhibited LPS-induced AMPK activation and IL-1β release via β-arrestin-2 in THP-1 cells. The exploration of these mechanisms may help optimize therapeutic agents targeting these pathways in inflammatory diseases. - Highlights: • β{sub 2}-AR agonist fenoterol exerts its protective effect on LPS-treated THP-1 cells. • Fenoterol inhibits LPS-induced AMPK activation and IL-1β production. • β-arrestin2 mediates fenoterol-inhibited AMPK activation and IL-1β release. • AMPKα1 is involved in LPS-induced NF-κB activation and IL-1β production.

  8. β-Arrestin-biased AT1R stimulation promotes cell survival during acute cardiac injury.

    PubMed

    Kim, Ki-Seok; Abraham, Dennis; Williams, Barbara; Violin, Jonathan D; Mao, Lan; Rockman, Howard A

    2012-10-15

    Pharmacological blockade of the ANG II type 1 receptor (AT1R) is a common therapy for treatment of congestive heart failure and hypertension. Increasing evidence suggests that selective engagement of β-arrestin-mediated AT1R signaling, referred to as biased signaling, promotes cardioprotective signaling. Here, we tested the hypothesis that a β-arrestin-biased AT1R ligand TRV120023 would confer cardioprotection in response to acute cardiac injury compared with the traditional AT1R blocker (ARB), losartan. TRV120023 promotes cardiac contractility, assessed by pressure-volume loop analyses, while blocking the effects of endogenous ANG II. Compared with losartan, TRV120023 significantly activates MAPK and Akt signaling pathways. These hemodynamic and biochemical effects were lost in β-arrestin-2 knockout (KO) mice. In response to cardiac injury induced by ischemia reperfusion injury or mechanical stretch, pretreatment with TRV120023 significantly diminishes cell death compared with losartan, which did not appear to be cardioprotective. This cytoprotective effect was lost in β-arrestin-2 KO mice. The β-arrestin-biased AT1R ligand, TRV120023, has cardioprotective and functional properties in vivo, which are distinct from losartan. Our data suggest that this novel class of drugs may provide an advantage over conventional ARBs by supporting cardiac function and reducing cellular injury during acute cardiac injury.

  9. Content Validity Studies of Licensing Examinations.

    ERIC Educational Resources Information Center

    Smith, I. Leon; Hambleton, Ronald K.

    1990-01-01

    Implementing measurement specialists' ideas about content validity with licensure examinations and the problem of court litigation are discussed. Validity issues surfacing when sponsors of national licensure examinations conduct validity investigations are considered. Issues include local versus national focus on content validity, job analysis,…

  10. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

    PubMed Central

    Zhou, X. Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W.; Suino-Powell, Kelly M.; Boutet, Sébastien; Williams, Garth J.; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N.; Spence, John C.H.; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C.; Cherezov, Vadim; Melcher, Karsten; Xu, H. Eric

    2016-01-01

    Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. PMID:27070998

  11. Arrestin translocation is stoichiometric to rhodopsin isomerization and accelerated by phototransduction in Drosophila photoreceptors

    PubMed Central

    Satoh, Akiko K.; Xia, Hongai; Yan, Limin; Liu, Che-Hsiung; Hardie, Roger C.; Ready, Donald F.

    2010-01-01

    Upon illumination visual arrestin translocates from photoreceptor cell bodies to rhodopsin and membrane-rich photosensory compartments - vertebrate outer segments or invertebrate rhabdomeres - where it quenches activated rhodopsin. Both the mechanism and function of arrestin translocation are unresolved and controversial. In dark-adapted photoreceptors of the fruitfly Drosophila, confocal immunocytochemistry shows arrestin (Arr2) associated with distributed photoreceptor endomembranes. Immunocytochemistry and live imaging of GFP-tagged Arr2 demonstrate rapid reversible translocation to stimulated rhabdomeres in stoichiometric proportion to rhodopsin photoisomerization. Translocation is very rapid in normal photoreceptors (time constant <10 s), and can also be resolved in the time course of electroretinogram recordings. Genetic elimination of key phototransduction proteins, including phospholipase C (PLC), Gq and the light-sensitive Ca2+ permeable TRP channels, slows translocation by 10-100 fold. Our results indicate that Arr2 translocation in Drosophila photoreceptors is driven by diffusion, but profoundly accelerated by phototransduction and Ca2+ influx. PMID:20869596

  12. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex.

    PubMed

    Zhou, X Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Suino-Powell, Kelly M; Boutet, Sébastien; Williams, Garth J; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

    2016-01-01

    Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.

  13. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex.

    PubMed

    Zhou, X Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Suino-Powell, Kelly M; Boutet, Sébastien; Williams, Garth J; Wang, Meitian; Li, Dianfan; Caffrey, Martin; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

    2016-01-01

    Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. PMID:27070998

  14. PGE2 Inhibits IL-10 Production via EP2-Mediated β-Arrestin Signaling in Neuroinflammatory Condition.

    PubMed

    Chu, Chun-Hsien; Chen, Shih-Heng; Wang, Qingshan; Langenbach, Robert; Li, Hong; Zeldin, Darryl; Chen, Shiou-Lan; Wang, Shijun; Gao, Huiming; Lu, Ru-Band; Hong, Jau-Shyong

    2015-08-01

    Regulatory mechanisms of the expression of interleukin-10 (IL-10) in brain inflammatory conditions remain elusive. To address this issue, we used multiple primary brain cell cultures to study the expression of IL-10 in lipopolysaccharide (LPS)-elicited inflammatory conditions. In neuron-glia cultures, LPS triggered well-orchestrated expression of various immune factors in the following order: tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and lastly IL-10, and these inflammatory mediators were mainly produced from microglia. While exogenous application of individual earlier-released pro-inflammatory factors (e.g., TNF-α, IL-1β, or PGE2) failed to induce IL-10 expression, removal of LPS from the cultures showed the requirement of continuing presence of LPS for IL-10 expression. Interestingly, genetic disruption of tnf-α, its receptors tnf-r1/r2, and cox-2 and pharmacological inhibition of COX-2 activity enhanced LPS-induced IL-10 production in microglia, which suggests negative regulation of IL-10 induction by the earlier-released TNF-α and PGE2. Further studies showed that negative regulation of IL-10 production by TNF-α is mediated by PGE2. Mechanistic studies indicated that PGE2-elicited suppression of IL-10 induction was eliminated by genetic disruption of the PGE2 receptor EP2 and was mimicked by the specific agonist for the EP2, butaprost, but not agonists for the other three EP receptors. Inhibition of cAMP-dependent signal transduction failed to affect PGE2-mediated inhibition of IL-10 production, suggesting that a G protein-independent pathway was involved. Indeed, deficiency in β-arrestin-1 or β-arrestin-2 abolished PGE2-elicited suppression of IL-10 production. In conclusion, we have demonstrated that COX-2-derived PGE2 inhibits IL-10 expression in brain microglia through a novel EP2- and β-arrestin-dependent signaling pathway.

  15. Role of receptor-attached phosphates in binding of visual and non-visual arrestins to G protein-coupled receptors.

    PubMed

    Gimenez, Luis E; Kook, Seunghyi; Vishnivetskiy, Sergey A; Ahmed, M Rafiuddin; Gurevich, Eugenia V; Gurevich, Vsevolod V

    2012-03-16

    Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in β-strand I was shown to disrupt the interaction of α-helix I, β-strand I, and the C-tail of visual arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins in which the K2A mutations had little effect on net BRET(max) values for the M2 muscarinic acetylcholine (M2R), β(2)-adrenergic (β(2)AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual arrestins normally, whereas phosphorylation-deficient β(2)AR mutants bound arrestins at 20-50% of the level of wild type β(2)AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. Although arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of arrestin-2 and -3 is much greater in the case of β(2)AR than M2R and D2 dopamine receptor.

  16. The physical association of the P2Y12 receptor with PAR4 regulates arrestin-mediated Akt activation.

    PubMed

    Khan, Aasma; Li, Dongjun; Ibrahim, Salam; Smyth, Emer; Woulfe, Donna S

    2014-07-01

    It is now well accepted that protease activated receptor (PAR) 1 and PAR4 have differential roles in platelet activation. PAR4, a low-affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12-dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form of PAR4, "PAR4SFT," by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and contributes to Gq-coupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent stabilization of platelet thrombi.

  17. The physical association of the P2Y12 receptor with PAR4 regulates arrestin-mediated Akt activation.

    PubMed

    Khan, Aasma; Li, Dongjun; Ibrahim, Salam; Smyth, Emer; Woulfe, Donna S

    2014-07-01

    It is now well accepted that protease activated receptor (PAR) 1 and PAR4 have differential roles in platelet activation. PAR4, a low-affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12-dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form of PAR4, "PAR4SFT," by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and contributes to Gq-coupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent stabilization of platelet thrombi. PMID:24723492

  18. Evidence of a role for S. cerevisiae α-arrestin Art1 (Ldb19) in mating projection and zygote formations.

    PubMed

    Choudhary, Pooja; Loewen, Michele C

    2016-01-01

    The discovery of arrestin-mediated biased signalling mechanisms for mammalian G-protein coupled receptors (GPCRs) has revolutionized the field over the last decade. Now, with the recent demonstration of a role for α-arrestins in internalization of the yeast pheromone GPCR, Ste2p, the possibility of arrestin-mediated alternate GPCR functionalities in yeast also follows. Here, the effects of knockout and complementation of yeast α-arrestin expression during mating are reported. Although minor effects on classical pheromone-related signalling are noted for a few arrestins, much stronger effects were observed downstream of cell cycle arrest, in particular linking Ldb19 (Art1) to mediation of zygote formation. Subsequent phenotypic observations linked this activity to more pronounced projection formation in an Art1 complemented noncuple α-arrestin knockout line, compared to the knockout-line alone, or either of the Art3 or Art6 complemented lines. Together with the observation of ligand-stimulated localization of Art-GFP to the mating projection, a possible role for this arrestin-like protein in projection formation is supported. While leaving the full mechanism of alternate Art1 functionality to be elucidated, together these findings implicate Art1 in selective regulation of mating events downstream of receptor internalization and cell cycle arrest, leading to schmoo, and ultimately zygote formation.

  19. Report on Content Definition Process in Social Studies Testing.

    ERIC Educational Resources Information Center

    House, Ernest R.; Lawrence, Nancy

    This content assessment project is designed to determine what social studies content should be tested on national standardized tests and how that content should be defined. Sixteen historians, political scientists, and social studies educators were interviewed to identify key concepts. In a second phase, the cultural literacy rationale for content…

  20. Beta-arrestin 1 is involved in the catabolic response stimulated by hyaluronan degradation in mouse chondrocytes.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-08-01

    Beta-arrestin-1 (β-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1β (IL-1β) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As β-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of β-arrestin-1 in a model of IL-1β-induced inflammatory response in mouse chondrocytes. IL-1β treatment significantly increases chondrocytes TLR-4, CD44, β-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1β-stimulated chondrocytes with β-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of β-arrestin-1. β-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1β-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1β-induced activation of β-arrestin-1. PMID:25673209

  1. Divergent β-arrestin-dependent signaling events are dependent upon sequences within G-protein-coupled receptor C termini.

    PubMed

    Pal, Kasturi; Mathur, Maneesh; Kumar, Puneet; DeFea, Kathryn

    2013-02-01

    β-Arrestins are multifunctional adaptor proteins that, upon recruitment to an activated G-protein-coupled receptor, can promote desensitization of G-protein signaling and receptor internalization while simultaneously eliciting an independent signal. The result of β-arrestin signaling depends upon the activating receptor. For example, activation of two Gα(q)-coupled receptors, protease-activated receptor-2 (PAR(2)) and neurokinin-1 receptor (NK1R), results in drastically different signaling events. PAR(2) promotes β-arrestin-dependent membrane-sequestered extracellular signal-regulated kinase (ERK1/2) activation, cofilin activation, and cell migration, whereas NK1R promotes nuclear ERK1/2 activation and proliferation. Using bioluminescence resonance energy transfer to monitor receptor/β-arrestin interactions in real time, we observe that PAR(2) has a higher apparent affinity for both β-arrestins than does NK1R, recruits them at a faster rate, and exhibits more rapid desensitization of the G-protein signal. Furthermore, recruitment of β-arrestins to PAR(2) does not require prior Gα(q) signaling events, whereas inhibition of Gα(q) signaling intermediates inhibits recruitment of β-arrestins to NK1R. Using chimeric receptors in which the C terminus of PAR(2) is fused to the N terminus of NK1R and vice versa and a critical Ser/Thr mutant of PAR(2), we demonstrate that interactions between β-arrestins and specific phosphoresidues in the C termini of each receptor are crucial for determining the rate and magnitude of β-arrestin recruitment as well as the ultimate signaling outcome.

  2. Beta-arrestin 1 is involved in the catabolic response stimulated by hyaluronan degradation in mouse chondrocytes.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; D'Ascola, Angela; Scuruchi, Michele; Calatroni, Alberto; Campo, Salvatore

    2015-08-01

    Beta-arrestin-1 (β-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1β (IL-1β) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As β-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of β-arrestin-1 in a model of IL-1β-induced inflammatory response in mouse chondrocytes. IL-1β treatment significantly increases chondrocytes TLR-4, CD44, β-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1β-stimulated chondrocytes with β-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of β-arrestin-1. β-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1β-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1β-induced activation of β-arrestin-1.

  3. Light-Dependent Phosphorylation of Bardet Biedl Syndrome 5 in Photoreceptor Cells Modulates its Interaction with Arrestin1

    PubMed Central

    Smith, Tyler S.; Spitzbarth, Benjamin; Li, Jian; Dugger, Donald R.; Stern-Schneider, Gabi; Sehn, Elisabeth; Bolch, Susan N.; McDowell, J. Hugh; Tipton, Jeremiah; Wolfrum, Uwe; Smith, W. Clay

    2013-01-01

    Arrestins are dynamic proteins which move between cell compartments triggered by stimulation of G-protein-coupled receptors. Even more dynamically in vertebrate photoreceptors, arrestin1 (Arr1) moves between the inner and outer segments according to the lighting conditions. Previous studies have shown that the light-driven translocation of Arr1 in rod photoreceptors is initiated by rhodopsin through a phospholipase C/protein kinase C (PKC) signaling cascade. The purpose of this study is to identify the PKC substrate that regulates the translocation of Arr1. Mass spectrometry was used to identify the primary phosphorylated proteins in extracts prepared from PKC-stimulated mouse eye cups, confirming the finding with in vitro phosphorylation assays. Our results show that BBS5 is the principal protein phosphorylated either by phorbol ester stimulation or by light stimulation of PKC. Via immunoprecipitation of BBS5 in rod outer segments, Arr1 was pulled down; phosphorylation of BBS5 reduced this co-precipitation of Arr1. Immunofluorescence and immunoelectron microscopy showed that BBS5 principally localizes along the axonemes of rods and cones, but also in photoreceptor inner segments, and synaptic regions. Our principal findings in this study are three-fold. First, we demonstrate that BBS5 is post-translationally regulated by phosphorylation via PKC, an event that is triggered by light in photoreceptor cells. Second, we find a direct interaction between BBS5 and Arr1, an interaction that is modulated by phosphorylation of BBS5. Finally, we show that BBS5 is distributed along the photoreceptor axoneme, co-localizing with Arr1 in the dark. These findings suggest a role for BBS5 in regulating light-dependent translocation of Arr1 and a model describing its role in Arr1 translocation is proposed. PMID:23817741

  4. Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light-evoked translocation in vivo.

    PubMed

    Berkowitz, Bruce A; Gorgis, Jawan; Patel, Ankit; Baameur, Faiza; Gurevich, Vsevolod V; Craft, Cheryl M; Kefalov, Vladimir J; Roberts, Robin

    2015-02-01

    Rod tetrameric arrestin 1 (tet-ARR1), stored in the outer nuclear layer/inner segments in the dark, modulates photoreceptor synaptic activity; light exposure stimulates a reduction via translocation to the outer segments for terminating G-protein coupled phototransduction signaling. Here, we test the hypothesis that intraretinal spin-lattice relaxation rate in the rotating frame (1/T1ρ), an endogenous MRI contrast mechanism, has high potential for evaluating rod tet-ARR1 and its reduction via translocation. Dark- and light-exposed mice (null for the ARR1 gene, overexpressing ARR1, diabetic, or wild type with or without treatment with Mn2+, a calcium channel probe) were studied using 1/T1ρ MRI. Immunohistochemistry and single-cell recordings of the retinas were also performed. In wild-type mice with or without treatment with Mn2+, 1/T1ρ of avascular outer retina (64% to 72% depth) was significantly (P < 0.05) greater in the dark than in the light; a significant (P < 0.05) but opposite pattern was noted in the inner retina (<50% depth). Light-evoked outer retina Δ1/T1ρ was absent in ARR1-null mice and supernormal in overexpressing mice. In diabetic mice, the outer retinal Δ1/T1ρ pattern suggested normal dark-to-light tet-ARR1 translocation and chromophore content, conclusions confirmed ex vivo. Light-stimulated Δ1/T1ρ in inner retina was linked to changes in blood volume. Our data support 1/T1ρ MRI for noninvasively assessing rod tet-ARR1 and its reduction via protein translocation, which can be combined with other metrics of retinal function in vivo. PMID:25351983

  5. GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

    PubMed

    Thomsen, Alex R B; Plouffe, Bianca; Cahill, Thomas J; Shukla, Arun K; Tarrasch, Jeffrey T; Dosey, Annie M; Kahsai, Alem W; Strachan, Ryan T; Pani, Biswaranjan; Mahoney, Jacob P; Huang, Liyin; Breton, Billy; Heydenreich, Franziska M; Sunahara, Roger K; Skiniotis, Georgios; Bouvier, Michel; Lefkowitz, Robert J

    2016-08-11

    Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs. PMID:27499021

  6. Recruitment of β-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization

    PubMed Central

    Green, Jill A.; Schmid, Cullen L.; Bley, Elizabeth; Monsma, Paula C.; Brown, Anthony; Bohn, Laura M.

    2015-01-01

    Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates β-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking β-arrestin 2. Together, our results implicate β-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for β-arrestin in the mediation of Sstr3 ciliary signaling. PMID:26503786

  7. GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

    PubMed

    Thomsen, Alex R B; Plouffe, Bianca; Cahill, Thomas J; Shukla, Arun K; Tarrasch, Jeffrey T; Dosey, Annie M; Kahsai, Alem W; Strachan, Ryan T; Pani, Biswaranjan; Mahoney, Jacob P; Huang, Liyin; Breton, Billy; Heydenreich, Franziska M; Sunahara, Roger K; Skiniotis, Georgios; Bouvier, Michel; Lefkowitz, Robert J

    2016-08-11

    Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

  8. Analysis of Arrestin Recruitment to Chemokine Receptors by Bioluminescence Resonance Energy Transfer.

    PubMed

    Bonneterre, J; Montpas, N; Boularan, C; Galés, C; Heveker, N

    2016-01-01

    Chemokine receptors recruit the multifunctional scaffolding protein beta arrestin in response to binding of their chemokine ligands. Given that arrestin recruitment represents a signaling axis that is in part independent from G-protein signaling, it has become a hallmark of G protein-coupled receptor functional selectivity. Therefore, quantification of arrestin recruitment has become a requirement for the delineation of chemokine and drug candidate activity along different signaling axes. Bioluminescence resonance energy transfer (BRET) techniques provide methodology for such quantification that can reveal differences between nonredundant chemokines binding the same receptor, and that can be upscaled for high-throughput testing. We here provide protocols for the careful setup of BRET-based arrestin recruitment assays, and examples for the application of such systems in dose-response or time-course experiments. Suggestions are given for troubleshooting, optimizing test systems, and the interpretation of results obtained with BRET-based assays, which indeed yield an intricate blend of quantitative and qualitative information.

  9. EGFR trans-activation by urotensin II receptor is mediated by β-arrestin recruitment and confers cardioprotection in pressure overload-induced cardiac hypertrophy.

    PubMed

    Esposito, Giovanni; Perrino, Cinzia; Cannavo, Alessandro; Schiattarella, Gabriele G; Borgia, Francesco; Sannino, Anna; Pironti, Gianluigi; Gargiulo, Giuseppe; Di Serafino, Luigi; Franzone, Anna; Scudiero, Laura; Grieco, Paolo; Indolfi, Ciro; Chiariello, Massimo

    2011-06-01

    Urotensin II (UTII) and its seven trans-membrane receptor (UTR) are up-regulated in the heart under pathological conditions. Previous in vitro studies have shown that UTII trans-activates the epidermal growth factor receptor (EGFR), however, the role of such novel signalling pathway stimulated by UTII is currently unknown. In this study, we hypothesized that EGFR trans-activation by UTII might exert a protective effect in the overloaded heart. To test this hypothesis, we induced cardiac hypertrophy by transverse aortic constriction (TAC) in wild-type mice, and tested the effects of the UTII antagonist Urantide (UR) on cardiac function, structure, and EGFR trans-activation. After 7 days of pressure overload, UR treatment induced a rapid and significant impairment of cardiac function compared to vehicle. In UR-treated TAC mice, cardiac dysfunction was associated with reduced phosphorylation levels of the EGFR and extracellular-regulated kinase (ERK), increased apoptotic cell death and fibrosis. In vitro UTR stimulation induced membrane translocation of β-arrestin 1/2, EGFR phosphorylation/internalization, and ERK activation in HEK293 cells. Furthermore, UTII administration lowered apoptotic cell death induced by serum deprivation, as shown by reduced TUNEL/Annexin V staining and caspase 3 activation. Interestingly, UTII-mediated EGFR trans-activation could be prevented by UR treatment or knockdown of β-arrestin 1/2. Our data show, for the first time in vivo, a new UTR signalling pathway which is mediated by EGFR trans-activation, dependent by β-arrestin 1/2, promoting cell survival and cardioprotection.

  10. Differential Phosphorylation Provides a Switch to Control How α-Arrestin Rod1 Down-regulates Mating Pheromone Response in Saccharomyces cerevisiae

    PubMed Central

    Alvaro, Christopher G.; Aindow, Ann; Thorner, Jeremy

    2016-01-01

    G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate stimulus-dependent activation of cognate heterotrimeric G-proteins, triggering ensuing downstream cellular responses. Tight regulation of GPCR-evoked pathways is required because prolonged stimulation can be detrimental to an organism. Ste2, a GPCR in Saccharomyces cerevisiae that mediates response of MATa haploids to the peptide mating pheromone α-factor, is down-regulated by both constitutive and agonist-induced endocytosis. Efficient agonist-stimulated internalization of Ste2 requires its association with an adaptor protein, the α-arrestin Rod1/Art4, which recruits the HECT-domain ubiquitin ligase Rsp5, allowing for ubiquitinylation of the C-terminal tail of the receptor and its engagement by the clathrin-dependent endocytic machinery. We previously showed that dephosphorylation of Rod1 by calcineurin (phosphoprotein phosphatase 2B) is required for optimal Rod1 function in Ste2 down-regulation. We show here that negative regulation of Rod1 by phosphorylation is mediated by two distinct stress-activated protein kinases, Snf1/AMPK and Ypk1/SGK1, and demonstrate both in vitro and in vivo that this phospho-regulation impedes the ability of Rod1 to promote mating pathway desensitization. These studies also revealed that, in the absence of its phosphorylation, Rod1 can promote adaptation independently of Rsp5-mediated receptor ubiquitinylation, consistent with recent evidence that α-arrestins can contribute to cargo recognition by both clathrin-dependent and clathrin-independent mechanisms. However, in cells lacking a component (formin Bni1) required for clathrin-independent entry, Rod1 derivatives that are largely unphosphorylated and unable to associate with Rsp5 still promote efficient adaptation, indicating a third mechanism by which this α-arrestin promotes desensitization of the pheromone-response pathway. PMID:26920760

  11. The β-arrestin-biased ligand TRV120023 inhibits angiotensin II-induced cardiac hypertrophy while preserving enhanced myofilament response to calcium.

    PubMed

    Monasky, Michelle M; Taglieri, Domenico M; Henze, Marcus; Warren, Chad M; Utter, Megan S; Soergel, David G; Violin, Jonathan D; Solaro, R John

    2013-09-15

    In the present study, we compared the cardioprotective effects of TRV120023, a novel angiotensin II (ANG II) type 1 receptor (AT1R) ligand, which blocks G protein coupling but stimulates β-arrestin signaling, against treatment with losartan, a conventional AT1R blocker in the treatment of cardiac hypertrophy and regulation of myofilament activity and phosphorylation. Rats were subjected to 3 wk of treatment with saline, ANG II, ANG II + losartan, ANG II + TRV120023, or TRV120023 alone. ANG II induced increased left ventricular mass compared with rats that received ANG II + losartan or ANG II + TRV120023. Compared with saline controls, ANG II induced a significant increase in pCa50 and maximum Ca(2+)-activated myofilament tension but reduced the Hill coefficient (nH). TRV120023 increased maximum tension and pCa50, although to lesser extent than ANG II. In contrast to ANG II, TRV120023 increased nH. Losartan blocked the effects of ANG II on pCa50 and nH and reduced maximum tension below that of saline controls. ANG II + TRV120023 showed responses similar to those of TRV120023 alone; compared with ANG II + losartan, ANG II + TRV120023 preserved maximum tension and increased both pCa50 and cooperativity. Tropomyosin phosphorylation was lower in myofilaments from saline-treated hearts compared with the other groups. Phosphorylation of cardiac troponin I was significantly reduced in ANG II + TRV120023 and TRV120023 groups versus saline controls, and myosin-binding protein C phosphorylation at Ser(282) was unaffected by ANG II or losartan but significantly reduced with TRV120023 treatment compared with all other groups. Our data indicate that TRV120023-related promotion of β-arrestin signaling and enhanced contractility involves a mechanism promoting the myofilament response to Ca(2+) via altered protein phosphorylation. Selective activation of β-arrestin-dependent pathways may provide advantages over conventional AT1R blockers.

  12. 23 CFR 650.117 - Content of design studies.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... BRIDGES, STRUCTURES, AND HYDRAULICS Location and Hydraulic Design of Encroachments on Flood Plains § 650.117 Content of design studies. (a) The detail of studies shall be commensurate with the risk... 23 Highways 1 2013-04-01 2013-04-01 false Content of design studies. 650.117 Section...

  13. 23 CFR 650.117 - Content of design studies.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... BRIDGES, STRUCTURES, AND HYDRAULICS Location and Hydraulic Design of Encroachments on Flood Plains § 650.117 Content of design studies. (a) The detail of studies shall be commensurate with the risk... 23 Highways 1 2014-04-01 2014-04-01 false Content of design studies. 650.117 Section...

  14. 23 CFR 650.117 - Content of design studies.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... BRIDGES, STRUCTURES, AND HYDRAULICS Location and Hydraulic Design of Encroachments on Flood Plains § 650.117 Content of design studies. (a) The detail of studies shall be commensurate with the risk... 23 Highways 1 2012-04-01 2012-04-01 false Content of design studies. 650.117 Section...

  15. Multiple Scaffolding Functions of β-Arrestins in the Degradation of G Protein-coupled Receptor Kinase 2*

    PubMed Central

    Nogués, Laura; Salcedo, Alicia; Mayor, Federico; Penela, Petronila

    2011-01-01

    G protein-coupled receptor kinase 2 (GRK2) plays a fundamental role in the regulation of G protein-coupled receptors (GPCRs), and changes in GRK2 expression levels can have an important impact on cell functions. GRK2 is known to be degraded by the proteasome pathway. We have shown previously that β-arrestins participate in enhanced kinase turnover upon GPCR stimulation by facilitating GRK2 phosphorylation by c-Src or by MAPK or by recruiting the Mdm2 E3 ubiquitin ligase to the receptor complex. In this report, we have investigated how such diverse β-arrestin scaffold functions are integrated to modulate GRK2 degradation. Interestingly, we found that in the absence of GPCR activation, β-arrestins do not perform an adaptor role for GRK2/Mdm2 association, but rather compete with GRK2 for direct Mdm2 binding to regulate basal kinase turnover. Upon agonist stimulation, β-arrestins-mediated phosphorylation of GRK2 at serine 670 by MAPK facilitates Mdm2-mediated GRK2 degradation, whereas c-Src-dependent phosphorylation would support the action of an undetermined β-arrestin-recruited ligase in the absence of GPCR activation. The ability of β-arrestins to play different scaffold functions would allow coordination of both Mdm2-dependent and -independent processes aimed at the specific modulation of GRK2 turnover in different signaling contexts. PMID:21081496

  16. β-Arrestin-1 protects against endoplasmic reticulum stress/p53-upregulated modulator of apoptosis-mediated apoptosis via repressing p-p65/inducible nitric oxide synthase in portal hypertensive gastropathy.

    PubMed

    Tan, Siwei; Li, Leijia; Chen, Tingting; Chen, Xiaoliang; Tao, Li; Lin, Xianyi; Tao, Jin; Huang, Xiaoli; Jiang, Jie; Liu, Huiling; Wu, Bin

    2015-10-01

    Portal hypertensive gastropathy (PHG) is a serious cause of bleeding in patients, and is associated with portal hypertension. β-Arrestins (β-arrestin-1 and β-arrestin-2) are well-established mediators of endocytosis of G-protein-coupled receptors (GPCRs), ubiquitination, and G-protein-independent signaling. The role of β-arrestin-1 (β-arr1) in mucosal apoptosis in PHG remains unclear. The aim of this study was to investigate the involvement of β-arr1 in PHG via its regulation of endoplasmic reticulum (ER) stress/p53-upregulated modulator of apoptosis (PUMA) apoptotic signaling. Gastric mucosal injury and apoptosis were studied in PHG patients and in PHG mouse models. The induction of β-arr1 and the ER stress/PUMA signaling pathway were investigated, and the mechanisms of β-arr1-regulated gastric mucosal apoptosis were analyzed in vivo and in vitro experiments. β-arr1 and ER stress/PUMA signaling elements were markedly induced in the gastric mucosa of PHG patients and mouse models. Blockage of ER stress demonstrably attenuated the mucosal apoptosis of PHG, while targeted deletion of β-arr1 significantly aggravated the injury and ER stress/PUMA-mediated apoptosis. β-arr1 limited the activation of p65 to repress TNF-α-induced inducible nitric oxide synthase (iNOS) expression and NO release, which could regulate ER stress/PUMA-mediated mucosal apoptosis in PHG. In vivo and in vitro experiments further demonstrated that β-arr1 protected against mucosal apoptosis by repressing TNF-α-induced iNOS expression via inhibiting the activation of p65. These results indicated that β-arr1 regulated ER stress/PUMA-induced mucosal epithelial apoptosis through suppression of the TNF-α/p65/iNOS signaling pathway activation and that β-arr1 is a potential therapeutic target for PHG.

  17. The induction of thioredoxin-1 by epinephrine withdraws stress via interaction with β-arrestin-1.

    PubMed

    Jia, Jin-Jing; Zeng, Xian-Si; Zhou, Xiao-Shuang; Li, Ye; Bai, Jie

    2014-01-01

    Stress regulates a panel of important physiological functions and disease states. Epinephrine is produced under stresses threaten to homeostasis. Thioredoxin-1(Trx-1) is a redox regulating protein which is induced to resist stresses and related with various diseases. Thus, it is important to examine whether Trx-1 is induced by epinephrine and to understand the underlying molecular mechanisms that Trx-1 modulates epinephrine stress. Here, we show that the expression of Trx-1 was induced by epinephrine via β-adrenergic receptor/Cyclic AMP/protein kinase A (PKA) signaling pathway in PC12 cells. The down-regulation of Trx-1 by siRNA aggravated accumulation of γ-H2AX and further decreased expression of p53 by epinephrine. Accordingly, Trx-1 overexpression alleviated accumulation of γ-H2AX and restored the expressions of p53 and C/EBP homologous protein (CHOP) in the cortex, hippocampus and thymus of mice. Moreover, Trx-1 overexpression reduced the malondialdehyde concentration by epinephrine. We further explored the mechanism on p53 and γ-H2AX regulated by Trx-1. We found that overexpression of Trx-1 suppressed β-arrestin-1 expression through interaction with β-arrestin-1. Consequently, the downregulation of β-arrestin-1 suppressed the cell viability and the expressions of γ-H2AX and cyclin D1, and increased p53 expression. Taken together, our data suggest that Trx-1/β-arrestin-1 interaction may represent a novel endogenous mechanism on protecting against stress. PMID:25486571

  18. Not just signal shutoff: the protective role of arrestin-1 in rod cells.

    PubMed

    Sommer, Martha E; Hofmann, Klaus Peter; Heck, Martin

    2014-01-01

    The retinal rod cell is an exquisitely sensitive single-photon detector that primarily functions in dim light (e.g., moonlight). However, rod cells must routinely survive light intensities more than a billion times greater (e.g., bright daylight). One serious challenge to rod cell survival in daylight is the massive amount of all-trans-retinal that is released by Meta II, the light-activated form of the photoreceptor rhodopsin. All-trans-retinal is toxic, and its condensation products have been implicated in disease. Our recent work has developed the concept that rod arrestin (arrestin-1), which terminates Meta II signaling, has an additional role in protecting rod cells from the consequences of bright light by limiting free all-trans-retinal. In this chapter we will elaborate upon the molecular mechanisms by which arrestin-1 serves as both a single-photon response quencher as well as an instrument of rod cell survival in bright light. This discussion will take place within the framework of three distinct functional modules of vision: signal transduction, the retinoid cycle, and protein translocation.

  19. GPR3 Stimulates Aβ Production via Interactions with APP and β-Arrestin2

    PubMed Central

    Nelson, Christopher D.; Sheng, Morgan

    2013-01-01

    The orphan G protein-coupled receptor (GPCR) GPR3 enhances the processing of Amyloid Precursor Protein (APP) to the neurotoxic beta-amyloid (Aβ) peptide via incompletely understood mechanisms. Through overexpression and shRNA knockdown experiments in HEK293 cells, we show that β-arrestin2 (βarr2), a GPCR-interacting scaffold protein reported to bind γ-secretase, is an essential factor for GPR3-stimulated Aβ production. For a panel of GPR3 receptor mutants, the degree of stimulation of Aβ production correlates with receptor-β-arrestin binding and receptor trafficking to endocytic vesicles. However, GPR3’s recruitment of βarr2 cannot be the sole explanation, because interaction with βarr2 is common to most GPCRs, whereas GPR3 is relatively unique among GPCRs in enhancing Aβ production. In addition to β-arrestin, APP is present in a complex with GPR3 and stimulation of Aβ production by GPR3 mutants correlates with their level of APP binding. Importantly, among a broader selection of GPCRs, only GPR3 and prostaglandin E receptor 2 subtype EP2 (PTGER2; another GPCR that increases Aβ production) interact with APP, and PTGER2 does so in an agonist-stimulated manner. These data indicate that a subset of GPCRs, including GPR3 and PTGER2, can associate with APP when internalized via βarr2, and thereby promote the cleavage of APP to generate Aβ. PMID:24069330

  20. Autophagy-associated alpha-arrestin signaling is required for conidiogenous cell development in Magnaporthe oryzae

    PubMed Central

    Dong, Bo; Xu, Xiaojin; Chen, Guoqing; Zhang, Dandan; Tang, Mingzhi; Xu, Fei; Liu, Xiaohong; Wang, Hua; Zhou, Bo

    2016-01-01

    Conidiation patterning is evolutionarily complex and mechanism concerning conidiogenous cell differentiation remains largely unknown. Magnaporthe oryzae conidiates in a sympodial way and uses its conidia to infect host and disseminate blast disease. Arrestins are multifunctional proteins that modulate receptor down-regulation and scaffold components of intracellular trafficking routes. We here report an alpha-arrestin that regulates patterns of conidiation and contributes to pathogenicity in M. oryzae. We show that disruption of ARRDC1 generates mutants which produce conidia in an acropetal array and ARRDC1 significantly affects expression profile of CCA1, a virulence-related transcription factor required for conidiogenous cell differentiation. Although germ tubes normally develop appressoria, penetration peg formation is dramatically impaired and Δarrdc1 mutants are mostly nonpathogenic. Fluorescent analysis indicates that EGFP-ARRDC1 puncta are well colocalized with DsRed2-Atg8, and this distribution profile could not be altered in Δatg9 mutants, suggesting ARRDC1 enters into autophagic flux before autophagosome maturation. We propose that M. oryzae employs ARRDC1 to regulate specific receptors in response to conidiation-related signals for conidiogenous cell differentiation and utilize autophagosomes for desensitization of conidiogenous receptor, which transmits extracellular signal to the downstream elements of transcription factors. Our investigation extends novel significance of autophagy-associated alpha-arrestin signaling to fungal parasites. PMID:27498554

  1. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex

    DOE PAGES

    Zhou, X. Edward; Gao, Xiang; Barty, Anton; Kang, Yanyong; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; White, Thomas A.; Yefanov, Oleksandr; Han, Gye Won; et al

    2016-04-12

    Here, serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solvedmore » with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.« less

  2. Autophagy-associated alpha-arrestin signaling is required for conidiogenous cell development in Magnaporthe oryzae.

    PubMed

    Dong, Bo; Xu, Xiaojin; Chen, Guoqing; Zhang, Dandan; Tang, Mingzhi; Xu, Fei; Liu, Xiaohong; Wang, Hua; Zhou, Bo

    2016-01-01

    Conidiation patterning is evolutionarily complex and mechanism concerning conidiogenous cell differentiation remains largely unknown. Magnaporthe oryzae conidiates in a sympodial way and uses its conidia to infect host and disseminate blast disease. Arrestins are multifunctional proteins that modulate receptor down-regulation and scaffold components of intracellular trafficking routes. We here report an alpha-arrestin that regulates patterns of conidiation and contributes to pathogenicity in M. oryzae. We show that disruption of ARRDC1 generates mutants which produce conidia in an acropetal array and ARRDC1 significantly affects expression profile of CCA1, a virulence-related transcription factor required for conidiogenous cell differentiation. Although germ tubes normally develop appressoria, penetration peg formation is dramatically impaired and Δarrdc1 mutants are mostly nonpathogenic. Fluorescent analysis indicates that EGFP-ARRDC1 puncta are well colocalized with DsRed2-Atg8, and this distribution profile could not be altered in Δatg9 mutants, suggesting ARRDC1 enters into autophagic flux before autophagosome maturation. We propose that M. oryzae employs ARRDC1 to regulate specific receptors in response to conidiation-related signals for conidiogenous cell differentiation and utilize autophagosomes for desensitization of conidiogenous receptor, which transmits extracellular signal to the downstream elements of transcription factors. Our investigation extends novel significance of autophagy-associated alpha-arrestin signaling to fungal parasites. PMID:27498554

  3. Desensitization of human CRF2(a) receptor signaling governed by agonist potency and βarrestin2 recruitment.

    PubMed

    Hauger, Richard L; Olivares-Reyes, J Alberto; Braun, Sandra; Hernandez-Aranda, Judith; Hudson, Christine C; Gutknecht, Eric; Dautzenberg, Frank M; Oakley, Robert H

    2013-09-10

    The primary goal was to determine agonist-specific regulation of CRF2(a) receptor function. Exposure of human retinoblastoma Y79 cells to selective (UCN2, UCN3 or stresscopins) and non-selective (UCN1 or sauvagine) agonists prominently desensitized CRF2(a) receptors in a rapid, concentration-dependent manner. A considerably slower rate and smaller magnitude of desensitization developed in response to the weak agonist CRF. CRF1 receptor desensitization stimulated by CRF, cortagine or stressin1-A had no effect on CRF2(a) receptor cyclic AMP signaling. Conversely, desensitization of CRF2(a) receptors by UCN2 or UCN3 did not cross-desensitize Gs-coupled CRF1 receptor signaling. In transfected HEK293 cells, activation of CRF2(a) receptors by UCN2, UCN3 or CRF resulted in receptor phosphorylation and internalization proportional to agonist potency. Neither protein kinase A nor casein kinases mediated CRF2(a) receptor phosphorylation or desensitization. Exposure of HEK293 or U2OS cells to UCN2 or UCN3 (100nM) produced strong βarrestin2 translocation and colocalization with membrane CRF2(a) receptors while CRF (1μM) generated only weak βarrestin2 recruitment. βarrestin2 did not internalize with the receptor, however, indicating that transient CRF2(a) receptor-arrestin complexes dissociate at or near the cell membrane. Since deletion of the βarrestin2 gene upregulated Gs-coupled CRF2(a) receptor signaling in MEF cells, a βarrestin2 mechanism restrains Gs-coupled CRF2(a) receptor signaling activated by urocortins. We further conclude that the rate and extent of homologous CRF2(a) receptor desensitization are governed by agonist-specific mechanisms affecting GRK phosphorylation, βarrestin2 recruitment, and internalization thereby producing unique signal transduction profiles that differentially affect the stress response. PMID:23820308

  4. Kappa Opioid Receptor Activation of p38 MAPK Is GRK3- and Arrestin-dependent in Neurons and Astrocytes*

    PubMed Central

    Bruchas, Michael R.; Macey, Tara A.; Lowe, Janet D.; Chavkin, Charles

    2007-01-01

    AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (γ-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism. PMID:16648139

  5. β-Arrestin-Selective G Protein-Coupled Receptor Agonists Engender Unique Biological Efficacy in Vivo

    PubMed Central

    Gesty-Palmer, Diane; Yuan, Ling; Martin, Bronwen; Wood, William H.; Lee, Mi-Hye; Janech, Michael G.; Tsoi, Lam C.; Zheng, W. Jim; Maudsley, Stuart

    2013-01-01

    Biased G protein-coupled receptor agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors. In vitro, d-Trp12,Tyr34-bPTH(7–34) [bPTH(7–34)], a biased agonist for the type 1 PTH receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling. In vivo, both bPTH(7–34) and the conventional agonist hPTH(1–34) stimulate anabolic bone formation. To understand how two PTH receptor ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 wk with vehicle, bPTH(7–34) or hPTH(1–34). Treatment of wild-type mice with bPTH(7–34) primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival, and migration. These responses were absent in β-arrestin2-null mice, identifying them as downstream targets of β-arrestin2-mediated signaling. In contrast, hPTH(1–34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization. hPTH(1–34) actions were less dependent on β-arrestin2, as might be expected of a ligand capable of G protein activation. In vitro, bPTH(7–34) slowed the rate of preosteoblast proliferation, enhanced osteoblast survival when exposed to an apoptotic stimulus, and stimulated cell migration in wild-type, but not β-arrestin2-null, calvarial osteoblasts. These results suggest that bPTH(7–34) and hPTH(1–34) affect bone mass in vivo through predominantly separate genomic mechanisms created by largely distinct receptor-signaling networks and demonstrate that functional selectivity can be exploited to change the quality of G protein-coupled receptor efficacy. PMID:23315939

  6. Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

    PubMed Central

    Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L.; Hille, Bertil

    2016-01-01

    Activated Gq protein–coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to

  7. Selectively engaging β-arrestins at the angiotensin II type 1 receptor reduces blood pressure and increases cardiac performance.

    PubMed

    Violin, Jonathan D; DeWire, Scott M; Yamashita, Dennis; Rominger, David H; Nguyen, Lisa; Schiller, Kevin; Whalen, Erin J; Gowen, Maxine; Lark, Michael W

    2010-12-01

    Biased G protein-coupled receptor ligands engage subsets of the receptor signals normally stimulated by unbiased agonists. However, it is unclear whether ligand bias can elicit differentiated pharmacology in vivo. Here, we describe the discovery of a potent, selective β-arrestin biased ligand of the angiotensin II type 1 receptor. TRV120027 (Sar-Arg-Val-Tyr-Ile-His-Pro-D-Ala-OH) competitively antagonizes angiotensin II-stimulated G protein signaling, but stimulates β-arrestin recruitment and activates several kinase pathways, including p42/44 mitogen-activated protein kinase, Src, and endothelial nitric-oxide synthase phosphorylation via β-arrestin coupling. Consistent with β-arrestin efficacy, and unlike unbiased antagonists, TRV120027 increased cardiomyocyte contractility in vitro. In rats, TRV120027 reduced mean arterial pressure, as did the unbiased antagonists losartan and telmisartan. However, unlike the unbiased antagonists, which decreased cardiac performance, TRV120027 increased cardiac performance and preserved cardiac stroke volume. These striking differences in vivo between unbiased and β-arrestin biased ligands validate the use of biased ligands to selectively target specific receptor functions in drug discovery.

  8. Unraveling the molecular architecture of a G protein-coupled receptor/β-arrestin/Erk module complex

    PubMed Central

    Bourquard, Thomas; Landomiel, Flavie; Reiter, Eric; Crépieux, Pascale; Ritchie, David W.; Azé, Jérôme; Poupon, Anne

    2015-01-01

    β-arrestins serve as signaling scaffolds downstream of G protein-coupled receptors, and thus play a crucial role in a plethora of cellular processes. Although it is largely accepted that the ability of β-arrestins to interact simultaneously with many protein partners is key in G protein-independent signaling of GPCRs, only the precise knowledge of these multimeric arrangements will allow a full understanding of the dynamics of these interactions and their functional consequences. However, current experimental procedures for the determination of the three-dimensional structures of protein-protein complexes are not well adapted to analyze these short-lived, multi-component assemblies. We propose a model of the receptor/β-arrestin/Erk1 signaling module, which is consistent with most of the available experimental data. Moreover, for the β-arrestin/Raf1 and the β-arrestin/ERK interactions, we have used the model to design interfering peptides and shown that they compete with both partners, hereby demonstrating the validity of the predicted interaction regions. PMID:26030356

  9. Insulin-Like Growth Factor-1 Contributes to Mucosal Repair by β-Arrestin2-Mediated Extracellular Signal-Related Kinase Signaling in Experimental Colitis.

    PubMed

    Chen, Tingting; Zheng, Fengping; Tao, Jin; Tan, Siwei; Zeng, Lixian; Peng, Xiaojie; Wu, Bin

    2015-09-01

    Insulin-like growth factor-1 (IGF-1) possesses the ability to attenuate intestinal damage and promote mucosal repair of colitis. β-Arrestins, as the scaffolding proteins of G protein-coupled receptors or non-G protein-coupled receptors signaling, can be involved in IGF-1-mediated signaling pathways. However, the interaction of IGF-1 and β-arrestin2 in the mucosal repair of experimental colitis remains unexplored. Ulcerative colitis was induced in β-arrestin2 wild-type mice and β-arrestin2 knockout littermates by using 3% dextran sulfate sodium for 5 days, followed by regular water consumption for 1, 2, 3, and 4 weeks to analyze the mucosal repair from experimental colitis. Disease activity index and histologic score analyses were performed. Apoptosis and proliferation were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Ki-67 staining, respectively. The expressions of β-arrestin2, phospho (p)-IGF-1R, and p-extracellular signal-regulated kinase (ERK)1/2 were examined. Furthermore, β-arrestin2 was overexpressed or altered in HCT116 cells by transfection before IGF-1 treatment in vitro. IGF-1 and β-arrestin2 expression was up-regulated in the repairing phase of experimental colitis. Targeted deletion of β-arrestin2 delayed the repair of colitis by inhibiting cell proliferation without affecting the levels of IGF-1 and p-IGF-1R. The β-arrestin2/ERK signaling pathway was involved in IGF-1-mediated mucosal repair through promoting epithelial cell and goblet cell regeneration from experimental colitis. These results indicate that IGF-1 contributes to the mucosal repair by β-arrestin2-mediated ERK signaling in experimental colitis.

  10. Agonist-biased signaling at the histamine H4 receptor: JNJ7777120 recruits β-arrestin without activating G proteins.

    PubMed

    Rosethorne, Elizabeth M; Charlton, Steven J

    2011-04-01

    The G(i/o)-coupled histamine H(4) receptor is highly expressed in hemopoietic cells and is a promising new target for the treatment of chronic inflammatory diseases. 1-[(5-Chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ7777120) has been described as a selective antagonist at the H(4) receptor and is widely used to characterize the physiological role of the H(4) receptor. We have investigated the pharmacological properties of JNJ7777120 using two distinct downstream signaling measurements, G protein activation and β-arrestin recruitment. The H(4) receptor agonists histamine and clobenpropit, but not JNJ7777120, were able to induce [(35)S]GTPγS binding in membranes prepared from U2OS-H(4) cells. Thioperamide, a dual H(3)/H(4) receptor antagonist, and JNJ7777120 were both able to inhibit the [(35)S]GTPγS binding induced by clobenpropit. Agonists and antagonists specific for other members of the histamine receptor family had no effect in this assay format. Histamine and clobenpropit increased β-arrestin recruitment to the H(4) receptor in a concentration-dependent manner. This β-arrestin recruitment could be inhibited by preincubation with thioperamide. We were surprised to find that preincubation with the H(4)-selective antagonist JNJ7777120 potentiated rather than antagonized the response to a submaximal concentration of clobenpropit. JNJ7777120 treatment alone resulted in an increase in β-arrestin recruitment, which again could be inhibited by preincubation with thioperamide. Schild analysis demonstrated competitive antagonism between thioperamide and both clobenpropit and JNJ7777120. Histamine and clobenpropit had comparable potencies for both [(35)S]GTPγS binding and β-arrestin recruitment, suggesting little difference in the levels of receptor reserve between the two assays. In conclusion, we have demonstrated that JNJ7777120 recruits β-arrestin to the H(4) receptor, independent of G protein activation.

  11. Faculty Across the Disciplines Respond to Study Skills Content.

    ERIC Educational Resources Information Center

    Boucher, Ellizabeth F.; Haynes, Ada F.

    1996-01-01

    Describes a study of faculty at Tennessee Technological University regarding their teaching styles and perceptions of student deficiencies and necessary study skills content. Indicates that faculty cited critical thinking, personal responsibility, time management, and problem-solving skills as essential and reported that students lacked critical…

  12. β-Arrestin2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis.

    PubMed

    Zeng, L X; Tao, J; Liu, H L; Tan, S W; Yang, Y D; Peng, X J; Liu, Z H; Jiang, J; Wu, B

    2015-05-01

    β-Arrestins (β-arrs) are regulators and mediators of G protein-coupled receptor signaling, and accumulating evidence suggests that they are functionally involved in inflammation and autoimmune diseases. However, the effect of β-arrs is unclear in inflammatory bowel disease (IBD), and the role of β-arr2 is unknown in ulcerative colitis (UC) and Crohn's disease (CD). The aim of this study is to investigate whether β-arr2 encourages inflammation-induced epithelial apoptosis through endoplasmic reticulum (ER) stress/p53-upregulated modulator of apoptosis (PUMA) in colitis. In the present study, the results showed that β-arr2 was increased in specimens from patients with UC or CD. Furthermore, a β-arr2 deficiency significantly repressed intestinal inflammation, ameliorated colitis, and alleviated mucosal apoptosis in mice. In addition, the targeted deletion of β-arr2 depressed ER stress, inhibited PUMA, and downregulated PUMA-mediated mitochondrial apoptotic signaling in colitis. β-Arr2, an important modulator of G protein-coupled receptor function, binds eIF2α to activate ER stress signaling. Furthermore, the knockdown of PUMA dramatically prevented β-arr2-induced apoptosis via alleviating ER stress in vitro. The results suggest that β-arr2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis and that β-arr2 is a potential therapeutic target for colitis.

  13. [Near infrared spectroscopy study on water content in turbine oil].

    PubMed

    Chen, Bin; Liu, Ge; Zhang, Xian-Ming

    2013-11-01

    Near infrared (NIR) spectroscopy combined with successive projections algorithm (SPA) was investigated for determination of water content in turbine oil. Through the 57 samples of different water content in turbine oil scanned applying near infrared (NIR) spectroscopy, with the water content in the turbine oil of 0-0.156%, different pretreatment methods such as the original spectra, first derivative spectra and differential polynomial least squares fitting algorithm Savitzky-Golay (SG), and successive projections algorithm (SPA) were applied for the extraction of effective wavelengths, the correlation coefficient (R) and root mean square error (RMSE) were used as the model evaluation indices, accordingly water content in turbine oil was investigated. The results indicated that the original spectra with different water content in turbine oil were pretreated by the performance of first derivative + SG pretreatments, then the selected effective wavelengths were used as the inputs of least square support vector machine (LS-SVM). A total of 16 variables selected by SPA were employed to construct the model of SPA and least square support vector machine (SPA-LS-SVM). There is 9 as The correlation coefficient was 0.975 9 and the root of mean square error of validation set was 2.655 8 x 10(-3) using the model, and it is feasible to determine the water content in oil using near infrared spectroscopy and SPA-LS-SVM, and an excellent prediction precision was obtained. This study supplied a new and alternative approach to the further application of near infrared spectroscopy in on-line monitoring of contamination such as water content in oil.

  14. Undergraduate Knowledge of Aging: A Comparative Study of Biopsychosocial Content

    ERIC Educational Resources Information Center

    Damron-Rodriguez, JoAnn; Funderburk, Brooke; Lee, Martin; Solomon, David H.

    2004-01-01

    This study assesses undergraduate knowledge of aging, distinguishing between types of deficits (ignorance vs. misinformation) and content areas as delineated by a biopsychosocial framework. Knowledge is examined as an outcome of taking an aging elective, while accounting for course rating and knowledge retention. A diverse body of UCLA…

  15. Mathematics Education Research in Turkey: A Content Analysis Study

    ERIC Educational Resources Information Center

    Ciltas, Alper; Guler, Gursel; Sozbilir, Mustafa

    2012-01-01

    In this study, a content analysis of research is aimed in the field of mathematics education of Turkish researchers. To this aim, the investigation of 359 article were made which were accessed from web in full text between 1987 and 2009 years and which were published in the field of mathematics education from 32 different journals. 27 of these…

  16. 23 CFR 650.117 - Content of design studies.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... reasons, when applicable, why FEMA criteria (44 CFR 60.3, formerly 24 CFR 1910.3) are demonstrably... 23 Highways 1 2011-04-01 2011-04-01 false Content of design studies. 650.117 Section 650.117... BRIDGES, STRUCTURES, AND HYDRAULICS Location and Hydraulic Design of Encroachments on Flood Plains §...

  17. Children's Content Interest--A Factor Analytic Study.

    ERIC Educational Resources Information Center

    Feeley, Joan T.

    Recognizing that interest is essential to motivation, this study was designed both to identify and describe the content interest patterns and media preferences (print and television) of middle-grade children and to determine any relationship between these interests and sex, race, or socioeconomic status (SES). An inventory was administered to 250…

  18. Integrating Language and Content Learning in the Social Studies Classroom.

    ERIC Educational Resources Information Center

    Kang, Hee-Won; LeSourd, Sandra J.

    This paper focuses on helping social studies teachers discover ways to help second language students comprehend, use, and learn language as well as content in the classroom. Activities conducive to this purpose include: providing contextual support such as pictures, globes, videotapes, diagrams, body and facial gestures, pantomime and role…

  19. Application of grounded theory to content definition: a case study.

    PubMed

    Audiss, D; Roth, T

    1999-02-01

    Successful implementation of a clinical information system requires clinician involvement throughout the process of content definition and system development to ensure acceptance of the automated care process. In these times of downsizing, however, clinicians are not always able to participate fully in the dontent definition phase of system development and often become frustrated with their inability to obtain the patient information they need from the system. The qualitative research principles of grounded theory afford clinicians the opportunity to participate in content definition for information systems. This article presents a case study of the application of grounded theory to develop systematically the content definition for a clinical information system in preparation for implementation on four medical-surgical units.

  20. Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins.

    PubMed

    Bouzo-Lorenzo, Monica; Santo-Zas, Icía; Lodeiro, Maria; Nogueiras, Rubén; Casanueva, Felipe F; Castro, Marian; Pazos, Yolanda; Tobin, Andrew B; Butcher, Adrian J; Camiña, Jesús P

    2016-03-03

    The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser(362), Ser(363) and Thr(366) residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr(350) and Ser(349) are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output.

  1. Distinct phosphorylation sites on the ghrelin receptor, GHSR1a, establish a code that determines the functions of ß-arrestins

    PubMed Central

    Bouzo-Lorenzo, Monica; Santo-Zas, Icía; Lodeiro, Maria; Nogueiras, Rubén; Casanueva, Felipe F.; Castro, Marian; Pazos, Yolanda; Tobin, Andrew B; Butcher, Adrian J.; Camiña, Jesús P.

    2016-01-01

    The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser362, Ser363 and Thr366 residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr350 and Ser349 are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output. PMID:26935831

  2. Mitragynine/Corynantheidine Pseudoindoxyls As Opioid Analgesics with Mu Agonism and Delta Antagonism, Which Do Not Recruit β-Arrestin-2.

    PubMed

    Váradi, András; Marrone, Gina F; Palmer, Travis C; Narayan, Ankita; Szabó, Márton R; Le Rouzic, Valerie; Grinnell, Steven G; Subrath, Joan J; Warner, Evelyn; Kalra, Sanjay; Hunkele, Amanda; Pagirsky, Jeremy; Eans, Shainnel O; Medina, Jessica M; Xu, Jin; Pan, Ying-Xian; Borics, Attila; Pasternak, Gavril W; McLaughlin, Jay P; Majumdar, Susruta

    2016-09-22

    Natural products found in Mitragyna speciosa, commonly known as kratom, represent diverse scaffolds (indole, indolenine, and spiro pseudoindoxyl) with opioid activity, providing opportunities to better understand opioid pharmacology. Herein, we report the pharmacology and SAR studies both in vitro and in vivo of mitragynine pseudoindoxyl (3), an oxidative rearrangement product of the corynanthe alkaloid mitragynine. 3 and its corresponding corynantheidine analogs show promise as potent analgesics with a mechanism of action that includes mu opioid receptor agonism/delta opioid receptor antagonism. In vitro, 3 and its analogs were potent agonists in [(35)S]GTPγS assays at the mu opioid receptor but failed to recruit β-arrestin-2, which is associated with opioid side effects. Additionally, 3 developed analgesic tolerance more slowly than morphine, showed limited physical dependence, respiratory depression, constipation, and displayed no reward or aversion in CPP/CPA assays, suggesting that analogs might represent a promising new generation of novel pain relievers. PMID:27556704

  3. Mitragynine/Corynantheidine Pseudoindoxyls As Opioid Analgesics with Mu Agonism and Delta Antagonism, Which Do Not Recruit β-Arrestin-2.

    PubMed

    Váradi, András; Marrone, Gina F; Palmer, Travis C; Narayan, Ankita; Szabó, Márton R; Le Rouzic, Valerie; Grinnell, Steven G; Subrath, Joan J; Warner, Evelyn; Kalra, Sanjay; Hunkele, Amanda; Pagirsky, Jeremy; Eans, Shainnel O; Medina, Jessica M; Xu, Jin; Pan, Ying-Xian; Borics, Attila; Pasternak, Gavril W; McLaughlin, Jay P; Majumdar, Susruta

    2016-09-22

    Natural products found in Mitragyna speciosa, commonly known as kratom, represent diverse scaffolds (indole, indolenine, and spiro pseudoindoxyl) with opioid activity, providing opportunities to better understand opioid pharmacology. Herein, we report the pharmacology and SAR studies both in vitro and in vivo of mitragynine pseudoindoxyl (3), an oxidative rearrangement product of the corynanthe alkaloid mitragynine. 3 and its corresponding corynantheidine analogs show promise as potent analgesics with a mechanism of action that includes mu opioid receptor agonism/delta opioid receptor antagonism. In vitro, 3 and its analogs were potent agonists in [(35)S]GTPγS assays at the mu opioid receptor but failed to recruit β-arrestin-2, which is associated with opioid side effects. Additionally, 3 developed analgesic tolerance more slowly than morphine, showed limited physical dependence, respiratory depression, constipation, and displayed no reward or aversion in CPP/CPA assays, suggesting that analogs might represent a promising new generation of novel pain relievers.

  4. Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling.

    PubMed

    Heitzler, Domitille; Durand, Guillaume; Gallay, Nathalie; Rizk, Aurélien; Ahn, Seungkirl; Kim, Jihee; Violin, Jonathan D; Dupuy, Laurence; Gauthier, Christophe; Piketty, Vincent; Crépieux, Pascale; Poupon, Anne; Clément, Frédérique; Fages, François; Lefkowitz, Robert J; Reiter, Eric

    2012-01-01

    Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on β-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.

  5. β-arrestin1 interacts with the G protein subunits β1γ2 and promotes β1γ2-dependent Akt signaling for NF-κB activation

    PubMed Central

    Yang, Ming; He, Rong L.; Benovic, Jeffrey L.; Ye, Richard D.

    2009-01-01

    SYNOPSIS β-Arrestins are known to regulate G protein signaling through interactions with their downstream effectors. Here, we report that β-arrestin1 associates with the G protein β1γ2 subunits in transfected cells, and purified β-arrestin1 interacts with Gβ1γ2 derived from in vitro translation. Deletion mutagenesis of β-arrestin1 led to the identification of a region, comprising amino acids 181-280, as being responsible for its interaction with Gβ1γ2. Overexpression of β-arrestin1 facilitates Gβ1γ2-mediated Akt phosphorylation, and inhibition of endogenous β-arrestin1 expression by small interfering RNA (siRNA) diminishes this effect. Through investigation of nuclear factor κB (NF-κB), a transcription factor regulated by Akt signaling, we have found that overexpression of β-arrestin1 significantly enhances Gβ1γ2-mediated nuclear translocation of NF-κB proteins and expression of a NF-κB-directed luciferase reporter. Overexpression of β-arrestin1 also promotes bradykinin-induced, Gβγ-mediated NF-κB luciferase reporter expression, which is reverted by silencing the endogenous β-arrestin1 with a specific siRNA. These results identify novel functions of β-arrestin1 in binding to the β1γ2 subunits of heterotrimeric G proteins and promoting Gβγ-mediated Akt signaling for NF-κB activation. PMID:18729826

  6. Stress conditions promote yeast Gap1 permease ubiquitylation and down-regulation via the arrestin-like Bul and Aly proteins.

    PubMed

    Crapeau, Myriam; Merhi, Ahmad; André, Bruno

    2014-08-01

    Gap1, the yeast general amino acid permease, is a convenient model for studying how the intracellular traffic of membrane transporters is regulated. Present at the plasma membrane under poor nitrogen supply conditions, it undergoes ubiquitylation, endocytosis, and degradation upon activation of the TORC1 kinase complex in response to an increase in internal amino acids. This down-regulation is stimulated by TORC1-dependent phosphoinhibition of the Npr1 kinase, resulting in activation by dephosphorylation of the arrestin-like Bul1 and Bul2 adaptors recruiting the Rsp5 ubiquitin ligase to Gap1. We report here that Gap1 is also down-regulated when cells are treated with the TORC1 inhibitor rapamycin or subjected to various stresses and that a lack of the Tco89 subunit of TORC1 causes constitutive Gap1 down-regulation. Both the Bul1 and Bul2 and the Aly1 and Aly2 arrestin-like adaptors of Rsp5 promote this down-regulation without undergoing dephosphorylation. Furthermore, they act via the C-terminal regions of Gap1 not involved in ubiquitylation in response to internal amino acids, whereas a Gap1 mutant altered in the N-terminal tail and resistant to ubiquitylation by internal amino acids is efficiently down-regulated under stress via the Bul and Aly adaptors. Although the Bul proteins mediate Gap1 ubiquitylation of two possible lysines, Lys-9 and Lys-16, the Aly proteins promote ubiquitylation of the Lys-16 residue only. This stress-induced pathway of Gap1 down-regulation targets other permeases as well, and it likely allows cells facing adverse conditions to retrieve amino acids from permease degradation.

  7. Using Bioluminescence Resonance Energy Transfer (BRET) to Characterize Agonist-Induced Arrestin Recruitment to Modified and Unmodified G Protein-Coupled Receptors.

    PubMed

    Donthamsetti, Prashant; Quejada, Jose Rafael; Javitch, Jonathan A; Gurevich, Vsevolod V; Lambert, Nevin A

    2015-09-01

    G protein-coupled receptors (GPCRs) represent ∼25% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which are involved in differential signaling pathways. Because functionally selective or biased ligands activate one of these two pathways, they may be superior medications for certain diseases states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for two bioluminescence resonance energy transfer (BRET)-based assays used to monitor arrestin recruitment to GPCRs. One assay requires modification of GPCRs by fusion to a BRET donor or acceptor moiety, whereas the other can detect arrestin recruitment to unmodified GPCRs.

  8. Using bioluminescent resonance energy transfer (BRET) to characterize agonist-induced arrestin recruitment to modified and unmodified G protein-coupled receptors (GPCRs)

    PubMed Central

    Donthamsetti, Prashant; Quejada, Jose Rafael; Javitch, Jonathan A.; Gurevich, Vsevolod V.; Lambert, Nevin A.

    2015-01-01

    G protein-coupled receptors (GPCRs) represent ~25% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which are involved in differential signaling pathways. Functionally selective or biased ligands activate one of these two pathways and may be superior medications for certain diseases states. The identification of these ligands requires robust drug screening assays for both G protein and arrestin activity. Here we describe in detail the technical aspects of two bioluminescence resonance energy (BRET)-based assays that can be used to monitor arrestin recruitment to GPCRs. One assay requires modification of GPCRs by fusion to a BRET donor or acceptor moiety, whereas the other can detect recruitment of arrestin to unmodified GPCRs. PMID:26331887

  9. The content of family practice: do we need more studies?

    PubMed

    Curry, L; Macintyre, K

    1982-01-01

    Having an accurate job description of family physicians is important to a number of audiences. There is a tendency to produce another content profile of family practice in response to every specific request for an accurate job description, rather than examining the existing profiles. We analyzed the amount of similarity and therefore redundancy in currently available profiles of family practice. Our findings indicate remarkable consistency across profile studies. We conclude that there is no need to continue producing profiles of family practice unless something significant occurs in the medical environment to suggest there might be a change in the profile.

  10. Genetic Deletion of β-Arrestin-2 and the Mitigation of Established Airway Hyperresponsiveness in a Murine Asthma Model.

    PubMed

    Chen, Minyong; Hegde, Akhil; Choi, Yeon Ho; Theriot, Barbara S; Premont, Richard T; Chen, Wei; Walker, Julia K L

    2015-09-01

    β-Arrestin-2 (βarr2) is a ubiquitously expressed cytosolic protein that terminates G protein-coupled receptor signaling and transduces G protein-independent signaling. We previously showed that mice lacking βarr2 do not develop an asthma phenotype when sensitized to, and challenged with, allergens. The current study evaluates if an established asthma phenotype can be mitigated by deletion of βarr2 using an inducible Cre recombinase. We sensitized and challenged mice to ovalbumin (OVA) and demonstrated that on Day (d) 24 the allergic asthma phenotype was apparent in uninduced βarr2 and wild-type (WT) mice. In a second group of OVA-treated mice, tamoxifen was injected on d24 to d28 to activate Cre recombinase, and OVA aerosol challenge was continued through d44. The asthma phenotype was assessed using lung mechanics measurements, bronchoalveolar lavage cell analysis, and histological assessment of mucin and airway inflammation. Compared with their respective saline-treated controls, OVA-treated WT mice and mice expressing the inducible Cre recombinase displayed a significant asthma phenotype at d45. Whereas tamoxifen treatment had no significant effect on the asthma phenotype in WT mice, it inhibited βarr2 expression and caused a significant reduction in airway hyper-responsiveness (AHR) in Cre-inducible mice. These findings suggest that βarr2 is actively required for perpetuation of the AHR component of the allergic asthma phenotype. Our finding that βarr2 participates in the perpetuation of AHR in an asthma model means that targeting βarr2 may provide immediate and potentially long-term relief from daily asthma symptoms due to AHR irrespective of inflammation.

  11. Non-Hematopoietic β-Arrestin1 Confers Protection Against Experimental Colitis.

    PubMed

    Lee, Taehyung; Lee, Eunhee; Arrollo, David; Lucas, Peter C; Parameswaran, Narayanan

    2016-05-01

    β-Arrestins are multifunctional scaffolding proteins that modulate G protein-coupled receptor (GPCR)-dependent and -independent cell signaling pathways in various types of cells. We recently demonstrated that β-arrestin1 (β-arr1) deficiency strikingly attenuates dextran sodium sulfate (DSS)-induced colitis in mice. Since DSS-induced colitis is in part dependent on gut epithelial injury, we examined the role of β-arr1 in intestinal epithelial cells (IECs) using a colon epithelial cell line, SW480 cells. Surprisingly, we found that knockdown of β-arr1 in SW480 cells enhanced epithelial cell death via a caspase-3-dependent process. To understand the in vivo relevance and potential cell type-specific role of β-arr1 in colitis development, we generated bone marrow chimeras with β-arr1 deficiency in either the hematopoietic or non-hematopoietic compartment. Reconstituted chimeric mice were then subjected to DSS-induced colitis. Similar to our previous findings, β-arr1 deficiency in the hematopoietic compartment protected mice from DSS-induced colitis. However, consistent with the role of β-arr1 in epithelial apoptosis in vitro, non-hematopoietic β-arr1 deficiency led to an exacerbated colitis phenotype. To further understand signaling mechanisms, we examined the effect of β-arr1 on TNF-α-mediated NFκB and MAPK pathways. Our results demonstrate that β-arr1 has a critical role in modulating ERK, JNK and p38 MAPK pathways mediated by TNF-α in IECs. Together, our results show that β-arr1-dependent signaling in hematopoietic and non-hematopoietic cells differentially regulates colitis pathogenesis and further demonstrates that β-arr1 in epithelial cells inhibits TNF-α-induced cell death pathways.

  12. Lipid G Protein-coupled Receptor Ligand Identification Using β-Arrestin PathHunter™ Assay

    PubMed Central

    Yin, Hong; Chu, Alan; Li, Wei; Wang, Bin; Shelton, Fabiola; Otero, Francella; Nguyen, Deborah G.; Caldwell, Jeremy S.; Chen, Yu Alice

    2009-01-01

    A growing number of orphan G-protein-coupled receptors (GPCRs) have been reported to be activated by lipid ligands, such as lysophosphatidic acid, sphingosine 1-phosphate (S1P), and cannabinoids, for which there are already well established receptors. These new ligand claims are controversial due to either lack of independent confirmations or conflicting reports. We used the β-arrestin PathHunter™ assay system, a newly developed, generic GPCR assay format that measures β-arrestin binding to GPCRs, to evaluate lipid receptor and ligand pairing. This assay eliminates interference from endogenous receptors on the parental cells because it measures a signal that is specifically generated by the tagged receptor and is immediately downstream of receptor activation. We screened a large number of newly “deorphaned” receptors (GPR23, GPR92, GPR55, G2A, GPR18, GPR3, GPR6, GPR12, and GPR63) and control receptors against a collection of ∼400 lipid molecules to try to identify the receptor ligand in an unbiased fashion. GPR92 was confirmed to be a lysophosphatidic acid receptor with weaker responses to farnesyl pyrophosphate and geranylgeranyl diphosphate. The putative cannabinoid receptor GPR55 responded strongly to AM251, rimonabant, and lysophosphatidylinositol but only very weakly to endocannabinoids. G2A receptor was confirmed to be an oxidized free fatty acid receptor. In addition, we discovered that 3,3′-diindolylmethane, a dietary molecule from cruciferous vegetables, which has known anti-cancer properties, to be a CB2 receptor partial agonist, with binding affinity around 1 μm. The anti-inflammatory effect of 3,3′-diindolylmethane in RAW264.7 cells was shown to be partially mediated by CB2. PMID:19286662

  13. Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell.

    PubMed Central

    Byk, T; Bar-Yaacov, M; Doza, Y N; Minke, B; Selinger, Z

    1993-01-01

    Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to the active form of metarhodopsin. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. On the other hand, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsin to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein. Binding of 49-kDa arrestin further quenches the activity of metarhodopsin and protects it from dephosphorylation. Light-dependent binding and release of 49-kDa arrestin from metarhodopsin- and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the rdgC phosphatase. We propose that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8446607

  14. Content and Workflow Management for Library Websites: Case Studies

    ERIC Educational Resources Information Center

    Yu, Holly, Ed.

    2005-01-01

    Using database-driven web pages or web content management (WCM) systems to manage increasingly diverse web content and to streamline workflows is a commonly practiced solution recognized in libraries today. However, limited library web content management models and funding constraints prevent many libraries from purchasing commercially available…

  15. Working experiences of Iranian retired nurses: a content analysis study.

    PubMed

    Nobahar, Monir; Ahmadi, Fazlollah; Alhani, Fatemah; Fallahi Khoshknab, Masood

    2013-10-01

    Understanding the experiences of retired nurses can be useful in increasing self-confidence, motivation to work and work enthusiasm among nurses. The purpose of this study was to explore the work experiences of Iranian retired nurses. A qualitative design was conducted using a content analysis approach. Purposive sampling was used to choose the study participants. Semi-structured interviews were held to collect the perspectives of 20 retired nurses (10 female and 10 male). Two main themes emerged in the data analysis: 'work problems and unpleasant experiences in a sense' with subthemes 'exhausting work', 'insufficient salary', 'inappropriate relation' and 'unsuitable social position'; and 'job satisfaction and pleasant experiences in a sense' with subthemes 'divine satisfaction and religious belief', 'satisfaction of patients and their companions' and 'love of nursing profession and relaxation experience'. The findings indicate the challenges that nurses face after retirement. These experiences will help nurse managers to adopt appropriate measures to support nurses after retirement. PMID:24093736

  16. Study of the detail content of Apollo orbital photography

    NASA Technical Reports Server (NTRS)

    Kinzly, R. E.

    1972-01-01

    The results achieved during a study of the Detail Content of Apollo Orbital Photography are reported. The effect of residual motion smear or image reproduction processes upon the detail content of lunar surface imagery obtained from the orbiting command module are assessed. Data and conclusions obtained from the Apollo 8, 12, 14 and 15 missions are included. For the Apollo 8, 12 and 14 missions, the bracket-mounted Hasselblad camera had no mechanism internal to the camera for motion compensation. If the motion of the command module were left totally uncompensated, these photographs would exhibit a ground smear varying from 12 to 27 meters depending upon the focal length of the lens and the exposure time. During the photographic sequences motion compensation was attempted by firing the attitude control system of the spacecraft at a rate to compensate for the motion relative to the lunar surface. The residual smear occurring in selected frames of imagery was assessed using edge analyses methods to obtain and achieved modulation transfer function (MTF) which was compared to a baseline MTF.

  17. Studying of tritium content in snowpack of Degelen mountain range.

    PubMed

    Turchenko, D V; Lukashenko, S N; Aidarkhanov, A O; Lyakhova, O N

    2014-06-01

    The paper presents the results of investigation of tritium content in the layers of snow located in the streambeds of the "Degelen" massif contaminated with tritium. The objects of investigation were selected watercourses Karabulak, Uzynbulak, Aktybai located beyond the "Degelen" site. We studied the spatial distribution of tritium relative to the streambed of watercourses and defined the borders of the snow cover contamination. In the centre of the creek watercourses the snow contamination in the surface layer is as high as 40 000 Bq/L. The values of the background levels of tritium in areas not related to the streambed, which range from 40 to 50 Bq/L. The results of snow cover measurements in different seasonal periods were compared. The main mechanisms causing tritium transfer in snow were examined and identified. The most important mechanism of tritium transfer in the streams is tritium emanation from ice or soil surface.

  18. A calcineurin-dependent switch controls the trafficking function of α-arrestin Aly1/Art6.

    PubMed

    O'Donnell, Allyson F; Huang, Laiqiang; Thorner, Jeremy; Cyert, Martha S

    2013-08-16

    Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5. However, the molecular mechanisms that control α-arrestin function are not well understood. Here, we show that α-arrestin Aly1/Art6 is a phosphoprotein that specifically interacts with and is dephosphorylated by the Ca(2+)- and calmodulin-dependent phosphoprotein phosphatase calcineurin/PP2B. Dephosphorylation of Aly1 by calcineurin at a subset of phospho-sites is required for Aly1-mediated trafficking of the aspartic acid and glutamic acid transporter Dip5 to the vacuole, but it does not alter Rsp5 binding, ubiquitinylation, or stability of Aly1. In addition, dephosphorylation of Aly1 by calcineurin does not regulate the ability of Aly1 to promote the intracellular sorting of the general amino acid permease Gap1. These results suggest that phosphorylation of Aly1 inhibits its vacuolar trafficking function and, conversely, that dephosphorylation of Aly1 by calcineurin serves as a regulatory switch to promote Aly1-mediated trafficking to the vacuole.

  19. The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein-coupled receptor lysosomal sorting.

    PubMed

    Dores, Michael R; Lin, Huilan; J Grimsey, Neil; Mendez, Francisco; Trejo, JoAnn

    2015-12-15

    The sorting of G protein-coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain-containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs.

  20. beta-Arrestin mediates beta1-adrenergic receptor-epidermal growth factor receptor interaction and downstream signaling.

    PubMed

    Tilley, Douglas G; Kim, Il-Man; Patel, Priyesh A; Violin, Jonathan D; Rockman, Howard A

    2009-07-24

    beta1-Adrenergic receptor (beta1AR) stimulation confers cardioprotection via beta-arrestin-de pend ent transactivation of epidermal growth factor receptors (EGFRs), however, the precise mechanism for this salutary process is unknown. We tested the hypothesis that the beta1AR and EGFR form a complex that differentially directs intracellular signaling pathways. beta1AR stimulation and EGF ligand can each induce equivalent EGFR phosphorylation, internalization, and downstream activation of ERK1/2, but only EGF ligand causes translocation of activated ERK to the nucleus, whereas beta1AR-stimulated/EGFR-transactivated ERK is restricted to the cytoplasm. beta1AR and EGFR are shown to interact as a receptor complex both in cell culture and endogenously in human heart, an interaction that is selective and undergoes dynamic regulation by ligand stimulation. Although catecholamine stimulation mediates the retention of beta1AR-EGFR interaction throughout receptor internalization, direct EGF ligand stimulation initiates the internalization of EGFR alone. Continued interaction of beta1AR with EGFR following activation is dependent upon C-terminal tail GRK phosphorylation sites of the beta1AR and recruitment of beta-arrestin. These data reveal a new signaling paradigm in which beta-arrestin is required for the maintenance of a beta1AR-EGFR interaction that can direct cytosolic targeting of ERK in response to catecholamine stimulation.

  1. The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein–coupled receptor lysosomal sorting

    PubMed Central

    Dores, Michael R.; Lin, Huilan; J. Grimsey, Neil; Mendez, Francisco; Trejo, JoAnn

    2015-01-01

    The sorting of G protein–coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain–containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs. PMID:26490116

  2. Studies on the energy content of pigeon feeds I. Determination of digestibility and metabolizable energy content.

    PubMed

    Hullar, I; Meleg, I; Fekete, S; Romvari, R

    1999-12-01

    The digestibility coefficient and metabolizable energy (ME) content of the most important pigeon feeds (corn, wheat, barley, red and white millet, sorghum, canary seed, peas, lentils, sunflower, and hemp) were determined. The experiment was carried out using 10 adult male homing pigeons. All feeds were fed alone, in a whole-grain form, ad libitum. Drinking water and grit were offered to the birds on a continuous basis. Each feedstuff was fed to five pigeons in 1-wk cycles. There was no significant difference between the values determined in pigeons and those reported in the literature for chickens among the digestibilities of the CP of the various feeds. For pigeons, the digestibility of carbohydrates (N-free extracts, NFE) was lower (e.g., 62.37 vs 83.00% for barley and 63.45 vs 77.00% for peas), whereas the ether extract (EE) was higher (e.g., 75.58 vs 61.00% for barley and 82.59 vs 80.00% for peas) in pigeons compared with chickens. As a result, the AMEn values determined in pigeons did not differ significantly from those reported for chickens but tended to be slightly higher. For feeds of high-oil content, that difference may be somewhat larger. The correlation between the CP, EE, crude fiber (CF), and NFE contents of the feeds and the ME values determined in this experiment were calculated by multivariate linear regression. It was concluded that it was more accurate to determine and tabulate the ME contents of other potential pigeon feeds directly by experimental methods rather than using an equation. PMID:10626652

  3. β-Arrestin interacts with the beta/gamma subunits of trimeric G-proteins and dishevelled in the Wnt/Ca(2+) pathway in xenopus gastrulation.

    PubMed

    Seitz, Katharina; Dürsch, Verena; Harnoš, Jakub; Bryja, Vitezslav; Gentzel, Marc; Schambony, Alexandra

    2014-01-01

    β-Catenin independent, non-canonical Wnt signaling pathways play a major role in the regulation of morphogenetic movements in vertebrates. The term non-canonical Wnt signaling comprises multiple, intracellularly divergent, Wnt-activated and β-Catenin independent signaling cascades including the Wnt/Planar Cell Polarity and the Wnt/Ca(2+) cascades. Wnt/Planar Cell Polarity and Wnt/Ca(2+) pathways share common effector proteins, including the Wnt ligand, Frizzled receptors and Dishevelled, with each other and with additional branches of Wnt signaling. Along with the aforementioned proteins, β-Arrestin has been identified as an essential effector protein in the Wnt/β-Catenin and the Wnt/Planar Cell Polarity pathway. Our results demonstrate that β-Arrestin is required in the Wnt/Ca(2+) signaling cascade upstream of Protein Kinase C (PKC) and Ca(2+)/Calmodulin-dependent Protein Kinase II (CamKII). We have further characterized the role of β-Arrestin in this branch of non-canonical Wnt signaling by knock-down and rescue experiments in Xenopus embryo explants and analyzed protein-protein interactions in 293T cells. Functional interaction of β-Arrestin, the β subunit of trimeric G-proteins and Dishevelled is required to induce PKC activation and membrane translocation. In Xenopus gastrulation, β-Arrestin function in Wnt/Ca(2+) signaling is essential for convergent extension movements. We further show that β-Arrestin physically interacts with the β subunit of trimeric G-proteins and Dishevelled, and that the interaction between β-Arrestin and Dishevelled is promoted by the beta/gamma subunits of trimeric G-proteins, indicating the formation of a multiprotein signaling complex.

  4. β-Arrestin-2 modulates radiation-induced intestinal crypt progenitor/stem cell injury

    PubMed Central

    Liu, Z; Tian, H; Jiang, J; Yang, Y; Tan, S; Lin, X; Liu, H; Wu, B

    2016-01-01

    Intestinal crypt progenitor/stem (ICPS) cell apoptosis and vascular endothelial cell apoptosis are responsible for the initiation and development of ionizing radiation (IR)-evoked gastrointestinal syndrome. The signaling mechanisms underlying IR-induced ICPS cell apoptosis remain largely unclear. Our findings provide evidence that β-arrestin-2 (βarr2)-mediated ICPS cell apoptosis is crucial for IR-stimulated intestinal injury. βArr2-deficient mice exhibited decreased ICPS cell and intestinal Lgr5+ (leucine-rich repeat-containing G-protein-coupled receptor 5-positive) stem cell apoptosis, promoted crypt proliferation and reproduction, and protracted survival following lethal doses of radiation. Radioprotection in the ICPS cells isolated from βarr2-deficient mice depended on prolonged nuclear factor-κB (NF-κB) activation via direct interaction of βarr2 with IκBα and subsequent inhibition of p53-upregulated modulator of apoptosis (PUMA)-mediated mitochondrial dysfunction. Unexpectedly, βarr2 deficiency had little effect on IR-induced intestinal vascular endothelial cell apoptosis in mice. Consistently, βarr2 knockdown also provided significant radioresistance by manipulating NF-κB/PUMA signaling in Lgr5+ cells in vitro. Collectively, these observations show that targeting the βarr2/NF-κB/PUMA novel pathway is a potential radiomitigator for limiting the damaging effect of radiotherapy on the gastrointestinal system. Significance statement: acute injury to the intestinal mucosa is a major dose-limiting complication of abdominal radiotherapy. The issue of whether the critical factor for the initiation of radiation-induced intestinal injury is intestinal stem cell apoptosis or endothelial cell apoptosis remains unresolved. βArrs have recently been found to be multifunctional adaptor of apoptosis. Here, we found that β-arrestin-2 (βarr2) deficiency was associated with decreased radiation-induced ICPS cell apoptosis, which prolonged survival in abdominally

  5. β-Arrestin-2 modulates radiation-induced intestinal crypt progenitor/stem cell injury.

    PubMed

    Liu, Z; Tian, H; Jiang, J; Yang, Y; Tan, S; Lin, X; Liu, H; Wu, B

    2016-09-01

    Intestinal crypt progenitor/stem (ICPS) cell apoptosis and vascular endothelial cell apoptosis are responsible for the initiation and development of ionizing radiation (IR)-evoked gastrointestinal syndrome. The signaling mechanisms underlying IR-induced ICPS cell apoptosis remain largely unclear. Our findings provide evidence that β-arrestin-2 (βarr2)-mediated ICPS cell apoptosis is crucial for IR-stimulated intestinal injury. βArr2-deficient mice exhibited decreased ICPS cell and intestinal Lgr5(+) (leucine-rich repeat-containing G-protein-coupled receptor 5-positive) stem cell apoptosis, promoted crypt proliferation and reproduction, and protracted survival following lethal doses of radiation. Radioprotection in the ICPS cells isolated from βarr2-deficient mice depended on prolonged nuclear factor-κB (NF-κB) activation via direct interaction of βarr2 with IκBα and subsequent inhibition of p53-upregulated modulator of apoptosis (PUMA)-mediated mitochondrial dysfunction. Unexpectedly, βarr2 deficiency had little effect on IR-induced intestinal vascular endothelial cell apoptosis in mice. Consistently, βarr2 knockdown also provided significant radioresistance by manipulating NF-κB/PUMA signaling in Lgr5(+) cells in vitro. Collectively, these observations show that targeting the βarr2/NF-κB/PUMA novel pathway is a potential radiomitigator for limiting the damaging effect of radiotherapy on the gastrointestinal system. Significance statement: acute injury to the intestinal mucosa is a major dose-limiting complication of abdominal radiotherapy. The issue of whether the critical factor for the initiation of radiation-induced intestinal injury is intestinal stem cell apoptosis or endothelial cell apoptosis remains unresolved. βArrs have recently been found to be multifunctional adaptor of apoptosis. Here, we found that β-arrestin-2 (βarr2) deficiency was associated with decreased radiation-induced ICPS cell apoptosis, which prolonged survival in

  6. G-Protein/β-Arrestin-Linked Fluctuating Network of G-Protein-Coupled Receptors for Predicting Drug Efficacy and Bias Using Short-Term Molecular Dynamics Simulation

    PubMed Central

    Ichikawa, Osamu; Fujimoto, Kazushi; Yamada, Atsushi; Okazaki, Susumu; Yamazaki, Kazuto

    2016-01-01

    The efficacy and bias of signal transduction induced by a drug at a target protein are closely associated with the benefits and side effects of the drug. In particular, partial agonist activity and G-protein/β-arrestin-biased agonist activity for the G-protein-coupled receptor (GPCR) family, the family with the most target proteins of launched drugs, are key issues in drug discovery. However, designing GPCR drugs with appropriate efficacy and bias is challenging because the dynamic mechanism of signal transduction induced by ligand—receptor interactions is complicated. Here, we identified the G-protein/β-arrestin-linked fluctuating network, which initiates large-scale conformational changes, using sub-microsecond molecular dynamics (MD) simulations of the β2-adrenergic receptor (β2AR) with a diverse collection of ligands and correlation analysis of their G protein/β-arrestin efficacy. The G-protein-linked fluctuating network extends from the ligand-binding site to the G-protein-binding site through the connector region, and the β-arrestin-linked fluctuating network consists of the NPxxY motif and adjacent regions. We confirmed that the averaged values of fluctuation in the fluctuating network detected are good quantitative indexes for explaining G protein/β-arrestin efficacy. These results indicate that short-term MD simulation is a practical method to predict the efficacy and bias of any compound for GPCRs. PMID:27187591

  7. β-arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism

    PubMed Central

    Fernández-Arenas, Elena; Calleja, Enrique; Martínez-Martín, Nadia; Gharbi, Severine I; Navajas, Rosana; García-Medel, Noel; Penela, Petronila; Alcamí, Antonio; Mayor, Federico; Albar, Juan P; Alarcón, Balbino

    2014-01-01

    T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen-presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β-arrestin-1 as a ligand of non-phosphorylated resting TCRs. Using dominant-negative and knockdown approaches we demonstrate that β-arrestin-1 is required for the internalization and downregulation of non-engaged bystander TCRs. Furthermore, TCR triggering provokes the β-arrestin-1-mediated downregulation of the G-protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β-arrestin-1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR-triggered PKC-mediated phosphorylation of β-arrestin-1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal. PMID:24502978

  8. Study of the detail content of Apollo orbital photography

    NASA Technical Reports Server (NTRS)

    Kinzly, R. E.

    1974-01-01

    The development and application of image evaluation methods for assessing the detail content of Apollo orbital photography was demonstrated. Edge analysis using shadow to sunlight edges interior to craters was successfully used to evaluate the fine detail content of Apollo 15, 16, and 17 imagery. A method for evaluating tone quality was developed using a gain factor as a function of object contrast and average exposure level that can be related to object detectability.

  9. A Study about the Level of a Teacher's Content Knowledge, Pedagogical Content Knowledge, Instructional Practices, and Demographics and Their Effects on Students' Literacy Achievement

    ERIC Educational Resources Information Center

    Brunsberg, Suzy LuAnn

    2013-01-01

    It might be assumed that teachers' content and pedagogical content knowledge affect student learning. However, most studies do not include observations of actual classroom instruction. This study provides empirical evidence that illustrates the significance of a teacher's content knowledge; a teacher's pedagogical content knowledge; instructional…

  10. Dependence of seismoelectric amplitudes on water content - a field study

    NASA Astrophysics Data System (ADS)

    Strahser, M. H. P.; Matthey, P.-D.; Jouniaux, L.; Sailhac, P.

    2009-04-01

    In porous saturated media, seismic compressional waves can cause seismoelectric and seismoelectromagnetic signals through electrokinetic coupling. It has been observed that these measureable signals also occur in partially saturated media, but the theory is largely unknown for these circumstances. Seismoelectromagnetic tomography is expected to combine the sensitivity of electrical properties to water-content and permeability, to the high spatial resolution of seismic surveys. A better understanding of the physical processes and a reliable quantification of the conversion between seismic and electric energy are necessary and need to take into account the effect of water-content, especially for shallow subsurface investigations. In order to quantify seismoelectric signals with changing water content, we repeated seismoelectric and seismic measurements on the same profile in the Vosges Mountains during several months. The electrical resistivity was also monitored to take into account the water-content variations. We show that an exponential relation can be established between the seismoelectric amplitudes normalized with the seismic amplitudes and the resistivity which in turn is related to the saturation: Increasing resistivity (decreasing water content) leads to decreasing normalized seismoelectric amplitudes. These results imply that the electrokinetic coefficient should increase with water-saturation, as measured in laboratory, but not predicted by theory. This work was funded by CNRS and Université Louis Pasteur de Strasbourg.

  11. Negative impact of β-arrestin-1 on post-myocardial infarction heart failure via cardiac and adrenal-dependent neurohormonal mechanisms.

    PubMed

    Bathgate-Siryk, Ashley; Dabul, Samalia; Pandya, Krunal; Walklett, Karlee; Rengo, Giuseppe; Cannavo, Alessandro; De Lucia, Claudio; Liccardo, Daniela; Gao, Erhe; Leosco, Dario; Koch, Walter J; Lymperopoulos, Anastasios

    2014-02-01

    β-Arrestin (βarr)-1 and β-arrestin-2 (βarrs) are universal G-protein-coupled receptor adapter proteins that negatively regulate cardiac β-adrenergic receptor (βAR) function via βAR desensitization and downregulation. In addition, they mediate G-protein-independent βAR signaling, which might be beneficial, for example, antiapoptotic, for the heart. However, the specific role(s) of each βarr isoform in cardiac βAR dysfunction, the molecular hallmark of chronic heart failure (HF), remains unknown. Furthermore, adrenal βarr1 exacerbates HF by chronically enhancing adrenal production and hence circulating levels of aldosterone and catecholamines. Herein, we sought to delineate specific roles of βarr1 in post-myocardial infarction (MI) HF by testing the effects of βarr1 genetic deletion on normal and post-MI cardiac function and morphology. We studied βarr1 knockout (βarr1KO) mice alongside wild-type controls under normal conditions and after surgical MI. Normal (sham-operated) βarr1KO mice display enhanced βAR-dependent contractility and post-MI βarr1KO mice enhanced overall cardiac function (and βAR-dependent contractility) compared with wild type. Post-MI βarr1KO mice also show increased survival and decreased cardiac infarct size, apoptosis, and adverse remodeling, as well as circulating catecholamines and aldosterone, compared with post-MI wild type. The underlying mechanisms, on one hand, improved cardiac βAR signaling and function, as evidenced by increased βAR density and procontractile signaling, via reduced cardiac βAR desensitization because of cardiac βarr1 absence, and, on the other hand, decreased production leading to lower circulating levels of catecholamines and aldosterone because of adrenal βarr1 absence. Thus, βarr1, via both cardiac and adrenal effects, is detrimental for cardiac structure and function and significantly exacerbates post-MI HF.

  12. β-Arrestin1 enhances hepatocellular carcinogenesis through inflammation-mediated Akt signalling.

    PubMed

    Yang, Yidong; Guo, Yunwei; Tan, Siwei; Ke, Bilun; Tao, Jin; Liu, Huiling; Jiang, Jie; Chen, Jianning; Chen, Guihua; Wu, Bin

    2015-01-01

    G-protein-coupled receptors (GPCR) constitute the largest known superfamily for signal transduction and transmission, and they control a variety of physiological and pathological processes. GPCR adaptor β-arrestins (ARRBs) play a role in cancerous proliferation. However, the effect of ARRBs in inflammation-mediated hepatocellular carcinogenesis is unknown. Here we show that ARRB1, but not ARRB2, is upregulated in inflammation-associated hepatocellular carcinoma (HCC) and paracancerous tissues in humans. A genotoxic carcinogen, diethylnitrosamine (DEN), significantly induces hepatic inflammation, TNF-α production and ARRB1 expression. Although ARRB1 deficiency does not affect hepatic inflammation and TNF-α production, it markedly represses hepatocellular carcinogenesis by suppressing malignant proliferation in DEN-treated mice. Furthermore, TNF-α directly induces hepatic ARRB1 expression and enhances ARRB1 interaction with Akt by binding to boost Akt phosphorylation, resulting in malignant proliferation of liver cells. Our data suggest that ARRB1 enhances hepatocellular carcinogenesis by inflammation-mediated Akt signalling and that ARRB1 may be a potential therapeutic target for HCC.

  13. Analyzing the roles of multi-functional proteins in cells: the case of arrestins and GRKs

    PubMed Central

    Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2016-01-01

    Most proteins have multiple functions. Obviously, conventional methods of manipulating the level of the protein of interest in the cell, such as over-expression, knockout or knockdown, affect all of its functions simultaneously. The key advantage of these methods is that over-expression, knockout or knockdown does not require any knowledge of the molecular mechanisms of the function(s) of the protein of interest. The disadvantage is that these approaches are inadequate to elucidate the role of an individual function of the protein in a particular cellular process. An alternative is the use of re-engineered proteins, in which a single function is eliminated or enhanced. The use of mono-functional elements of a multi-functional protein can also yield cleaner answers. This approach requires detailed knowledge of the structural basis of each function of the protein in question. Thus, a lot of preliminary structure-function work is necessary to make it possible. However, when this information is available, replacing the protein of interest with a mutant in which individual functions are modified can shed light on the biological role of those particular functions. Here we illustrate this point using the example of protein kinases, most of which have additional non-enzymatic functions, as well as arrestins, known multi-functional signaling regulators in the cell. PMID:26453028

  14. Is the content of confabulation positive? An experimental study.

    PubMed

    Fotopoulou, Aikaterini; Conway, Martin A; Tyrer, Stephen; Birchall, Daniel; Griffiths, Philippa; Solms, Mark

    2008-01-01

    There has been little experimental work investigating the emotional content of confabulation, despite clinical descriptions of self-serving and affectively positive biases. False memories were elicited in 10 amnesic confabulating patients, 10 healthy controls and four amnesic control patients without confabulation. Memory protocols of the interviews with these groups were presented to naïve raters who were asked to rate the emotional valence of the listed confabulations. The false memories of the confabulating patients were found to distort previous experiences in ways significantly more pleasant and self-enhancing than those of controls. It was also found paradoxically that the more depressed the patients' mood the more positive the content of their confabulations. These findings suggest that the content of confabulation is mostly positive. The results have implications for the role of emotion and motivation in confabulation, as well as for the clinical management of confabulating patients.

  15. Searching for Educational Content in the For-Profit Internet: Case Study and Analysis.

    ERIC Educational Resources Information Center

    Fabos, Bettina

    This case study investigates the commercialized nature of Internet content and the ways educators and students negotiate and talk about such content. In factoring in the economic and historical context of educational Internet content, this case study also addresses educators' evolving attitudes towards commercialism in the classroom. A survey was…

  16. Family Studies I. Course Objectives, Content Analysis, Supporting Objectives and Content Generalizations.

    ERIC Educational Resources Information Center

    McKenzie, Sharon, Comp.; Martin, Joan, Ed.

    Family Studies I represents the first part of a consumer and homemaking program focusing on individual and family development, parenting, and the daily tasks of living in a family setting. The course is designed to help students study the family as it exists in day-to-day living and to aid them in understanding themselves in relation to their…

  17. Optical sensing of vegetation water content: A synthesis study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vegetation Water Content (VWC) plays an important role in parameterizing the vegetation influence on microwave soil moisture retrieval. During the past decade, researchers have developed relationships between VWC and vegetation indices available from satellite optical sensors in order to create larg...

  18. The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors.

    PubMed

    Oakley, Robert H; Hudson, Christine C; Cruickshank, Rachael D; Meyers, Diane M; Payne, Richard E; Rhem, Shay M; Loomis, Carson R

    2002-11-01

    G protein-coupled receptors (GPCRs) have proven to be a rich source of therapeutic targets; therefore, finding compounds that regulate these receptors is a critical goal in drug discovery. The Transfluor technology utilizes the redistribution of fluorescently labeled arrestins from the cytoplasm to agonist-occupied receptors at the plasma membrane to monitor quantitatively the activation or inactivation of GPCRs. Here, we show that the Transfluor technology can be quantitated on the INCell Analyzer system (INCAS) using the vasopressin V(2) receptor (V(2)R), which binds arrestin with high affinity, and the beta(2)-adrenergic receptor (beta(2)AR), which binds arrestin with low affinity. U2OS cells stably expressing an arrestin-green fluorescent protein conjugate and either the V(2)R or the beta(2)AR were plated in 96-well plastic plates and analyzed by the INCAS at a screening rate of 5 min per plate. Agonist dose-response and antagonist dose-inhibition curves revealed signal-to-background ratios of approximately 25:1 and 8:1 for the V(2)R and beta(2)AR, respectively. EC(50) values agreed closely with K(d) values reported in the literature for the different receptor agonists. In addition, small amounts of arrestin translocation induced by sub-EC(50) doses of agonist were distinguished from the background noise of untreated cells. Furthermore, differences in the magnitude of arrestin translocation distinguished partial agonists from full agonists, and Z' values for these ligands were >0.5. These data show that the Transfluor technology, combined with an automated image analysis system, provides a direct, robust, and universal assay for high throughput screening of known and orphan GPCRs.

  19. β-Arrestin 1’s Interaction with TC45 Attenuates Stat signaling by dephosphorylating Stat to inhibit antimicrobial peptide expression

    PubMed Central

    Sun, Jie-Jie; Yang, Hui-Ting; Niu, Guo-Juan; Feng, Xiao-Wu; Lan, Jiang-Feng; Zhao, Xiao-Fan; Wang, Jin-Xing

    2016-01-01

    Impaired phosphatase activity leads to the persistent activation of signal transducers and activators of transcription (Stat). In mammals, Stat family members are often phosphorylated or dephosphorylated by the same enzymes. To date, only one Stat similar to mammalian Stat5a/b has been found in crustaceans and there have been few studies in Stat signal regulation in crustaceans. Here, we report that β-arrestin1 interacts with TC45 (45-kDa form of T cell protein tyrosine phosphatase) in the nucleus to attenuate Stat signaling by promoting dephosphorylation of Stat. Initially, we showed that Stat translocates into the nucleus to induce antimicrobial peptide (AMP) expression after bacterial infection. βArr1 enters the nucleus of hemocytes and recruits TC45 to form the βarr1-TC45-Stat complex, which dephosphorylates Stat efficiently. The interaction of TC45 with Stat decreased and Stat phosphorylation increased in βarr1-silenced shrimp (Marsupenaeus japonicus) after challenge with Vibrio anguillarum. βArr1 directly interacts with Stat in nucleus and accelerates Stat dephosphorylation by recruiting TC45 after V. anguillarum challenge. Further study showed that βarr1 and TC45 also affect AMP expression, which is regulated by Stat. Therefore, βarr1 and TC45 are involved in the anti-V. anguillarum immune response by regulating Stat activity negatively to decrease AMP expression in shrimp. PMID:27782165

  20. A content validity study of the defense mechanism inventory.

    PubMed

    Blacha, M D; Fancher, R E

    1977-08-01

    The content validity of the Defense Mechanism Inventory was tested by 20 raters who evaluated each item in terms of which 15 defenses and three ego threats it represented. Items purportedly measuring the global defense categories of principalization, turning against self, and reversal, achieved relatively high rater agreement (over 70%) while projection and turning against Object fared poorly (29% and 39% respectively). Differential content validity was found in the levels and areas of the Inventory, indicating that the context in which items appear affect their representativeness of defensive behaviors. The individual defense mechanisms were disproportionately represented by the Inventory. Ratings suggested that aggressiveness was the major ego threat being measured by the items. Most of the problems appear correctable through rewriting many of the items.

  1. Prostaglandin E2 promotes lung cancer cell migration via EP4-betaArrestin1-c-Src signalsome.

    PubMed

    Kim, Jae Il; Lakshmikanthan, Vijayabaskar; Frilot, Nicole; Daaka, Yehia

    2010-04-01

    Many human cancers express elevated levels of cyclooxygenase-2 (COX-2), an enzyme responsible for the biosynthesis of prostaglandins. Available clinical data establish the protective effect of COX-2 inhibition on human cancer progression. However, despite these encouraging outcomes, the appearance of unwanted side effects remains a major hurdle for the general application of COX-2 inhibitors as effective cancer drugs. Hence, a better understanding of the molecular signals downstream of COX-2 is needed for the elucidation of drug targets that may improve cancer therapy. Here, we show that the COX-2 product prostaglandin E(2) (PGE(2)) acts on cognate receptor EP4 to promote the migration of A549 lung cancer cells. Treatment with PGE(2) enhances tyrosine kinase c-Src activation, and blockade of c-Src activity represses the PGE(2)-mediated lung cancer cell migration. PGE(2) affects target cells by activating four receptors named EP1 to EP4. Use of EP subtype-selective ligand agonists suggested that EP4 mediates prostaglandin-induced A549 lung cancer cell migration, and this conclusion was confirmed using a short hairpin RNA approach to specifically knock down EP4 expression. Proximal EP4 effectors include heterotrimeric Gs and betaArrestin proteins. Knockdown of betaArrestin1 expression with shRNA significantly impaired the PGE(2)-induced c-Src activation and cell migration. Together, these results support the idea that increased expression of the COX-2 product PGE(2) in the lung tumor microenvironment may initiate a mitogenic signaling cascade composed of EP4, betaArrestin1, and c-Src which mediates cancer cell migration. Selective targeting of EP4 with a ligand antagonist may provide an efficient approach to better manage patients with advanced lung cancer.

  2. β-Arrestin recruitment and G protein signaling by the atypical human chemokine decoy receptor CCX-CKR.

    PubMed

    Watts, Anne O; Verkaar, Folkert; van der Lee, Miranda M C; Timmerman, Claudia A W; Kuijer, Martien; van Offenbeek, Jody; van Lith, Lambertus H C J; Smit, Martine J; Leurs, Rob; Zaman, Guido J R; Vischer, Henry F

    2013-03-01

    Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric Gi proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with β-arrestin2-GFP translocation. Moreover, recruitment of β-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate Gi signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the Gi inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive Gi proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold β-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways.

  3. MOISTURE CONTENT AND POROSITY OF CONCRETE RUBBLE STUDY.

    SciTech Connect

    Phifer, M

    2005-10-07

    Tritium contaminated concrete rubble from the 232-F Tritium Facility was disposed in the Slit 1 Trenches 1 and 2 in 1997. A Special Analysis (SA) has been performed to evaluate any impact this disposal may have on the groundwater. The SA assumed that the disposed concrete rubble was fully saturated at the time of disposal, however if the concrete was less than fully saturated, migration of tritium out of the concrete would be slower than under fully saturated conditions. Therefore if the concrete at disposal was less than fully saturated, the PA assumption of full saturation would be a conservative assumption. In order to evaluate whether the PA assumption resulted in a conservative analysis from the standpoint of the concrete saturation, concrete rubble samples were collected from various facilities being demolished at the Savannah River Site (SRS) and evaluated for in-field moisture content, absorbable moisture, and water exchangeable porosity. The purpose of this task was to collect concrete rubble samples from various demolished SRS facilities for the purpose of determining in-field moisture content, absorbable moisture, and water exchangeable porosity. Since moisture content testing for concrete rubble is not typical, existing ASTM Standards were reviewed for method and procedure development.

  4. Real-time imaging of leukotriene B₄ mediated cell migration and BLT1 interactions with β-arrestin.

    PubMed

    Jala, Venkatakrishna R; Haribabu, Bodduluri

    2010-01-01

    G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins(1-2). Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of β-arrestins(3). β-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization (4-6). Leukotriene B(4;) (LTB(4;)) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB(4;) receptor 1 (BLT1; a high affinity receptor) and LTB(4;) receptor 2 (BLT2; a low affinity receptor)(7-9). The LTB(4;)-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis(10-17). The current paper describes the methodologies developed to monitor LTB(4;)-induced leukocyte migration and the interactions of BLT1 with β-arrestin and , receptor translocation in live cells using microscopy imaging techniques(18-19). Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described (20-21). These cells were tested in live cell imaging methods to demonstrate LTB(4;) induced cell migration. The human BLT1 was tagged

  5. Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5

    PubMed Central

    Becuwe, Michel; Léon, Sébastien

    2014-01-01

    After endocytosis, membrane proteins can recycle to the cell membrane or be degraded in lysosomes. Cargo ubiquitylation favors their lysosomal targeting and can be regulated by external signals, but the mechanism is ill-defined. Here, we studied the post-endocytic trafficking of Jen1, a yeast monocarboxylate transporter, using microfluidics-assisted live-cell imaging. We show that the ubiquitin ligase Rsp5 and the glucose-regulated arrestin-related trafficking adaptors (ART) protein Rod1, involved in the glucose-induced internalization of Jen1, are also required for the post-endocytic sorting of Jen1 to the yeast lysosome. This new step takes place at the trans-Golgi network (TGN), where Rod1 localizes dynamically upon triggering endocytosis. Indeed, transporter trafficking to the TGN after internalization is required for their degradation. Glucose removal promotes Rod1 relocalization to the cytosol and Jen1 deubiquitylation, allowing transporter recycling when the signal is only transient. Therefore, nutrient availability regulates transporter fate through the localization of the ART/Rsp5 ubiquitylation complex at the TGN. DOI: http://dx.doi.org/10.7554/eLife.03307.001 PMID:25380227

  6. Central HIV-1 Tat exposure elevates anxiety and fear conditioned responses of male mice concurrent with altered mu-opioid receptor-mediated G-protein activation and β-arrestin 2 activity in the forebrain.

    PubMed

    Hahn, Yun K; Paris, Jason J; Lichtman, Aron H; Hauser, Kurt F; Sim-Selley, Laura J; Selley, Dana E; Knapp, Pamela E

    2016-08-01

    Co-exposure to opiates and HIV/HIV proteins results in enhanced CNS morphological and behavioral deficits in HIV(+) individuals and in animal models. Opiates with abuse liability, such as heroin and morphine, bind preferentially to and have pharmacological actions through μ-opioid-receptors (MORs). The mechanisms underlying opiate-HIV interactions are not understood. Exposure to the HIV-1 transactivator of transcription (Tat) protein causes neurodegenerative outcomes that parallel many aspects of the human disease. We have also observed that in vivo exposure to Tat results in apparent changes in morphine efficacy, and thus have hypothesized that HIV proteins might alter MOR activation. To test our hypothesis, MOR-mediated G-protein activation was determined in neuroAIDS-relevant forebrain regions of transgenic mice with inducible CNS expression of HIV-1 Tat. G-protein activation was assessed by MOR agonist-stimulated [(35)S]guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPγS) autoradiography in brain sections, and in concentration-effect curves of MOR agonist-stimulated [(35)S]GTPγS binding in membranes isolated from specific brain regions. Comparative studies were done using the MOR-selective agonist DAMGO ([D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin) and a more clinically relevant agonist, morphine. Tat exposure reduced MOR-mediated G-protein activation in an agonist, time, and regionally dependent manner. Levels of the GPCR regulatory protein β-arrestin-2, which is involved in MOR desensitization, were found to be elevated in only one affected brain region, the amygdala; amygdalar β-arrestin-2 also showed a significantly increased association with MOR by co-immunoprecipitation, suggesting decreased availability of MOR. Interestingly, this correlated with changes in anxiety and fear-conditioned extinction, behaviors that have substantial amygdalar input. We propose that HIV-1 Tat alters the intrinsic capacity of MOR to signal in response to agonist binding

  7. Multi-Core Processor Memory Contention Benchmark Analysis Case Study

    NASA Technical Reports Server (NTRS)

    Simon, Tyler; McGalliard, James

    2009-01-01

    Multi-core processors dominate current mainframe, server, and high performance computing (HPC) systems. This paper provides synthetic kernel and natural benchmark results from an HPC system at the NASA Goddard Space Flight Center that illustrate the performance impacts of multi-core (dual- and quad-core) vs. single core processor systems. Analysis of processor design, application source code, and synthetic and natural test results all indicate that multi-core processors can suffer from significant memory subsystem contention compared to similar single-core processors.

  8. Content Metadata Standards for Marine Science: A Case Study

    USGS Publications Warehouse

    Riall, Rebecca L.; Marincioni, Fausto; Lightsom, Frances L.

    2004-01-01

    The U.S. Geological Survey developed a content metadata standard to meet the demands of organizing electronic resources in the marine sciences for a broad, heterogeneous audience. These metadata standards are used by the Marine Realms Information Bank project, a Web-based public distributed library of marine science from academic institutions and government agencies. The development and deployment of this metadata standard serve as a model, complete with lessons about mistakes, for the creation of similarly specialized metadata standards for digital libraries.

  9. Deliberate presleep control of dream content: an experimental study.

    PubMed

    Griffin, M L; Folkes, D

    1977-10-01

    29 subjects attempted, over a period of 10 nights, to influence their dream using techniques described in Garfield's book, Creative Dreaming (1974). A target suggestion was selected from a list of six suggestions compiled by, or for, each subject. Subjects kept daily records during the experiment both of their efforts at dream influence and of the dreams they recalled. Four judges attempted to identify from the dream material the target suggestion on each subject's suggestion list. The results indicated that the judges were unable to do so at better than chance levels. Thus analysis indicated no reliable evidence that conscious presleep suggestions become incorporated into dream content.

  10. Melanocortin 5 receptor signaling and internalization: role of MAPK/ERK pathway and β-arrestins 1/2.

    PubMed

    Rodrigues, Adriana R; Almeida, Henrique; Gouveia, Alexandra M

    2012-09-25

    The Melanocortin 5 receptor (MC5R) is a G-protein coupled receptor (GPCR) that exhibits high affinity for α-MSH. Here we present evidence for MC5R-GFP internalization and subsequent recycling to cell surface, in α-MSH-stimulated HeLa cells. This melanocortin induces a biphasic activation of ERK1/2 with an early peak at 15min, a G(i)-protein driven, β-arrestins 1/2 independent process, and a late sustained activation that is regulated by β-arrestins 1/2. ERK1/2 lead to downstream phosphorylation of 90-kDa ribosomal S6 kinases (p90RSK) and mitogen- and stress-activated protein kinase 1 (MSK1). Only a small fraction (10%) of phosphorylated p90RSK and ERK1/2 translocates to the nucleus inducing c-Fos expression. α-MSH also activates CREB through cAMP/PKA pathway. In 3T3-L1 adipocytes, where MC5R is endogenously expressed, α-MSH also induces phosphorylation and cytosolic retention of the same signaling molecules. These findings provide new evidence on the signaling mechanisms underlying MC5R biological response to α-MSH.

  11. Smoothened determines β-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium.

    PubMed

    Pal, Kasturi; Hwang, Sun-Hee; Somatilaka, Bandarigoda; Badgandi, Hemant; Jackson, Peter K; DeFea, Kathryn; Mukhopadhyay, Saikat

    2016-03-28

    Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves β-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161-β-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment. PMID:27002170

  12. A unique PDZ domain and arrestin-like fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer

    PubMed Central

    Gallon, Matthew; Clairfeuille, Thomas; Steinberg, Florian; Mas, Caroline; Ghai, Rajesh; Sessions, Richard B.; Teasdale, Rohan D.; Collins, Brett M.; Cullen, Peter J.

    2014-01-01

    The sorting nexin 27 (SNX27)-retromer complex is a major regulator of endosome-to-plasma membrane recycling of transmembrane cargos that contain a PSD95, Dlg1, zo-1 (PDZ)-binding motif. Here we describe the core interaction in SNX27-retromer assembly and its functional relevance for cargo sorting. Crystal structures and NMR experiments reveal that an exposed β-hairpin in the SNX27 PDZ domain engages a groove in the arrestin-like structure of the vacuolar protein sorting 26A (VPS26A) retromer subunit. The structure establishes how the SNX27 PDZ domain simultaneously binds PDZ-binding motifs and retromer-associated VPS26. Importantly, VPS26A binding increases the affinity of the SNX27 PDZ domain for PDZ- binding motifs by an order of magnitude, revealing cooperativity in cargo selection. With disruption of SNX27 and retromer function linked to synaptic dysfunction and neurodegenerative disease, our work provides the first step, to our knowledge, in the molecular description of this important sorting complex, and more broadly describes a unique interaction between a PDZ domain and an arrestin-like fold. PMID:25136126

  13. Constrained novelty search: a study on game content generation.

    PubMed

    Liapis, Antonios; Yannakakis, Georgios N; Togelius, Julian

    2015-01-01

    Novelty search is a recent algorithm geared toward exploring search spaces without regard to objectives. When the presence of constraints divides a search space into feasible space and infeasible space, interesting implications arise regarding how novelty search explores such spaces. This paper elaborates on the problem of constrained novelty search and proposes two novelty search algorithms which search within both the feasible and the infeasible space. Inspired by the FI-2pop genetic algorithm, both algorithms maintain and evolve two separate populations, one with feasible and one with infeasible individuals, while each population can use its own selection method. The proposed algorithms are applied to the problem of generating diverse but playable game levels, which is representative of the larger problem of procedural game content generation. Results show that the two-population constrained novelty search methods can create, under certain conditions, larger and more diverse sets of feasible game levels than current methods of novelty search, whether constrained or unconstrained. However, the best algorithm is contingent on the particularities of the search space and the genetic operators used. Additionally, the proposed enhancement of offspring boosting is shown to enhance performance in all cases of two-population novelty search.

  14. Moral content and assignment marking: an exploratory study.

    PubMed

    Lipscomb, Martin; Snelling, Paul C

    2006-08-01

    This small scale exploratory survey investigates the relation between the moral content of student assignments and potential marking behaviour by lecturers. A questionnaire containing a number of deliberately discriminatory statements was sent to a sample of nurse academics, and participants were asked to indicate whether the inclusion of these statements might affect the mark awarded. The sample was too small to undertake tests of statistical significance. However, the results tentatively suggest that a majority of participants would penalise assignments that contain morally questionable statements, and many would cite the code of professional conduct in justification. There appeared to be little difference in the response to statements that offered opinions (normative) or described actions (descriptive). However, differences between responses to statements discriminating against different groups were apparent, with racist statements being penalised more often and with greater severity than other categories. Qualitative data support the quantitative data. Several areas for discussion are identified, including the academic status of nursing, the use of the code of professional conduct, the moral claims made for nurses, a potential 'hierarchy of wrongness', student self censorship, and inconsistency in marking. Further research is justified. PMID:16458999

  15. Strategic and Organisational Considerations in Planning Content and Language Integrated Learning: A Study on the Coordination between Content and Language Teachers

    ERIC Educational Resources Information Center

    Pavón Vázquez, Víctor; Ávila López, Javier; Gallego Segador, Arturo; Espejo Mohedano, Roberto

    2015-01-01

    Content and language integrated learning (CLIL) is generally recognised as a fruitful example of bilingual education. However, success in CLIL may not be straightforward and may require the establishment of coordination between content and language teachers. The aim of this study is to investigate if content and language teachers are able to plan…

  16. I, Pronoun: A Study of Formality in Online Content

    ERIC Educational Resources Information Center

    Thayer, Alexander; Evans, Mary B.; McBride, Alicia A.; Queen, Matt; Spyridakis, Jan H.

    2010-01-01

    This article presents the results of a study that investigated readers' perceptions of tone formality in online text passages. The study found that readers perceived text passages to be less formal when they contained personal pronouns, active voice verbs, informal punctuation, or verb contractions. The study reveals that professional…

  17. Receptor sequestration in response to β-arrestin-2 phosphorylation by ERK1/2 governs steady-state levels of GPCR cell-surface expression

    PubMed Central

    Paradis, Justine S.; Ly, Stevenson; Blondel-Tepaz, Élodie; Galan, Jacob A.; Beautrait, Alexandre; Scott, Mark G. H.; Enslen, Hervé; Marullo, Stefano; Roux, Philippe P.; Bouvier, Michel

    2015-01-01

    MAPKs are activated in response to G protein-coupled receptor (GPCR) stimulation and play essential roles in regulating cellular processes downstream of these receptors. However, very little is known about the reciprocal effect of MAPK activation on GPCRs. To investigate possible crosstalk between the MAPK and GPCRs, we assessed the effect of ERK1/2 on the activity of several GPCR family members. We found that ERK1/2 activation leads to a reduction in the steady-state cell-surface expression of many GPCRs because of their intracellular sequestration. This subcellular redistribution resulted in a global dampening of cell responsiveness, as illustrated by reduced ligand-mediated G-protein activation and second-messenger generation as well as blunted GPCR kinases and β-arrestin recruitment. This ERK1/2-mediated regulatory process was observed for GPCRs that can interact with β-arrestins, such as type-2 vasopressin, type-1 angiotensin, and CXC type-4 chemokine receptors, but not for the prostaglandin F receptor that cannot interact with β-arrestin, implicating this scaffolding protein in the receptor’s subcellular redistribution. Complementation experiments in mouse embryonic fibroblasts lacking β-arrestins combined with in vitro kinase assays revealed that β-arrestin-2 phosphorylation on Ser14 and Thr276 is essential for the ERK1/2-promoted GPCR sequestration. This previously unidentified regulatory mechanism was observed after constitutive activation as well as after receptor tyrosine kinase- or GPCR-mediated activation of ERK1/2, suggesting that it is a central node in the tonic regulation of cell responsiveness to GPCR stimulation, acting both as an effector and a negative regulator. PMID:26324936

  18. A National Study of Training Content and Activities for Faculty Development for Online Teaching

    ERIC Educational Resources Information Center

    Meyer, Katrina A.; Murrell, Vicki S.

    2014-01-01

    This article presents the results of a national study of 39 higher education institutions that collected information about their practices for faculty development for online teaching and particularly the content and training activities used during 2011-2012. This study found that the most frequently offered training content (97% of the…

  19. Connecting Content, Context, and Communication in a Sixth-Grade Social Studies Class through Political Cartoons

    ERIC Educational Resources Information Center

    Gallavan, Nancy P.; Webster-Smith, Angela; Dean, Sheila S.

    2012-01-01

    Sixth-grade students are challenged in understanding social studies content relevant to particular contexts, then connecting the content and context to their contemporary lives while communicating new knowledge to peers and teachers. Using political cartoons published after September 11, 2001, one sixth-grade social studies teacher designed…

  20. Preliminary assestment of lint cotton water content in gin-drying temperature studies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prior studies to measure total water (free and bound) in lint cotton by Karl Fischer Titration showed the method is more accurate and precise than moisture content by standard oven drying. The objective of the current study was to compare the moisture and total water contents from five cultivars de...

  1. A Comparative Study of Six Decades of General Science Textbooks: Evaluating the Evolution of Science Content

    ERIC Educational Resources Information Center

    Lewis, Anna

    2008-01-01

    This study examined science textbooks over time to better understand the "science content" expectations that the U.S. educational system deems appropriate for 8th and 9th grade science students. The study attempted to answer the questions: (1) What specific science content has been presented via the textbook from 1952 to 2008? (2) Within which…

  2. ICT Student Teachers' Pedagogical Content Knowledge: A Case Study

    ERIC Educational Resources Information Center

    Yilmaz, Nuray Parlak

    2016-01-01

    This study aimed to identify the difficulties that information technology student teachers have in teaching concepts. This qualitative study was carried out with 12 student teachers. The student teachers were fourth-year students enrolled in the Special Teaching Methods II course in the spring semester of the 2010-2011 academic year. The research…

  3. Content Analysis of Turkish Studies about the Multiple Intelligences Theory

    ERIC Educational Resources Information Center

    Saban, Ahmet

    2009-01-01

    Recently, there has been a significant increase in the number of multiple intelligences (MI) studies in Turkey. Consequently, a systematic analysis of these studies is crucial in order to be able to see the present situation and future trends in the field of education. By this way, it is also hoped that the current analysis will offer an avenue…

  4. Studying the information content of TMDs using Monte Carlo generators

    SciTech Connect

    Avakian, H.; Matevosyan, H.; Pasquini, B.; Schweitzer, P.

    2015-02-05

    Theoretical advances in studies of the nucleon structure have been spurred by recent measurements of spin and/or azimuthal asymmetries worldwide. One of the main challenges still remaining is the extraction of the parton distribution functions, generalized to describe transverse momentum and spatial distributions of partons from these observables with no or minimal model dependence. In this topical review we present the latest developments in the field with emphasis on requirements for Monte Carlo event generators, indispensable for studies of the complex 3D nucleon structure, and discuss examples of possible applications.

  5. Virtual Worlds in Distance Education: A Content Analysis Study

    ERIC Educational Resources Information Center

    Wang, Feihong; Lockee, Barbara B.

    2010-01-01

    The three-dimensional (3D) virtual world is one of the current innovations that has been discussed extensively as the potential medium for online distance education. Despite the heated discussion on the possible applications of 3D virtual worlds for distance education, empirical studies were hard to locate, and little has been written about how…

  6. Will More Diversified Staffs Diversify Newspaper Content? A Pilot Study.

    ERIC Educational Resources Information Center

    Fedler, Fred; and Others

    A pilot study asked 94 students enrolled in introductory newswriting classes at three separate universities to evaluate 18 news stories. About half the stories concerned topics that proponents of multiculturalism have suggested would receive more emphasis if newspapers employed more women and minorities: topics such as breast cancer, divorce,…

  7. 23 CFR 650.117 - Content of design studies.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... reasons, when applicable, why FEMA criteria (44 CFR 60.3, formerly 24 CFR 1910.3) are demonstrably... BRIDGES, STRUCTURES, AND HYDRAULICS Location and Hydraulic Design of Encroachments on Flood Plains § 650...) Studies by highway agencies shall contain: (1) The hydrologic and hydraulic data and design...

  8. Secondary Social Studies: Arkansas Public School Course Content Guide.

    ERIC Educational Resources Information Center

    Arkansas State Dept. of Education, Little Rock.

    This guide is offered as a framework on which a comprehensive curriculum can be built. Within each subject area and grade level, skills have been identified at three instructional levels: basic, developmental, and extension. The study of political and economic systems, citizenship rights and responsibilities, and the foundations of the U.S.…

  9. Integration of Basic Skills into Social Studies Content.

    ERIC Educational Resources Information Center

    Lunstrum, John P.; Irvin, Judith L.

    1981-01-01

    A basic skills model is presented which stresses the skills of writing, reading, study, and research for elementary school pupils. The model focuses on lesson background, the purpose of the reading, independent reading, follow-up discussion, developing related skills, and extending and applying ideas. A lesson about the 1910 British expedition to…

  10. A Quantitative Study of High School Yearbook Content.

    ERIC Educational Resources Information Center

    Krepel, Wayne J.; DuVall, Charles R.

    The purpose of this study was to analyze high school yearbooks, relative to quantitative page allotments, when classified by the size of the community, the type of socioeconomic environment of the school, and the size of the graduating class. A normative survey was conducted using a questionnaire requesting the respondent to furnish a copy of the…

  11. Content analysis of 186 descriptive case studies of hospitalized children.

    PubMed

    Barnes, C M; Bandak, A G; Beardslee, C I

    1990-01-01

    One hundred eighty-six single case studies of sick infants, children, and adolescents were analyzed to provide a substantial descriptive data base of children's responses to illness, hospitalization, and treatment over a span of more than 20 years. Units of theory of coping behavior are identified as a first phase for adding new knowledge to the field of pediatric nursing. Indicators of the significance of the data base are included.

  12. A Trend Study of Advertising Content Used by Banks Before, During and After a Banking Collapse.

    ERIC Educational Resources Information Center

    De Riemer, Cynthia; Baxter, Richard L.

    To examine the content of newspaper advertisements used by banks before, during, and after a major bank collapse, issues of the Knoxville (Tennessee) "News Sentinel" from 1982, 1983, and 1984 were analyzed for bank sponsored product and nonproduct advertisements. These advertisements were studied for type, size, and content relating to categories…

  13. New Histories for a New State: A Study of History Textbook Content in Northern Ireland

    ERIC Educational Resources Information Center

    Terra, Luke

    2014-01-01

    This study examined the changing content of history textbooks in Northern Ireland, drawing on a sample of 15 textbooks published from 1968 to 2010. Findings from the content and narrative analysis indicated that following the introduction of the Northern Ireland Curriculum in 1991, history textbooks shifted from a narrative to source-driven…

  14. Content-Based Instruction in Primary and Secondary School Settings. Case Studies in TESOL Practice Series

    ERIC Educational Resources Information Center

    Kaufman, Dorit, Ed.; Crandall, JoAnn, Ed.

    2005-01-01

    Content-based instruction (CBI) challenges English language educators to teach English using materials that learners encounter in their regular subject-area classes. This volume helps ESL and EFL teachers meet that challenge by providing them with creative ways to integrate English language learning with the content that students study at primary…

  15. A Case Study of Pre-Service Chemistry Teachers' Pedagogical Content Knowledge Development.

    ERIC Educational Resources Information Center

    Tuan, Hsiao-lin; And Others

    The goals of secondary preservice science teacher education are to prepare future science teachers with competency in both science content knowledge and science teaching ability. The study reported in this paper investigated the development of pedagogical content knowledge of three preservice chemistry teachers in Taiwan during one year of a…

  16. Evaluation of the Use of Team Teaching for Delivering Sensitive Content: A Pilot Study

    ERIC Educational Resources Information Center

    Kerridge, Joanna; Kyle, Gaye; Marks-Maran, Diane

    2009-01-01

    Many programmes in further and higher education contain sensitive areas of content, such as diversity, racism, power and privilege, breaking bad news, counselling, sex education and ethical decision making. Team teaching may be a useful method for delivering sensitive areas of course content. This article presents a pilot study that was undertaken…

  17. β-Arrestin1 and distinct CXCR4 structures are required for stromal derived factor-1 to downregulate CXCR4 cell-surface levels in neuroblastoma.

    PubMed

    Clift, Ian C; Bamidele, Adebowale O; Rodriguez-Ramirez, Christie; Kremer, Kimberly N; Hedin, Karen E

    2014-04-01

    CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) located on the cell surface that signals upon binding the chemokine stromal derived factor-1 (SDF-1; also called CXCL 12). CXCR4 promotes neuroblastoma proliferation and chemotaxis. CXCR4 expression negatively correlates with prognosis and drives neuroblastoma growth and metastasis in mouse models. All functions of CXCR4 require its expression on the cell surface, yet the molecular mechanisms that regulate CXCR4 cell-surface levels in neuroblastoma are poorly understood. We characterized CXCR4 cell-surface regulation in the related SH-SY5Y and SK-N-SH human neuroblastoma cell lines. SDF-1 treatment caused rapid down-modulation of CXCR4 in SH-SY5Y cells. Pharmacologic activation of protein kinase C similarly reduced CXCR4, but via a distinct mechanism. Analysis of CXCR4 mutants delineated two CXCR4 regions required for SDF-1 treatment to decrease cell-surface CXCR4 in neuroblastoma cells: the isoleucine-leucine motif at residues 328 and 329 and residues 343-352. In contrast, and unlike CXCR4 regulation in other cell types, serines 324, 325, 338, and 339 were not required. Arrestin proteins can bind and regulate GPCR cell-surface expression, often functioning together with kinases such as G protein-coupled receptor kinase 2 (GRK2). Using SK-N-SH cells which are naturally deficient in β-arrestin1, we showed that β-arrestin1 is required for the CXCR4 343-352 region to modulate CXCR4 cell-surface expression following treatment with SDF-1. Moreover, GRK2 overexpression enhanced CXCR4 internalization, via a mechanism requiring both β-arrestin1 expression and the 343-352 region. Together, these results characterize CXCR4 structural domains and β-arrestin1 as critical regulators of CXCR4 cell-surface expression in neuroblastoma. β-Arrestin1 levels may therefore influence the CXCR4-driven metastasis of neuroblastoma as well as prognosis. PMID:24452472

  18. Prolonged stimulation of μ-opioid receptors produces β-arrestin-2-mediated heterologous desensitization of α(2)-adrenoceptor function in locus ceruleus neurons.

    PubMed

    Dang, Vu C; Chieng, Billy C; Christie, MacDonald J

    2012-09-01

    Prolonged agonist stimulation of the μ-opioid receptor (MOR) initiates receptor regulatory events that rapidly attenuate receptor-mediated signaling (homologous desensitization). Emerging evidence suggests that persistent MOR stimulation can also reduce responsiveness of effectors to other G-protein-coupled receptors, termed heterologous desensitization. However, the mechanisms by which heterologous desensitization is triggered by MOR stimulation are unclear. This study used whole-cell patch-clamp recordings of ligand activated G-protein-activated inwardly rectifying potassium channel currents in mouse brain slices containing locus ceruleus (LC) neurons to determine the effects of prolonged stimulation of MOR on α(2)-adrenoceptor (α(2)-AR) function. The results show distinct and sequential development of homologous and heterologous desensitization during persistent stimulation of MOR in LC neurons with Met(5)-enkephalin (ME). ME stimulation of MOR promoted rapid homologous desensitization that reached a steady state after 5 min and partially recovered over 30 min. Longer stimulation of MOR (10 min) induced heterologous desensitization of α(2)-AR function that exhibited slower recovery than homologous desensitization. Heterologous (but not homologous) desensitization required β-arrestin-2 (βarr-2) because it was nearly abolished in βarr-2-knockout (ko) mice. Heterologous (but not homologous) desensitization was also prevented by inhibition of ERK1/2 and c-Src signaling in wild-type (wt) mouse LC neurons. Heterologous desensitization may be physiologically relevant during exposure to high doses of opioids because α(2)-AR-mediated slow inhibitory postsynaptic currents were depressed in wt but not βarr-2 ko LC neurons after prolonged exposure to opioids. Together, these findings demonstrate a novel mechanism by which βarr-2 can regulate postsynaptic responsiveness to neurotransmitter release.

  19. The Stem Cell-Expressed Receptor Lgr5 Possesses Canonical and Functionally Active Molecular Determinants Critical to β-arrestin-2 Recruitment

    PubMed Central

    Snyder, Joshua C.; Rochelle, Lauren K.; Barak, Larry S.; Caron, Marc G.

    2013-01-01

    Lgr5 is a membrane protein related to G protein-coupled receptors (GPCR)s whose expression identifies stem cells in multiple tissues and is strongly correlated with cancer. Despite the recent identification of endogenous ligands for Lgr5, its mode of signaling remains enigmatic. The ability to couple to G proteins and βarrestins are classical molecular behaviors of GPCRs that have yet to be observed for Lgr5. Therefore, the goal of this study was to determine if Lgr5 can engage a classical GPCR behavior and elucidate the molecular determinants of this process. Structural analysis of Lgr5 revealed several motifs consistent with its ability to recruit βarr2. Among them, a “SSS” serine cluster located at amino acid position 873-875 within the C-terminal tail (C-tail), is in a region consistent with other GPCRs that bind βarr2 with high-affinity. To test its functionality, a ligand-independent βarr2 translocation assay was implemented. We show that Lgr5 recruits βarr2 and that the “SSS” amino acids (873-875) are absolutely critical to this process. We also demonstrate that for full efficacy, this cluster requires other Lgr5 C-tail serines that were previously shown to be important for constitutive and βarr2 independent internalization of Lgr5. These data are proof of principle that a classical GPCR behavior can be manifested by Lgr5. The existence of alternative ligands or missing effectors of Lgr5 that scaffold this classical GPCR behavior and the downstream signaling pathways engaged should be considered. Characterizing Lgr5 signaling will be invaluable for assessing its role in tissue maintenance, repair, and disease. PMID:24386388

  20. Teachers' Professional Development: A Content Analysis about the Tendencies in Studies

    ERIC Educational Resources Information Center

    Yurtseven, Nihal; Bademcioglu, Mehtap

    2016-01-01

    The purpose of this study is to carry out a content analysis about the studies on teachers' professional development and to determine the tendencies in these studies. Within this scope, 60 studies that were registered to Turkish National Thesis Centre and ProQuest database between the years 2005-2015 were examined. Of the 60 studies, 37 of them…

  1. 78 FR 42085 - Draft Guidance for Industry on Pediatric Study Plans: Content of and Process for Submitting...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-15

    ...: Content of and Process for Submitting Initial Pediatric Study Plans and Amended Pediatric Study Plans... Plans: Content of and Process for Submitting Initial Pediatric Study Plans and Amended Pediatric Study... draft guidance for industry entitled ``Pediatric Study Plans: Content of and Process for...

  2. β-arrestin-2-biased agonism of delta opioid receptors sensitizes transient receptor potential vanilloid type 1 (TRPV1) in primary sensory neurons.

    PubMed

    Rowan, Matthew P; Szteyn, Kalina; Doyle, Allison P; Gomez, Ruben; Henry, Michael A; Jeske, Nathaniel A

    2014-01-01

    Despite advances in understanding the signaling mechanisms involved in the development and maintenance of chronic pain, the pharmacologic treatment of chronic pain has seen little advancement. Agonists at the mu opioid receptor (MOPr) continue to be vital in the treatment of many forms of chronic pain, but side-effects limit their clinical utility and range from relatively mild, such as constipation, to major, such as addiction and dependence. Additionally, chronic activation of MOPr results in pain hypersensitivity known as opioid-induced hyperalgesia (OIH), and we have shown recently that recruitment of β-arrestin2 to MOPr, away from transient potential vanilloid eceptor type 1 (TRPV1) in primary sensory neurons contributes to this phenomenon. The delta opioid receptor (DOPr) has become a promising target for the treatment of chronic pain, but little is known about the effects of chronic activation of DOPr on nociceptor sensitivity and OIH. Here we report that chronic activation of DOPr by the DOPr-selective agonist, SNC80, results in the sensitization of TRPV1 and behavioral signs of OIH via β-arrestin2 recruitment to DOPr and away from TRPV1. Conversely, chronic treatment with ARM390, a DOPr-selective agonist that does not recruit β-arrestin2, neither sensitized TRPV1 nor produced OIH. Interestingly, the effect of SNC80 to sensitize TRPV1 is species-dependent, as rats developed OIH but mice did not. Taken together, the reported data identify a novel side-effect of chronic administration of β-arrestin2-biased DOPr agonists and highlight the importance of potential species-specific effects of DOPr agonists.

  3. Detecting mineral content in turbid medium using nonlinear Raman imaging: feasibility study

    PubMed Central

    Arora, Rajan; Petrov, Georgi I.; Noojin, Gary D.; Thomas, Patrick A.; Denton, Michael L.; Rockwell, Benjamin A.; Thomas, Robert J.; Yakovlev, Vladislav V.

    2012-01-01

    Osteoporosis is a bone disease characterized by reduced mineral content with resulting changes in bone architecture, which in turn increases the risk of bone fracture. Raman spectroscopy has an intrinsic sensitivity to the chemical content of the bone, but its application to study bones in vivo is limited due to strong optical scattering in tissue. It has been proposed that Raman excitation with photoacoustic detection can successfully address the problem of chemically specific imaging in deep tissue. In this report, the principal possibility of photoacoustic imaging for detecting mineral content is evaluated. PMID:22337734

  4. A study of the metal content of municipal solid waste. Final report

    SciTech Connect

    Churney, K.L.; Domalski, E.S.

    1998-01-01

    Knowledge of the content of toxic components, so called pollutant precursors, in the municipal solid waste (MSW) stream is essential to development of the strategies for source reduction and reuse, recycling, composting and disposal. Data are scarce; trends in composition for any locality even more so. In a previous study the total and water soluble chlorine content of the components of municipal solid waste were determined from sampling studies at two sites, Baltimore County, MD, and Brooklyn, NY, each for a five day period. The total sulfur content of the combined combustible components was also determined. Because of the scarcity of data and synergistic effects, it seemed appropriate to determine the heavy metal content of the preceding material prior to its disposal. The metals chosen were the so-called priority pollutant metals (PPM): antimony, arsenic, beryllium, cadmium, chromium, copper, lead, mercury, nickel, selenium, silver, thallium, and zinc.

  5. Mitochondrial DNA content contributes to healthy aging in Chinese: a study from nonagenarians and centenarians.

    PubMed

    He, Yong-Han; Lu, Xiang; Wu, Huan; Cai, Wang-Wei; Yang, Li-Qin; Xu, Liang-You; Sun, Hong-Peng; Kong, Qing-Peng

    2014-07-01

    Mitochondrial DNA (mtDNA) content plays an important role in energy production and sustaining normal physiological function. A decline in the mtDNA content and subsequent dysfunction cause various senile diseases, with decreasing mtDNA content observed in the elderly individuals with age-related diseases. In contrast, the oldest old individuals, for example, centenarians, have a delayed or reduced prevalence of these diseases, suggesting centenarians may have a different pattern of the mtDNA content, enabling them to keep normal mitochondrial functions to help delay or escape senile diseases. To test this hypothesis, a total of 961 subjects, consisting of 424 longevity subjects and 537 younger control subjects from Hainan and Sichuan provinces of China, were recruited for this study. The mtDNA content was found to be inversely associated with age among the age of group 40-70 years. Surprisingly, no reduction of mtDNA content was observed in nonagenarians and centenarians; instead, these oldest old showed a significant increase than the elderly people aged between 50 and 70 years. The results suggest the higher mtDNA content may convey a beneficial effect to the longevity of people through assuring sufficient energy supply.

  6. The Relationship Between Sexual Content on Mass Media and Social Media: A Longitudinal Study.

    PubMed

    Vandenbosch, Laura; van Oosten, Johanna M F; Peter, Jochen

    2015-12-01

    The goal of this study was to investigate whether exposure to sexual reality television content and Internet pornography (IP) is related to sexual self-presentation on social media. Based on a two-wave panel survey among 1,765 adolescents aged 13-17 years, we found that watching sexual reality television content stimulated adolescents to produce and distribute sexual images of themselves on social media. In turn, sexual self-presentation on social media led adolescents to watch sexual reality television content more frequently. These relationships were similar among boys and girls. No reciprocal relationship between exposure to IP and boys' and girls' sexual self-presentation on social media was found. The results suggest that sexual content in mainstream mass media may predict adolescents' sexually oriented behavior on social media and vice versa. Moreover, adolescents seem to differentiate between types of sexual content (i.e., mainstream versus more explicit sexual content) when incorporating sexual media content in their sexual behavior online.

  7. The Relationship Between Sexual Content on Mass Media and Social Media: A Longitudinal Study.

    PubMed

    Vandenbosch, Laura; van Oosten, Johanna M F; Peter, Jochen

    2015-12-01

    The goal of this study was to investigate whether exposure to sexual reality television content and Internet pornography (IP) is related to sexual self-presentation on social media. Based on a two-wave panel survey among 1,765 adolescents aged 13-17 years, we found that watching sexual reality television content stimulated adolescents to produce and distribute sexual images of themselves on social media. In turn, sexual self-presentation on social media led adolescents to watch sexual reality television content more frequently. These relationships were similar among boys and girls. No reciprocal relationship between exposure to IP and boys' and girls' sexual self-presentation on social media was found. The results suggest that sexual content in mainstream mass media may predict adolescents' sexually oriented behavior on social media and vice versa. Moreover, adolescents seem to differentiate between types of sexual content (i.e., mainstream versus more explicit sexual content) when incorporating sexual media content in their sexual behavior online. PMID:26588715

  8. Making Connections among Student Learning, Content, and Teaching: Teacher Talk Paths in Elementary Mathematics Lesson Study

    ERIC Educational Resources Information Center

    Murata, Aki; Bofferding, Laura; Pothen, Bindu E.; Taylor, Megan W.; Wischnia, Sarah

    2012-01-01

    This study investigated how elementary teachers in a mathematics lesson study made sense of student learning, teaching, and content, as related to using representations in teaching multidigit subtraction, and how changes occurred over time in their talk and practice. The lesson-study process paved a group talk path along which teacher talk shifted…

  9. Preliminary study: fibre content in pet rabbit diets, crude fibre versus total dietary fibre.

    PubMed

    Molina, J; Martorell, J; Hervera, M; Pérez-Accino, J; Fragua, V; Villaverde, C

    2015-04-01

    Fibre is an important nutrient for rabbit health, and, on commercial pet rabbit packaging, it is labelled as crude fibre (CF). In several species, it is considered that CF is not an accurate representation of the fibre content in feedstuffs. The objective of this study was to compare the CF stated on the label (CFL) with laboratory analysis of CF (CFA) and the analysed content of total dietary fibre (TDF) in different commercial pet rabbit feeds. We selected 15 commercial diets and analysed CF and TDF. A mixed model was used to evaluate differences between CFL, CFA and TDF, and linear regression was performed to study the correlation between CFL and CFA with TDF. CFA and CFL were not significantly different (p = 0.836) in the feeds studied, and both were lower than TDF (p < 0.001). The correlations between TDF and both CFA and CFL were significant (p < 0.001 and p = 0.02, respectively), but the correlation was better with CFA (R = 0.86) than with CFL (R = 0.53). As expected, TDF content was higher than CF content, an average of two times. These results suggest that the CF content in rabbit diets reported on the label is not an appropriate indicator of their total fibre content, although further work with a larger sample size is required to confirm these results.

  10. [The Study of the Spectral Model for Estimating Pigment Contents of Tobacco Leaves in Field].

    PubMed

    Ren, Xiao; Lao, Cai-lian; Xu, Zhao-li; Jin, Yan; Guo, Yan; Li, Jun-hui; Yang, Yu-hong

    2015-06-01

    Fast and non-destructive measurements of tobacco leaf pigment contents by spectroscopy in situ in the field has great significance in production guidance for nutrient diagnosis and growth monitoring of tobacco in vegetative growth stage, and it is also very important for the quality evaluation of tobacco leaves in mature stage. The purpose of this study is to estimate the chlorophyll and carotenoid contents of tobacco leaves using tobacco leaf spectrum collected in the field. Reflectance spectrum of tobacco leaves in vegetative growth stage and mature stage were collected in situ in the field and the pigment contents of tobacco leaf samples were measured in this study, taking the tobacco leaf samples collected in each and both stages as modeling sets respectively, and using the methods of support vector machine (SVM) and spectral indice to establish the pigment content estimation models, and then compare the prediction performance of the models built by different methods. The study results indicated that the difference of estimation performance by each stage or mixed stages is not significant. For chlorophyll content, SVM and spectral indice modeling methods can both have a well estimation performance, while for carotenoid content, SVM modeling method has a better estimation performance than spectral indice. The coefficient of determination and the root mean square error of SVM model for estimating tobacco leaf chlorophyll content by each stage were 0.867 6 and 0.014 7, while the coefficient of determination and the root mean square error of SVM model for estimating tobacco leaf chlorophyll content by mixed stages were 0.898 6 and 0.012 3; The coefficient of determination and the root mean square error for estimating tobacco leaf carotenoid content by each stage were 0.861 4 and 0.002 5, while the coefficient of determination and the root mean square error of SVM model for estimating tobacco leaf carotenoid content by mixed stages were 0.839 9 and 0.002 5. The

  11. The studying of washing of arsenic and sulfur from coals having different ranges of arsenic contents

    USGS Publications Warehouse

    Wang, M.; Song, D.; Zheng, B.; Finkelman, R.B.; ,

    2008-01-01

    To study the effectiveness of washing in removal of arsenic and sulfur from coals with different ranges of arsenic concentration, coal was divided into three groups on the basis of arsenic content: 0-5.5 mg/kg, 5.5 mg/kg-8.00 mg/kg, and over 8.00 mg/kg. The result shows that the arsenic in coals with higher arsenic content occurs mainly in an inorganic state and can be relatively easily removed. Arsenic removal is very difficult and less complete when the arsenic content is lower than 5.5 mg/kg because most of this arsenic is in an organic state. There is no relationship between washing rate of total sulfur and arsenic content, but the relationship between the washing rate of total sulfur and percent of organic sulfur is very strong. ?? 2008 New York Academy of Sciences.

  12. The studying of washing of arsenic and sulfur from coals having different ranges of arsenic contents

    SciTech Connect

    Mingshi Wang; Dangyu Song; Baoshan Zheng; R.B. Finkelman

    2008-10-15

    To study the effectiveness of washing in removal of arsenic and sulfur from coals with different ranges of arsenic concentration, coal was divided into three groups on the basis of arsenic content: 0-5.5 mg/kg, 5.5 mg/kg-8.00 mg/kg, and over 8.00 mg/kg. The result shows that the arsenic in coals with higher arsenic content occurs mainly in an inorganic state and can be relatively easily removed. Arsenic removal is very difficult and less complete when the arsenic content is lower than 5.5 mg/kg because most of this arsenic is in an organic state. There is no relationship between washing rate of total sulfur and arsenic content, but the relationship between the washing rate of total sulfur and percent of organic sulfur is very strong.

  13. Developing fair tests for mathematics curriculum comparison studies: the role of content analyses

    NASA Astrophysics Data System (ADS)

    Chávez, Óscar; Papick, Ira; Ross, Daniel J.; Grouws, Douglas A.

    2011-12-01

    This article describes the process of development of assessment instruments for a three-year longitudinal comparative study that focused on evaluating American high school students' mathematics learning from two distinct approaches to content organization: curriculum built around a sequence of three full-year courses (Algebra 1, Geometry, and Algebra 2) and a sequence of integrated mathematics courses (algebra and geometry content, together with functions, data analysis, and discrete mathematics is integrated each year). The study was conducted in six school districts in five states involving over 4,000 students from schools that were using both curricular approaches but with different groups of students. In order to develop assessment instruments that were not biased towards either of the two curriculum programs (Fair Tests), an iterative process of content analyses, identification of common topics, internal and external reviews, pilot tests, and revisions was followed, resulting in five tests that were used in the three years of the study. Results indicate that these tests have solid discrimination properties and address adequately mathematics content common to both secondary curriculum programs. The corresponding scoring rubrics are highly reliable, with interrater reliability above 94% for all tests. Mathematics education researchers involved in curriculum comparison studies need to conduct content analyses of the curriculum materials under study in order to identify salient relationships between curriculum programs and student outcomes.

  14. Teaching cause-effect text structure through social studies content to at-risk second graders.

    PubMed

    Williams, Joanna P; Nubla-Kung, Abigail M; Pollini, Simonne; Stafford, K Brooke; Garcia, Amaya; Snyder, Anne E

    2007-01-01

    This study evaluated the effectiveness of a comprehension program integrated with social studies instruction designed for at-risk second graders. The program included instruction in cause-effect text structure, emphasizing clue words, generic questions, graphic organizers, and the close analysis of specially constructed cause-effect target paragraphs. This program was compared (a) to a content-only program that focused only on social studies and did not include text structure instruction and (b) to a no-instruction control. Fifteen classroom teachers, randomly assigned to treatment, provided the instruction. The program improved the comprehension of instructional cause-effect texts, and there were transfer effects on some comprehension measures. The performance of the 2 instructed groups did not differ on any of the content measures, indicating that such integrated instruction can be accomplished without a loss in the amount of content acquired. This study supports our previous findings on the effectiveness of explicit instruction at the primary-grade level.

  15. 17beta-estradiol-mediated neuroprotection and ERK activation require a pertussis toxin-sensitive mechanism involving GRK2 and beta-arrestin-1.

    PubMed

    Dominguez, Reymundo; Hu, Eric; Zhou, Miou; Baudry, Michel

    2009-04-01

    17-beta-Estradiol (E2) is a steroid hormone involved in numerous bodily functions, including several brain functions. In particular, E2 is neuroprotective against excitotoxicity and other forms of brain injuries, a property that requires the extracellular signal-regulated kinase (ERK) pathway and possibly that of other signaling molecules. The mechanism and identity of the receptor(s) involved remain unclear, although it has been suggested that E2 receptor alpha (ERalpha) and G proteins are involved. We, therefore, investigated whether E2-mediated neuroprotection and ERK activation were linked to pertussis toxin (PTX)-sensitive G-protein-coupled effector systems. Biochemical and image analysis of organotypic hippocampal slices and cortical neuronal cultures showed that E2-mediated neuroprotection as well as E2-induced ERK activation were sensitive to PTX. The sensitivity to PTX suggested a possible role of G-protein- and beta-arrestin-mediated mechanisms. Western immunoblots from E2-treated cortical neuronal cultures revealed an increase in phosphorylation of both G-protein-coupled receptor-kinase 2 and beta-arrestin-1, a G-protein-coupled receptor adaptor protein. Transfection of neurons with beta-arrestin-1 small interfering RNA prevented E2-induced ERK activation. Coimmunoprecipitation experiments indicated that E2 increased the recruitment of beta-arrestin-1 and c-Src to ERalpha. These findings suggested that ERalpha is regulated by a mechanism associated with receptor desensitization and downregulation. In support of this idea, we found that E2 treatment of cortical synaptoneurosomes resulted in internalization of ERalpha, whereas treatment of cortical neurons with the ER agonists E-6-BSA-FITC [beta-estradiol-6-(O-carboxymethyl)oxime-bovine serum albumin conjugated with fluorescein isothiocyanate] and E-6-biotin [1,3,5(10)-estratrien-3,17beta-diol-6-one-6-carboxymethloxime-NH-propyl-biotin] resulted in agonist internalization. These results demonstrate that E2

  16. Genome-Wide Association Study Identifies Candidate Genes for Starch Content Regulation in Maize Kernels.

    PubMed

    Liu, Na; Xue, Yadong; Guo, Zhanyong; Li, Weihua; Tang, Jihua

    2016-01-01

    Kernel starch content is an important trait in maize (Zea mays L.) as it accounts for 65-75% of the dry kernel weight and positively correlates with seed yield. A number of starch synthesis-related genes have been identified in maize in recent years. However, many loci underlying variation in starch content among maize inbred lines still remain to be identified. The current study is a genome-wide association study that used a set of 263 maize inbred lines. In this panel, the average kernel starch content was 66.99%, ranging from 60.60 to 71.58% over the three study years. These inbred lines were genotyped with the SNP50 BeadChip maize array, which is comprised of 56,110 evenly spaced, random SNPs. Population structure was controlled by a mixed linear model (MLM) as implemented in the software package TASSEL. After the statistical analyses, four SNPs were identified as significantly associated with starch content (P ≤ 0.0001), among which one each are located on chromosomes 1 and 5 and two are on chromosome 2. Furthermore, 77 candidate genes associated with starch synthesis were found within the 100-kb intervals containing these four QTLs, and four highly associated genes were within 20-kb intervals of the associated SNPs. Among the four genes, Glucose-1-phosphate adenylyltransferase (APS1; Gene ID GRMZM2G163437) is known as an important regulator of kernel starch content. The identified SNPs, QTLs, and candidate genes may not only be readily used for germplasm improvement by marker-assisted selection in breeding, but can also elucidate the genetic basis of starch content. Further studies on these identified candidate genes may help determine the molecular mechanisms regulating kernel starch content in maize and other important cereal crops. PMID:27512395

  17. Genome-Wide Association Study Identifies Candidate Genes for Starch Content Regulation in Maize Kernels

    PubMed Central

    Liu, Na; Xue, Yadong; Guo, Zhanyong; Li, Weihua; Tang, Jihua

    2016-01-01

    Kernel starch content is an important trait in maize (Zea mays L.) as it accounts for 65–75% of the dry kernel weight and positively correlates with seed yield. A number of starch synthesis-related genes have been identified in maize in recent years. However, many loci underlying variation in starch content among maize inbred lines still remain to be identified. The current study is a genome-wide association study that used a set of 263 maize inbred lines. In this panel, the average kernel starch content was 66.99%, ranging from 60.60 to 71.58% over the three study years. These inbred lines were genotyped with the SNP50 BeadChip maize array, which is comprised of 56,110 evenly spaced, random SNPs. Population structure was controlled by a mixed linear model (MLM) as implemented in the software package TASSEL. After the statistical analyses, four SNPs were identified as significantly associated with starch content (P ≤ 0.0001), among which one each are located on chromosomes 1 and 5 and two are on chromosome 2. Furthermore, 77 candidate genes associated with starch synthesis were found within the 100-kb intervals containing these four QTLs, and four highly associated genes were within 20-kb intervals of the associated SNPs. Among the four genes, Glucose-1-phosphate adenylyltransferase (APS1; Gene ID GRMZM2G163437) is known as an important regulator of kernel starch content. The identified SNPs, QTLs, and candidate genes may not only be readily used for germplasm improvement by marker-assisted selection in breeding, but can also elucidate the genetic basis of starch content. Further studies on these identified candidate genes may help determine the molecular mechanisms regulating kernel starch content in maize and other important cereal crops. PMID:27512395

  18. Comparative Study of Teaching Content in Teacher Education Programmes in Canada, Denmark, Finland and Singapore

    ERIC Educational Resources Information Center

    Rasmussen, Jens; Bayer, Martin

    2014-01-01

    This article presents the results of a comparative study of the content in selected teacher education programmes for primary and lower secondary teachers in Canada, Denmark, Finland and Singapore. First and foremost, the study is a comparison between teacher education programmes in, on the one hand, Canada, Finland and Singapore, all of which…

  19. The Internet and Some International Regulatory Issues Relating to Content: A Pilot Comparative Study.

    ERIC Educational Resources Information Center

    Australian Broadcasting Authority.

    In December 1996 UNESCO commissioned the Australian Broadcasting Authority to conduct a pilot study which considered a range of online issues; this report outlines the findings of the pilot study, based on data collected between February and May 1997 and updated in July 1997. The objective is to identify the main types of Internet content which…

  20. Strengthening the Conceptualization of Mathematics Pedagogical Content Knowledge for International Studies: A Taiwanese Perspective

    ERIC Educational Resources Information Center

    Hsieh, Feng-Jui

    2013-01-01

    This paper discusses different conceptual frameworks for measuring mathematics pedagogical content knowledge (MPCK) in international comparison studies. Two large-scale international comparative studies, "Mathematics Teaching in the Twenty-First Century" (MT21; Schmidt et al., 2011) and the "Teacher Education and Development Study…

  1. When Teaching Makes a Difference: Developing Science Teachers' Pedagogical Content Knowledge through Learning Study

    ERIC Educational Resources Information Center

    Nilsson, Pernilla

    2014-01-01

    It is a common view that developing teachers' competence to restructure or reframe their knowledge and beliefs is inevitably a complex challenge. This paper reports on a research project with the aim to develop science teachers' pedagogical content knowledge (PCK) through their participation in a learning study. A learning study is a…

  2. Promoting Acceleration of Comprehension and Content through Text in High School Social Studies Classes

    ERIC Educational Resources Information Center

    Wanzek, Jeanne; Swanson, Elizabeth A.; Roberts, Greg; Vaughn, Sharon; Kent, Shawn C.

    2015-01-01

    The purpose of this study was to evaluate the efficacy of Promoting Acceleration of Comprehension and Content Through Text intervention implemented with 11th-grade students enrolled in U.S. History classes. Using a within-teacher randomized design, the study was conducted in 41 classes (23 treatment classes) with 14 teachers providing the…

  3. The Six Remaining Facts: Social Studies Content Knowledge and Elementary Preservice Teachers

    ERIC Educational Resources Information Center

    Sanchez, Rebecca M.

    2010-01-01

    This article explores and examines the social studies content knowledge that preservice teachers have about commonly studies historical figures. The data indicate that preservice teachers often have shallow and decontextualized understandings of historical individuals such as Christopher Columbus and Helen Keller, despite their being repeatedly…

  4. Is Pedagogical Content Knowledge (PCK) Necessary for Reformed Science Teaching?: Evidence from an Empirical Study

    ERIC Educational Resources Information Center

    Park, Soonhye; Jang, Jeong-Yoon; Chen, Ying-Chih; Jung, Jinhong

    2011-01-01

    This study tested a hypothesis that focused on whether or not teachers' pedagogical content knowledge (PCK) is a necessary body of knowledge for reformed science teaching. This study utilized a quantitative research method to investigate the correlation between a teacher's PCK level as measured by the PCK rubric (Park et al. 2008) and the degree…

  5. Student and Professional Attitudes Regarding Advertising Influence on Broadcast News Content: A Comparative Study.

    ERIC Educational Resources Information Center

    Brown, Hubert W.; Barnes, Beth E.

    Students studying Broadcast Journalism or Advertising and professionals working in those fields were surveyed on their attitudes regarding advertising influence on broadcast news content. This study compares the attitudes of the students and practitioners in the respective professions. While students and professionals agreed on a majority of…

  6. Seven Principles of Instructional Content Design for a Remote Laboratory: A Case Study on ERRL

    ERIC Educational Resources Information Center

    Cagiltay, N. E.; Aydin, E.; Aydin, C. C.; Kara, A.; Alexandru, M.

    2011-01-01

    This paper discusses the results of a study of the requirements for developing a remote radio frequency (RF) laboratory for electrical engineering students. It investigates students' preferred usage of the technical content of a state-of-the-art RF laboratory. The results of this study are compared to previous findings, which dealt with other user…

  7. Stability studies of expired tablets of metoprolol tartrate and propranolol hydrochloride. Part 1. Content determination.

    PubMed

    Jasińska, Magdalena; Karwowski, Bolesław; Orszulak-Michalak, Daria; Kurczewska, Urszula

    2009-01-01

    In recent years the growing interest in drug stability problem has been observed. The stability of pharmaceutical products seems to play an important role from the economical point of view. However, there are not many studies that reported about the stability of drugs past their expiration dates. The objective of the current study was to determine tablet content of expired tablets and tablets with expiry date has not been exceeded. The analyzed tablets contained metoprolol tartrate (50 mg) and propranolol hydrochloride (10 mg), respectively. Content determination was performed using HPLC method with UV detection. The proposed method was validated with regard to linearity, sensitivity, intermediate accuracy and precision. No discrepancies between the results of determination and the declared values range for all the analyzed tablets were observed. The results of performed study might suggest that storage of analyzed batches of tablets over time period exceeding the expiry date given by the manufacturer did not influence their contents. PMID:20050534

  8. Effect of bread gluten content on gastrointestinal function: a crossover MRI study on healthy humans.

    PubMed

    Coletta, Marina; Gates, Fred K; Marciani, Luca; Shiwani, Henna; Major, Giles; Hoad, Caroline L; Chaddock, Gemma; Gowland, Penny A; Spiller, Robin C

    2016-01-14

    Gluten is a crucial functional component of bread, but the effect of increasing gluten content on gastrointestinal (GI) function remains uncertain. Our aim was to investigate the effect of increasing gluten content on GI function and symptoms in healthy participants using the unique capabilities of MRI. A total of twelve healthy participants completed this randomised, mechanistic, open-label, three-way crossover study. On days 1 and 2 they consumed either gluten-free bread (GFB), or normal gluten content bread (NGCB) or added gluten content bread (AGCB). The same bread was consumed on day 3, and MRI scans were performed every 60 min from fasting baseline up to 360 min after eating. The appearance of the gastric chime in the images was assessed using a visual heterogeneity score. Gastric volumes, the small bowel water content (SBWC), colonic volumes and colonic gas content and GI symptoms were measured. Fasting transverse colonic volume after the 2-d preload was significantly higher after GFB compared with NGCB and AGCB with a dose-dependent response (289 (SEM 96) v. 212 (SEM 74) v. 179 (SEM 87) ml, respectively; P=0·02). The intragastric chyme heterogeneity score was higher for the bread with increased gluten (AGCB 6 (interquartile range (IQR) 0·5) compared with GFB 3 (IQR 0·5); P=0·003). However, gastric half-emptying time was not different between breads nor were study day GI symptoms, postprandial SBWC, colonic volume and gas content. This MRI study showed novel mechanistic insights in the GI responses to different breads, which are poorly understood notwithstanding the importance of this staple food.

  9. Effect of bread gluten content on gastrointestinal function: a crossover MRI study on healthy humans.

    PubMed

    Coletta, Marina; Gates, Fred K; Marciani, Luca; Shiwani, Henna; Major, Giles; Hoad, Caroline L; Chaddock, Gemma; Gowland, Penny A; Spiller, Robin C

    2016-01-14

    Gluten is a crucial functional component of bread, but the effect of increasing gluten content on gastrointestinal (GI) function remains uncertain. Our aim was to investigate the effect of increasing gluten content on GI function and symptoms in healthy participants using the unique capabilities of MRI. A total of twelve healthy participants completed this randomised, mechanistic, open-label, three-way crossover study. On days 1 and 2 they consumed either gluten-free bread (GFB), or normal gluten content bread (NGCB) or added gluten content bread (AGCB). The same bread was consumed on day 3, and MRI scans were performed every 60 min from fasting baseline up to 360 min after eating. The appearance of the gastric chime in the images was assessed using a visual heterogeneity score. Gastric volumes, the small bowel water content (SBWC), colonic volumes and colonic gas content and GI symptoms were measured. Fasting transverse colonic volume after the 2-d preload was significantly higher after GFB compared with NGCB and AGCB with a dose-dependent response (289 (SEM 96) v. 212 (SEM 74) v. 179 (SEM 87) ml, respectively; P=0·02). The intragastric chyme heterogeneity score was higher for the bread with increased gluten (AGCB 6 (interquartile range (IQR) 0·5) compared with GFB 3 (IQR 0·5); P=0·003). However, gastric half-emptying time was not different between breads nor were study day GI symptoms, postprandial SBWC, colonic volume and gas content. This MRI study showed novel mechanistic insights in the GI responses to different breads, which are poorly understood notwithstanding the importance of this staple food. PMID:26522233

  10. [Study on hyperspectral estimation of pigment contents in leaves of cotton under disease stress].

    PubMed

    Chen, Bing; Li, Shao-Kun; Wang, Ke-Ru; Wang, Fang-Yong; Xiao, Chun-Hua; Pan, Wen-Chao

    2010-02-01

    The spectrum reflectance and pigment contents of cotton leaves infected with Verticillium wilt were measured in cotton disease nursery and field in different growth phases, and severity level of Verticillium wilt was investigated. The correlation between pigment contents of cotton leaves with Verticillium wilt and spectra reflectance, the first derivative of reflectance and spectral characteristic parameters were analyzed respectively. The estimation models about leaves pigment contents of disease cotton were established and tested. The results indicated that the correlations were best significant between chlorophyll a, b and a+b contents of leaves and spectral reflectance in visible wave bands, the first derivative spectrum at the wavelength regions of blue edge, yellow edge and red edge, and all spectral characteristic parameters (excluding red edge swing Dr). The models of transformed chlorophyll absorption in reflectance index (TCAR) and the new model of normalized difference vegetation index (NDVI [702,758]) had best estimated precision, and the relative errors were in average within 1.3%. Given that the model of NDVI [702,758] is very simple and practical, it was commended as a best model to estimate chlorophyll a, b and a+b contents of disease cotton. This study shows that leaves hyperspectra data can be used to estimate the pigment contents of cotton leaves quantitatively. This conclusion has also great practice and application value for monitoring the growth state and disease influence evaluation on cotton by using hyperspectral remote sensing.

  11. Consumers’ estimation of calorie content at fast food restaurants: cross sectional observational study

    PubMed Central

    Condon, Suzanne K; Kleinman, Ken; Mullen, Jewel; Linakis, Stephanie; Rifas-Shiman, Sheryl; Gillman, Matthew W

    2013-01-01

    Objective To investigate estimation of calorie (energy) content of meals from fast food restaurants in adults, adolescents, and school age children. Design Cross sectional study of repeated visits to fast food restaurant chains. Setting 89 fast food restaurants in four cities in New England, United States: McDonald’s, Burger King, Subway, Wendy’s, KFC, Dunkin’ Donuts. Participants 1877 adults and 330 school age children visiting restaurants at dinnertime (evening meal) in 2010 and 2011; 1178 adolescents visiting restaurants after school or at lunchtime in 2010 and 2011. Main outcome measure Estimated calorie content of purchased meals. Results Among adults, adolescents, and school age children, the mean actual calorie content of meals was 836 calories (SD 465), 756 calories (SD 455), and 733 calories (SD 359), respectively. A calorie is equivalent to 4.18 kJ. Compared with the actual figures, participants underestimated calorie content by means of 175 calories (95% confidence interval 145 to 205), 259 calories (227 to 291), and 175 calories (108 to 242), respectively. In multivariable linear regression models, underestimation of calorie content increased substantially as the actual meal calorie content increased. Adults and adolescents eating at Subway estimated 20% and 25% lower calorie content than McDonald’s diners (relative change 0.80, 95% confidence interval 0.66 to 0.96; 0.75, 0.57 to 0.99). Conclusions People eating at fast food restaurants underestimate the calorie content of meals, especially large meals. Education of consumers through calorie menu labeling and other outreach efforts might reduce the large degree of underestimation. PMID:23704170

  12. Socialization Content in Schools and Education for Sustainable Development--I. A Study of Teachers' Selective Traditions

    ERIC Educational Resources Information Center

    Sund, Per; Wickman, Per-Olof

    2011-01-01

    This article studies content issues by examining teachers' communicated socialization content. The value-laden socialization content constitutes the educational context for the teaching of integrated subject matter and has not yet been thoroughly studied empirically in environmental education research. The implications of the results can be…

  13. Systematic Study of the Content of Phytochemicals in Fresh and Fresh-Cut Vegetables.

    PubMed

    Alarcón-Flores, María Isabel; Romero-González, Roberto; Vidal, José Luis Martínez; Frenich, Antonia Garrido

    2015-01-01

    Vegetables and fruits have beneficial properties for human health, because of the presence of phytochemicals, but their concentration can fluctuate throughout the year. A systematic study of the phytochemical content in tomato, eggplant, carrot, broccoli and grape (fresh and fresh-cut) has been performed at different seasons, using liquid chromatography coupled to triple quadrupole mass spectrometry. It was observed that phenolic acids (the predominant group in carrot, eggplant and tomato) were found at higher concentrations in fresh carrot than in fresh-cut carrot. However, in the case of eggplant, they were detected at a higher content in fresh-cut than in fresh samples. Regarding tomato, the differences in the content of phenolic acids between fresh and fresh-cut were lower than in other matrices, except in winter sampling, where this family was detected at the highest concentration in fresh tomato. In grape, the flavonols content (predominant group) was higher in fresh grape than in fresh-cut during all samplings. The content of glucosinolates was lower in fresh-cut broccoli than in fresh samples in winter and spring sampling, although this trend changes in summer and autumn. In summary, phytochemical concentration did show significant differences during one-year monitoring, and the families of phytochemicals presented different behaviors depending on the matrix studied. PMID:26783709

  14. A comparative study of six decades of general science textbooks: Evaluating the evolution of science content

    NASA Astrophysics Data System (ADS)

    Lewis, Anna

    This study examined science textbooks over time to better understand the science content expectations that the U.S. educational system deems appropriate for 8th and 9th grade science students. The study attempted to answer the questions: (1) What specific science content has been presented via the textbook from 1952 to 2008? (2) Within which areas and in what way does the science content change? (3) Are new scientific findings reflected in 8th and 9th grade U.S. general science textbooks? Twenty-six themes were identified which reflect five areas in science: Chemistry, Physics, Earth Science, Biology, and Process of Science. Trends in science content in U.S. 8th and 9th grade general science textbooks, as revealed by this data sample, indicated no statistically significant change in depth of coverage in Physics and Process of Science over the past 60 years, no significant change in depth of coverage in Earth Science and Biology in the last 40 years, and no significant change in coverage in Chemistry over the last 30 years. Additionally, a total of sixteen new discoveries were found in the textbook sample. For classroom teachers this information may alert them to the necessity of going beyond the textbook in preparing students for life in a global society. In educational practice, this research supports and reinforces the need for inquiry learning and socioscientific curricula. It may also influence educators to challenge assumptions regarding the value and selection of the traditional classic science content.

  15. Analysis of Pedagogical Content Knowledge Studies in the Context of Mathematics Education in Turkey: A Meta-Synthesis Study

    ERIC Educational Resources Information Center

    Simsek, Nurullah; Boz, Nihat

    2016-01-01

    Studies that explore pedagogical content knowledge (PCK) in the field of mathematics education date back to the turn of the century in Turkey. In recent years, studies on PCK have gained momentum. Master's theses and doctoral dissertations have been written on PCK. In this context, there is a need to analyze the studies on PCK in Turkey to…

  16. Technology-Based Content through Virtual and Physical Modeling: A National Research Study

    ERIC Educational Resources Information Center

    Ernst, Jeremy V.; Clark, Aaron C.

    2009-01-01

    Visualization is becoming more prevalent as an application in science, engineering, and technology related professions. The analysis of static and dynamic graphical visualization provides data solutions and understandings that go beyond traditional forms of communication. The study of technology-based content and the application of conceptual…

  17. Family Peer Advocates: A Pilot Study of the Content and Process of Service Provision

    ERIC Educational Resources Information Center

    Wisdom, Jennifer P.; Olin, Serene; Shorter, Priscilla; Burton, Geraldine; Hoagwood, Kimberly

    2011-01-01

    Professional family peer advocates are increasingly employed by public mental health systems to deliver family-to-family support that reduces barriers families face in accessing children's mental health care. These services, however, are neither uniformly available nor standardized. This pilot study describes the process, content and context of…

  18. A Content and Methodological Review of Self-Advocacy Intervention Studies

    ERIC Educational Resources Information Center

    Test, David W.; Fowler, Catherine H.; Brewer, Denise M.; Wood, Wendy M.

    2005-01-01

    A content and methodological review of the literature of 25 self-advocacy intervention studies was conducted. First, each article was analyzed in terms of purpose, participants, design, dependent variable(s), independent variable(s), and results. Second, each manuscript was reviewed in terms of the quality indicators for single subject (n = 11),…

  19. Content Analysis of Meta-Analytic Studies from I/O Psychology.

    ERIC Educational Resources Information Center

    Cornwell, John M.

    The use of meta-analysis in industrial and organizational psychology has become quite common. Unfortunately, the understanding and research necessary to ensure appropriate application of the technique have not been as widespread. As part of a larger study, a content analysis of meta-analyses from the industrial and organizational psychological…

  20. The Structure of Mixed Method Studies in Educational Research: A Content Analysis

    ERIC Educational Resources Information Center

    Bryant, Lauren H.

    2011-01-01

    Educational researchers are beginning to use mixed methods designs to answer complex research questions. This content analysis investigates the structure and use of mixed methods in educational research in order to work toward a more standardized presentation. I used a concurrent mixed methods approach to analyze 30 studies from three prominent…

  1. 42 CFR 456.243 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 4 2014-10-01 2014-10-01 false Content of medical care evaluation studies. 456.243 Section 456.243 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS UTILIZATION CONTROL Utilization Control: Mental...

  2. 42 CFR 456.243 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 4 2010-10-01 2010-10-01 false Content of medical care evaluation studies. 456.243 Section 456.243 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS UTILIZATION CONTROL Utilization Control: Mental...

  3. 42 CFR 456.243 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 4 2012-10-01 2012-10-01 false Content of medical care evaluation studies. 456.243 Section 456.243 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS UTILIZATION CONTROL Utilization Control: Mental...

  4. 42 CFR 456.243 - Content of medical care evaluation studies.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 4 2011-10-01 2011-10-01 false Content of medical care evaluation studies. 456.243 Section 456.243 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL ASSISTANCE PROGRAMS UTILIZATION CONTROL Utilization Control: Mental...

  5. A Study of the Structure and Content of Principal Selection Interviews in Pennsylvania

    ERIC Educational Resources Information Center

    Weber, Elizabeth A.

    2012-01-01

    The principal plays a key role in student success. The employment interview is a critical element in the principal selection process. This study examined the interview structure and the content of the interview questions that districts used in their principal search for the 2011-2012 school year. The research-based practices for interview…

  6. Plasma phosphatidylcholine docosahexaenoic acid content and risk of dementia and Alzheimer disease: the Framingham Heart Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our aim in carrying out this analysis, was to assess the predictive value of plasma phosphatidylcholine (PC) DHA content, DHA intake, and fish intake for the risk of developing dementia in the Framingham Heart Study. A cohort of 899 subjects free of dementia was followed to assess the onset of incid...

  7. Teaching Science with Technology: Case Studies of Science Teachers' Development of Technology, Pedagogy, and Content Knowledge

    ERIC Educational Resources Information Center

    Guzey, S. Selcen; Roehrig, Gillian H.

    2009-01-01

    This study examines the development of technology, pedagogy, and content knowledge (TPACK) in four in-service secondary science teachers as they participated in a professional development program focusing on technology integration into K-12 classrooms to support science as inquiry teaching. In the program, probeware, mind-mapping tools (CMaps),…

  8. Developing Storypath Units for Integrating Social Studies and the Michigan Content Standards.

    ERIC Educational Resources Information Center

    Scott, Renay

    2000-01-01

    Describes "Storypath," a lesson plan framework that integrates social studies curriculum with the basic elements of a story. Outlines a three-week history unit, "The Coming of the American Revolution," created with "Storypath." Discusses the correlation between Storypath and the Michigan content standards and considers the assessments used. (CMK)

  9. Programme Content Orientation in Vocational Education and Training and Life Chances--A Comparative Study

    ERIC Educational Resources Information Center

    Kap, Hrvoje

    2014-01-01

    Comparative studies of vocational education and training systems rarely conduct systematic comparisons of the content of educational programmes, partly because of methodological difficulties. Yet, comparing the organisation of curricula can increase our understanding of how programme design reflects orientation towards various life chances in…

  10. Developing Fair Tests for Mathematics Curriculum Comparison Studies: The Role of Content Analyses

    ERIC Educational Resources Information Center

    Chavez, Oscar; Papick, Ira; Ross, Daniel J.; Grouws, Douglas A.

    2011-01-01

    This article describes the process of development of assessment instruments for a three-year longitudinal comparative study that focused on evaluating American high school students' mathematics learning from two distinct approaches to content organization: curriculum built around a sequence of three full-year courses (Algebra 1, Geometry, and…

  11. β-arrestin-2 regulates NMDA receptor function in spinal lamina II neurons and duration of persistent pain.

    PubMed

    Chen, Gang; Xie, Rou-Gang; Gao, Yong-Jing; Xu, Zhen-Zhong; Zhao, Lin-Xia; Bang, Sangsu; Berta, Temugin; Park, Chul-Kyu; Lay, Mark; Chen, Wei; Ji, Ru-Rong

    2016-01-01

    Mechanisms of acute pain transition to chronic pain are not fully understood. Here we demonstrate an active role of β-arrestin 2 (Arrb2) in regulating spinal cord NMDA receptor (NMDAR) function and the duration of pain. Intrathecal injection of the mu-opioid receptor agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin produces paradoxical behavioural responses: early-phase analgesia and late-phase mechanical allodynia which requires NMDAR; both phases are prolonged in Arrb2 knockout (KO) mice. Spinal administration of NMDA induces GluN2B-dependent mechanical allodynia, which is prolonged in Arrb2-KO mice and conditional KO mice lacking Arrb2 in presynaptic terminals expressing Nav1.8. Loss of Arrb2 also results in prolongation of inflammatory pain and neuropathic pain and enhancement of GluN2B-mediated NMDA currents in spinal lamina IIo not lamina I neurons. Finally, spinal over-expression of Arrb2 reverses chronic neuropathic pain after nerve injury. Thus, spinal Arrb2 may serve as an intracellular gate for acute to chronic pain transition via desensitization of NMDAR. PMID:27538456

  12. The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain

    PubMed Central

    Shi, Hang; Rojas, Raul; Bonifacino, Juan S.; Hurley, James H.

    2006-01-01

    The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29, and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1Å resolution reveals two curvedβ -sandwich domains connected by a polar core and a flexible linker. Vps26 has an unexpected structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235–246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a Gly in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting. PMID:16732284

  13. Muscarinic Acetylcholine Receptor M3 Modulates Odorant Receptor Activity via Inhibition of β-Arrestin-2 Recruitment

    PubMed Central

    Jiang, Yue; Li, Yun Rose; Tian, Huikai; Ma, Minghong; Matsunami, Hiroaki

    2015-01-01

    The olfactory system in rodents serves a critical function in social, reproductive, and survival behaviors. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odor-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odors. Here, we show that an M3-R antagonist attenuates odor-induced responses in OSNs from wild-type, but not M3-R-null mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odor-induced response in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system. PMID:25800153

  14. Recruitment of β-Arrestin 1 and 2 to the β2-Adrenoceptor: Analysis of 65 Ligands

    PubMed Central

    Littmann, Timo; Göttle, Martin; Reinartz, Michael T.; Kälble, Solveig; Wainer, Irving W.; Ozawa, Takeaki

    2015-01-01

    Beyond canonical signaling via Gαs and cAMP, the concept of functional selectivity at β2-adrenoceptors (β2ARs) describes the ability of adrenergic drugs to stabilize ligand-specific receptor conformations to initiate further signaling cascades comprising additional G-protein classes or β-arrestins (βarr). A set of 65 adrenergic ligands including 40 agonists and 25 antagonists in either racemic or enantiopure forms was used for βarr recruitment experiments based on a split-luciferase assay in a cellular system expressing β2AR. Many agonists showed only (weak) partial agonism regarding βarr recruitment. Potencies and/or efficacies increased depending on the number of chirality centers in (R) configuration; no (S)-configured distomer was more effective at inducing βarr recruitment other than the eutomer. βarr2 was recruited more effectively than βarr1. The analysis of antagonists revealed no significant effects on βarr recruitment. Several agonists showed preference for activation of Gαs GTPase relative to βarr recruitment, and no βarr-biased ligand was identified. In conclusion: 1) agonists show strong bias for Gαs activation relative to βarr recruitment; 2) agonists recruit βarr1 and βarr2 with subtle differences; and 3) there is no evidence for βarr recruitment by antagonists. PMID:26306764

  15. Structural and Biochemical Basis for Ubiquitin Ligase Recruitment by Arrestin-related Domain-containing Protein-3 (ARRDC3)*

    PubMed Central

    Qi, Shiqian; O'Hayre, Morgan; Gutkind, J. Silvio; Hurley, James H.

    2014-01-01

    After protracted stimulation, the β2-adrenergic receptor and many other G-protein-coupled receptors are ubiquitinated and down-regulated. Arrestin-related domain-containing protein-3 (ARRDC3) has been proposed to recruit the ubiquitin ligase Nedd4 to the β2-adrenergic receptor. ARRDC3 contains two PPXY motifs that could potentially interact with any of the four WW domains of Nedd4. Here we dissect the interaction determinants. ARRDC3 PPXY-Nedd4 WW dissociation constants vary from unmeasurable to Kd = 3 μm for the third WW domain of Nedd4 binding to the first PPXY motif of ARRDC3. Structures of the uncomplexed and PPXY1-bound WW3 domain were determined at 1.1 and 1.7 Å resolution. The structures revealed conformational changes upon binding and the hydrogen bonding network in exquisite detail. Tight packing of ARRDC3 Val-352′, part of a 310 helix at the C terminus of PPXY1, is important for high affinity binding to WW3. Although no single WW domain is strictly essential for the binding of Nedd4 and ARRDC3 expressed in HEK293 cells, high affinity binding of full-length ARRDC3 and Nedd4 is driven by the avid interaction of both PPXY motifs with either the WW2-WW3 or WW3-WW4 combinations, with Kd values as low as 300 nm. PMID:24379409

  16. Agonist-Activated Bombyx Corazonin Receptor Is Internalized via an Arrestin-Dependent and Clathrin-Independent Pathway.

    PubMed

    Yang, Jingwen; Shen, Zhangfei; Jiang, Xue; Yang, Huipeng; Huang, Haishan; Jin, Lili; Chen, Yajie; Shi, Liangen; Zhou, Naiming

    2016-07-19

    Agonist-induced internalization plays a key role in the tight regulation of the extent and duration of G protein-coupled receptor signaling. Previously, we have shown that the Bombyx corazonin receptor (BmCrzR) activates both Gαq- and Gαs-dependent signaling cascades. However, the molecular mechanisms involved in the regulation of the internalization and desensitization of BmCrzR remain to be elucidated. Here, vectors for expressing BmCrzR fused with enhanced green fluorescent protein (EGFP) at the C-terminal end were used to further characterize BmCrzR internalization. We found that the BmCrzR heterologously expressed in HEK-293 and BmN cells was rapidly internalized from the plasma membrane into the cytoplasm in a concentration- and time-dependent manner via a β-arrestin (Kurtz)-dependent and clathrin-independent pathway in response to agonist challenge. While most of the internalized receptors were recycled to the cell surface via early endosomes, some others were transported to lysosomes for degradation. Assays using RNA interference revealed that both GRK2 and GRK5 were essentially involved in the regulation of BmCrzR phosphorylation and internalization. Further investigations indicated that the identified cluster of Ser/Thr residues ((411)TSS(413)) was responsible for GRK-mediated phosphorylation and internalization. This is the first detailed investigation of the internalization and trafficking of Bombyx corazonin receptors. PMID:27348044

  17. [Cytologic study of glandular tumors in maxillofacial regions-- diagnostic application of nuclear DNA content].

    PubMed

    Kawasaki, T; Kimura, H; Shingaki, S; Saito, R; Nakajima, T; Mizutani, H; Mori, M; Ishiki, T

    1983-12-01

    We studied whether the DNA contents may be useful in the differential diagnosis of glandular tumors in the maxillofacial regions. In 25 of these tumors, the DNA contents were measured by microspectrophotometry, using 4 normal salivary glands as controls. We found that: (1) DNA histgram patterns were useful in differential diagnosis only in special cases. (2) The criteria were based on our decision and these tumors were classified into 3 types (a) Benign, (b) Low-grade malignant, (c) High-grade malignant. Our classification seemed to offer an objective means for differentiating between Benign & Malignant tumors of these types.

  18. Reading in the Social Studies and Natural Science Content Area: A Phenomenological Study of the Beliefs, Attitudes, and Strategies Sixth and Seventh Grade Content Area Teachers Use to Teach below Grade Level Readers

    ERIC Educational Resources Information Center

    Clark, Lisa A.

    2011-01-01

    The purposes of this study were to identify the attitudes and beliefs content teachers have concerning teaching reading in the content area to below level readers and to identify specific instructional strategies that are used to teach students who are below grade level the content area material. Twelve participants were selected, using maximum…

  19. Comparative study on mannose 6-phosphate residue contents of recombinant lysosomal enzymes.

    PubMed

    Togawa, Tadayasu; Takada, Masaru; Aizawa, Yoshiaki; Tsukimura, Takahiro; Chiba, Yasunori; Sakuraba, Hitoshi

    2014-03-01

    As most recombinant lysosomal enzymes are incorporated into cells via mannose 6-phosphate (M6P) receptors, the M6P content is important for effective enzyme replacement therapy (ERT) for lysosomal diseases. However, there have been no comprehensive reports of the M6P contents of lysosomal enzymes. We developed an M6P assay method comprising three steps, i.e., acid hydrolysis of glycoproteins, derivatization of M6P, and high-performance liquid chromatography, and determined the M6P contents of six recombinant lysosomal enzymes now available for ERT and one in the process of development. The assay is easy, specific, and reproducible. The results of the comparative study revealed that the M6P contents of agalsidase alfa, agalsidase beta, modified α-N-acetylgalactosaminidase, alglucosidase alfa, laronidase, idursulfase, and imiglucerase are 2.1, 2.9, 5.9, 0.7, 2.5, 3.2, and <0.3 mol/mol enzyme, respectively. The results were correlated with those of the biochemical analyses previously performed and that of the binding assay of exposed M6P of the enzymes with the domain 9 of the cation-independent M6P receptor. This assay method is useful for comparison of the M6P contents of recombinant lysosomal enzymes for ERT. PMID:24439675

  20. Comparative study of goal contents and goal characteristics between medical and business students

    PubMed Central

    Park, Soowon; Kim, Ji Eun; Lee, Jun-Young; Shin, Jongho

    2016-01-01

    Purpose: Medical and business are one of the most popular majors among students, and both fields require intensive training to reach certain level of expertise. During the development of professionalism, goal can become a crucial role in psychological impetus. The purpose of this study is to compare goal contents, goal characteristics, and effect of goal characteristics on student’s major satisfaction between medical and business. Methods: A total of 193 undergraduate students (97 medical students, 96 business students) answered survey questions including goal contents, goal characteristics (goal autonomy, goal attainability, social value of goal) and satisfaction on their majors. Qualitative analysis of goal contents and quantitative analysis of goal characteristics, and their effects on student major satisfaction were performed. Results: Goal content analysis showed percentage of social concern goal was higher in medical students (25.8%) than business students (6.3%), whereas percentage of wealth goal was higher business students (24.0%) than medical students (3.1%). Among goal characteristics, goal attainability and social value of goal were higher in medical students than business students. In both groups, social value of goal was significantly predict major satisfaction. Conclusion: Goal contents and goal characteristics are different between medical and business students. Curriculum and educational interventions that concerning students’ goal and developing programs to enhance students’ social value of goal is necessary. PMID:26838564

  1. Study of Polyphenol Content and Antioxidant Capacity of Myrianthus Arboreus (Cecropiaceae) Root Bark Extracts.

    PubMed

    Kasangana, Pierre Betu; Haddad, Pierre Selim; Stevanovic, Tatjana

    2015-01-01

    In order to evaluate the therapeutic potential of polyphenolic extracts from root bark of M. arboreus, we have determined the content of various polyphenols in aqueous and ethanol (EtOH) extract as well as two sub-fractions of the latter: ethyl acetate (EAc) and hexane (Hex). The total phenols, flavonoids, hydroxycinnamic acids and proanthocyanidins have been determined for all studied extracts/fractions by spectrophotometric methods. Both TP content (331.5 ± 2.5 mg GAE/g) and HCA content (201 ± 1.5 mg CAE/g) were determined to be the highest in EAc fraction of EtOH extract. All studied extracts were however determined to have a low content in flavonoids. The determination of antioxidant capacities of the studied extracts has also been performed by the following in vitro antioxidant tests: DPPH scavenging, phosphomolybdenum method and oxygen radical absorbance (ORACFl and ORACPRG) assay. The results of the DPPH free radical and ORACFl assays showed that there is no significant difference between the EAc fraction and Oligopin(®), but the EAc fraction exhibited the highest antioxidant capacity as determined by the phosphomolybdenium method. In addition, the EtOH extract was determined to have the same antioxidant efficiency as the synthetic antioxidant BHT or commercial extract Oligopin(®) by phosphomolybdenum method. On the other hand, a positive correlation (r < 0.6) was found between different classes of polyphenols and the results of the phosphomolybdenum method, ORACFl as well as ORACPRG, except for the DPPH assay, for which a negative correlation was indicated (r < 0.62). Interestingly, it seems that the content in hydroxycinnamic acids played a big role in all assays with r < 0.9. According to the present study, EAc fraction and EtOH extract should be further studied for the potential use in the pharmaceutical and food industry. PMID:26783713

  2. Study of Polyphenol Content and Antioxidant Capacity of Myrianthus Arboreus (Cecropiaceae) Root Bark Extracts

    PubMed Central

    Kasangana, Pierre Betu; Haddad, Pierre Selim; Stevanovic, Tatjana

    2015-01-01

    In order to evaluate the therapeutic potential of polyphenolic extracts from root bark of M. arboreus, we have determined the content of various polyphenols in aqueous and ethanol (EtOH) extract as well as two sub-fractions of the latter: ethyl acetate (EAc) and hexane (Hex). The total phenols, flavonoids, hydroxycinnamic acids and proanthocyanidins have been determined for all studied extracts/fractions by spectrophotometric methods. Both TP content (331.5 ± 2.5 mg GAE/g) and HCA content (201 ± 1.5 mg CAE/g) were determined to be the highest in EAc fraction of EtOH extract. All studied extracts were however determined to have a low content in flavonoids. The determination of antioxidant capacities of the studied extracts has also been performed by the following in vitro antioxidant tests: DPPH scavenging, phosphomolybdenum method and oxygen radical absorbance (ORACFl and ORACPRG) assay. The results of the DPPH free radical and ORACFl assays showed that there is no significant difference between the EAc fraction and Oligopin®, but the EAc fraction exhibited the highest antioxidant capacity as determined by the phosphomolybdenium method. In addition, the EtOH extract was determined to have the same antioxidant efficiency as the synthetic antioxidant BHT or commercial extract Oligopin® by phosphomolybdenum method. On the other hand, a positive correlation (r < 0.6) was found between different classes of polyphenols and the results of the phosphomolybdenum method, ORACFl as well as ORACPRG, except for the DPPH assay, for which a negative correlation was indicated (r < 0.62). Interestingly, it seems that the content in hydroxycinnamic acids played a big role in all assays with r < 0.9. According to the present study, EAc fraction and EtOH extract should be further studied for the potential use in the pharmaceutical and food industry. PMID:26783713

  3. Deletion of β-Arrestin2 in Mice Limited Pancreatic β-Cell Expansion under Metabolic Stress through Activation of the JNK Pathway

    PubMed Central

    Lin, Ziwei; Zhao, Yu; Song, Lige; Mu, Kaida; Zhang, Mingliang; Liu, Hongxia; Li, Xiaowen; Zhao, Jian; Wang, Chen; Jia, Weiping

    2016-01-01

    β-Arrestin2 (βarr2) is an adaptor protein that interacts with numerous signaling molecules and regulates insulin sensitivity. We reported previously that βarr2 was abundantly expressed in mouse pancreatic β-cells, and loss of βarr2 leads to impairment of acute- and late-phase insulin secretion. In the present study, we examined the dynamic changes of β-cell mass in βarr2-deficient (βarr2–/–) mice in vivo and explored the underlying mechanisms involved. βarr2–/– mice with exclusively luciferase overexpression in β-cells were generated and fed a high-fat diet (HFD). β-Cell mass was determined by in vivo noninvasive bioluminescence imaging from 4 to 20 wks of age. Proliferation was measured by 5-bromo-2-deoxyuridine (BrdU) incorporation and fluorescence-activated cell sorter analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting were conducted for gene and protein expression. We found that β-cell mass was reduced dramatically in βarr2–/– mice at 12 wks old compared with that of their respective HFD-fed controls. The percentage of BrdU- and Ki67-positive cells reduced in islets from βarr2–/– mice. Exposure of βarr2–/– islets to high levels of glucose and free fatty acids (FFAs) exacerbated cell death, which was associated with upregulation of the JNK pathway in these islets. Conversely, overexpression of βarr2 amplified β-cell proliferation with a concomitant increase in cyclinD2 expression and a decrease in p21 expression and protected β-cells from glucose- and FFA-induced cell death through JNK-activation inhibition. In conclusion, βarr2 plays roles in regulation of pancreatic β-cell mass through the modulation of cell cycle regulatory genes and the inhibition of JNK activation induced by glucolipotoxity, which implicates a role for βarr2 in the development of type 2 diabetes. PMID:26954469

  4. Contextual and conceptual content analysis in the study of foreign policy decision making

    SciTech Connect

    Schlagheck, D.M.

    1985-01-01

    This dissertation focuses upon two related questions in the study of foreign policy decision making at the individual level: (1) How does a decision maker define the situation he/she confronts. and, (2) How can the research reliably establish that definition. The problem of context and how a decision maker defines it is shown to be a common thread running throughout the foreign policy literature, brought together in a manageable form by the operational code. The operational code is used to guide the application of a new, contextual and conceptual content analysis program in a case study of Henry A. Kissinger's definition of the situations he faced in the Vietnam and arms control negotiations. Kissinger's verbal behavior is examined, including his academic writing; speeches and interviews he gave while in office; his memoirs; and, addresses he has made since leaving public service. The content analysis program (Minnesota Contextural Content Analysis, MCCA) analyzes an individual's understanding of context based on her/his choice of language, and scores verbal behavior in four context categories: pragmatic (rational), analytical, emotional, and traditional. Results of the content analysis of Kissinger's definition of the Vietnam War and arms control talks are analyzed in terms of COPDAB events data to determine whether Kissinger's verbal behavior was events dependent; results are also evaluated in terms of other psycho-biographical and operational studies of Kissinger, as well.

  5. The Social Studies Basic Skills Connection: Practical Strategies for Teaching Basic Skills in Conjunction with Social Studies Content.

    ERIC Educational Resources Information Center

    Missouri State Dept. of Education, Jefferson City.

    Arranged in two parts, this guide introduces elementary and secondary social studies teachers to a variety of methods for integrating social studies content and basic skills instruction. Chapter I defines basic skills as the skills an individual needs to become a self-directed learner, communicate clearly, and make reasoned decisions, and presents…

  6. Studying dream content using the archive and search engine on DreamBank.net.

    PubMed

    Domhoff, G William; Schneider, Adam

    2008-12-01

    This paper shows how the dream archive and search engine on DreamBank.net, a Web site containing over 22,000 dream reports, can be used to generate new findings on dream content, some of which raise interesting questions about the relationship between dreaming and various forms of waking thought. It begins with studies that draw dream reports from DreamBank.net for studies of social networks in dreams, and then demonstrates the usefulness of the search engine by employing word strings relating to religious and sexual elements. Examples from two lengthy individual dream series are used to show how the dreams of one person can be studied for characters, activities, and emotions. A final example shows that accurate inferences about a person's religious beliefs can be made on the basis of reading through dreams retrieved with a few keywords. The overall findings are similar to those in studies using traditional forms of content analysis. PMID:18682331

  7. "Didaktik" and the Selection of Content as Points of Departure for Studying the Quality of Teaching and Learning

    ERIC Educational Resources Information Center

    Johansen, Geir

    2007-01-01

    A critical look at the content we select for teaching and the criteria for that selection may help us to establish a theoretical framework for studying the quality of teaching and learning. The discussion of such criteria is central to the German and Nordic didaktik tradition. The criteria for content selection reveal how educational content is…

  8. High Water Contents in the Siberian Cratonic Mantle: An FTIR Study of Udachnaya Peridotite Xenoliths

    NASA Technical Reports Server (NTRS)

    Doucet, Luc S.; Peslier, Anne H.; Ionov, Dimitri A.; Brandon, Alan D.; Golovin, Alexander V.; Ashchepkov, Igor V.

    2013-01-01

    Water is believed to be a key factor controlling the long-term stability of cratonic lithosphere, but mechanisms responsible for the water content distribution in the mantle remain poorly constrained. Water contents were obtained by FTIR in olivine, pyroxene and garnet for 20 well-characterized peridotite xenoliths from the Udachnaya kimberlite (central Siberian craton) and equilibrated at 2-7 GPa. Water contents in minerals do not appear to be related to interaction with the host kimberlite. Diffusion modeling indicates that the core of olivines preserved their original water contents. The Udachnaya peridotites show a broad range of water contents in olivine (6.5 +/- 1.1 to 323 +- 65 ppm H2O (2 sigma)), and garnet (0 - 23 +/- 6 ppm H2O). The water contents of olivine and garnet are positively correlated with modal clinopyroxene, garnet and FeO in olivine. Water-rich garnets are also rich in middle rare earth elements. This is interpreted as the result of interaction between residual peridotites and water rich-melts, consistent with modal and cryptic metasomatism evidenced in the Siberian cratonic mantle. The most water-rich Udachnaya minerals contain 2 to 3 times more water than those from the Kaapvaal craton, the only craton with an intact mantle root for which water data is available. The highest water contents in olivine and orthopyroxene in this study (>= 300 ppm) are found at the bottom of the lithosphere (> 6.5 GPa). This is in contrast with the Kaapvaal craton where the olivines of peridotites equilibrated at > 6.4 GPa have < 1 ppm H2O. The latter "dry" olivine may make the base of the Kaapvaal cratonic root strong and thus protects it from erosion by the convective mantle The calculated viscosity for water-rich Udachnaya peridotites at > 6 GPa is lower or similar (8.4× 10(exp 16) to 8.0× 10(exp 18) Pa./s) to that of the asthenosphere (<= 3.7x10(exp 18) Pa./s ). Such lithologies would not be able to resist delamination by the convecting asthenosphere

  9. Multidimensional fluorescence studies of the phenolic content of dissolved organic carbon in humic substances.

    PubMed

    Pagano, Todd; Ross, Annemarie D; Chiarelli, Joseph; Kenny, Jonathan E

    2012-03-01

    Indicators suggest that the amount of dissolved organic carbon (DOC) in natural waters may be increasing. Climate change has been proposed as a potential contributor to the trend, and under such a mechanism, the phenolic content of DOC may also be increasing. This study explores the assessment of the phenolic character of DOC using multidimensional fluorescence spectroscopy as a more convenient alternative to traditional wet chemistry methods. Parallel factor analysis (PARAFAC) is applied to fluorescence excitation emission matrices (EEMs) of humic samples to analyze inherent phenolic content. The PARAFAC results are correlated with phenol concentrations derived from the Folin-Ciocalteau reagent-based method. The reagent-based method reveals that the phenolic content of five International Humic Substance Society (IHSS) samples varies from approximately 5.2 to 22 ppm Tannic Acid Equivalents (TAE). A four-component PARAFAC fit is applied to the EEMs of the IHSS sample dataset and it is determined by PARAFAC score correlations with phenol concentrations from the reagent-based method that components C2, C3, and C4 have the highest probability of containing phenolic groups. The results show the potential for PARAFAC analysis of multidimensional fluorescence data for monitoring the phenolic content of DOC.

  10. The influence of informed consent content on study participants’ contraceptive knowledge and concerns

    PubMed Central

    Stephenson, Rob; Grabbe, Kristina; Vwalika, Bellington; Ahmed, Yusuf; Vwalika, Cheswa; Haworth, Alan; Fuller, Laurie; Liu, Fong; Chomba, Elwyn; Allen, Susan

    2011-01-01

    Objectives Little is known about how the information presented in the informed consent process influences study outcomes among participants. This paper examines how the content of the informed consent document may influence baseline levels of knowledge among study participants. Methods We examined the influence of the content of informed consent documents on reported contraceptive knowledge and concerns among two groups of sero-discordant and HIV concordant positive couples enrolled in research projects at an HIV research center in Lusaka, Zambia. Results We found significant differences in the reporting of both contraceptive knowledge and concerns between couples viewing consent forms that included detailed information on contraception and those viewing consent forms without the detailed information. Conclusions The protection of human subjects is vital. The future challenge, however, lies in creating informed consent documents that can find the balance between ensuring that participants give truly informed consent, and educating participants in ways that compromise the assessment of the impact of behavioral interventions. PMID:21331352

  11. Middle school students' earthquake content and preparedness knowledge - A mixed method study

    NASA Astrophysics Data System (ADS)

    Henson, Harvey, Jr.

    The purpose of this study was to assess the effect of earthquake instruction on students' earthquake content and preparedness for earthquakes. This study used an innovative direct instruction on earthquake science content and concepts with an inquiry-based group activity on earthquake safety followed by an earthquake simulation and preparedness video to help middle school students understand and prepare for the regional seismic threat. A convenience sample of 384 sixth and seventh grade students at two small middle schools in southern Illinois was used in this study. Qualitative information was gathered using open-ended survey questions, classroom observations, and semi-structured interviews. Quantitative data were collected using a 21 item content questionnaire administered to test students' General Earthquake Knowledge, Local Earthquake Knowledge, and Earthquake Preparedness Knowledge before and after instruction. A pre-test and post-test survey Likert scale with 21 items was used to collect students' perceptions and attitudes. Qualitative data analysis included quantification of student responses to the open-ended questions and thematic analysis of observation notes and interview transcripts. Quantitative datasets were analyzed using descriptive and inferential statistical methods, including t tests to evaluate the differences in means scores between paired groups before and after interventions and one-way analysis of variance (ANOVA) to test for differences between mean scores of the comparison groups. Significant mean differences between groups were further examined using a Dunnett's C post hoc statistical analysis. Integration and interpretation of the qualitative and quantitative results of the study revealed a significant increase in general, local and preparedness earthquake knowledge among middle school students after the interventions. The findings specifically indicated that these students felt most aware and prepared for an earthquake after an

  12. Content Area Reading Instruction for Secondary Teacher Candidates: A Case Study of a State-Required Online Content Area Reading Course

    ERIC Educational Resources Information Center

    Biggs, Brad

    2014-01-01

    This dissertation examined in a state-required, online preservice teacher course in content area reading instruction (CARI) at a large land-grant university in Minnesota. Few studies have been published to date on revitalized literacy teacher preparation efforts in CARI (See Vagle, Dillon, Davison-Jenkins, & LaDuca, 2005; Dillon, O'Brien,…

  13. Content-related interactions and methods of reasoning within self-initiated organic chemistry study groups

    NASA Astrophysics Data System (ADS)

    Christian, Karen Jeanne

    2011-12-01

    Students often use study groups to prepare for class or exams; yet to date, we know very little about how these groups actually function. This study looked at the ways in which undergraduate organic chemistry students prepared for exams through self-initiated study groups. We sought to characterize the methods of social regulation, levels of content processing, and types of reasoning processes used by students within their groups. Our analysis showed that groups engaged in predominantly three types of interactions when discussing chemistry content: co-construction, teaching, and tutoring. Although each group engaged in each of these types of interactions at some point, their prevalence varied between groups and group members. Our analysis suggests that the types of interactions that were most common depended on the relative content knowledge of the group members as well as on the difficulty of the tasks in which they were engaged. Additionally, we were interested in characterizing the reasoning methods used by students within their study groups. We found that students used a combination of three content-relevant methods of reasoning: model-based reasoning, case-based reasoning, or rule-based reasoning, in conjunction with one chemically-irrelevant method of reasoning: symbol-based reasoning. The most common way for groups to reason was to use rules, whereas the least common way was for students to work from a model. In general, student reasoning correlated strongly to the subject matter to which students were paying attention, and was only weakly related to student interactions. Overall, results from this study may help instructors to construct appropriate tasks to guide what and how students study outside of the classroom. We found that students had a decidedly strategic approach in their study groups, relying heavily on material provided by their instructors, and using the reasoning strategies that resulted in the lowest levels of content processing. We suggest

  14. A content validity study of signs, symptoms and diseases/health problems expressed in LIBRAS1

    PubMed Central

    Aragão, Jamilly da Silva; de França, Inacia Sátiro Xavier; Coura, Alexsandro Silva; de Sousa, Francisco Stélio; Batista, Joana D'arc Lyra; Magalhães, Isabella Medeiros de Oliveira

    2015-01-01

    Objectives: to validate the content of signs, symptoms and diseases/health problems expressed in LIBRAS for people with deafness Method: methodological development study, which involved 36 people with deafness and three LIBRAS specialists. The study was conducted in three stages: investigation of the signs, symptoms and diseases/health problems, referred to by people with deafness, reported in a questionnaire; video recordings of how people with deafness express, through LIBRA, the signs, symptoms and diseases/health problems; and validation of the contents of the recordings of the expressions by LIBRAS specialists. Data were processed in a spreadsheet and analyzed using univariate tables, with absolute frequencies and percentages. The validation results were analyzed using the Content Validity Index (CVI). Results: 33 expressions in LIBRAS, of signs, symptoms and diseases/health problems were evaluated, and 28 expressions obtained a satisfactory CVI (1.00). Conclusions: the signs, symptoms and diseases/health problems expressed in LIBRAS presented validity, in the study region, for health professionals, especially nurses, for use in the clinical anamnesis of the nursing consultation for people with deafness. PMID:26625991

  15. Marker-trait association study for protein content in chickpea (Cicer arietinum L.).

    PubMed

    Jadhav, A A; Rayate, S J; Mhase, L B; Thudi, M; Chitikineni, A; Harer, P N; Jadhav, A S; Varshney, R K; Kulwal, P L

    2015-06-01

    Chickpea (Cicer arietinum L.) is the second most important cool season food legume cultivated in arid and semiarid regions of the world. The objective of the present study was to study variation for protein content in chickpea germplasm, and to find markers associated with it. A set of 187 genotypes comprising both international and exotic collections, and representing both desi and kabuli types with protein content ranging from 13.25% to 26.77% was used. Twenty-three SSR markers representing all eight linkage groups (LG) amplifying 153 loci were used for the analysis. Population structure analysis identified three subpopulations, and corresponding Q values of principal components were used to take care of population structure in the analysis which was performed using general linear and mixed linear models. Marker-trait association (MTA) analysis identified nine significant associations representing four QTLs in the entire population. Subpopulation analyses identified ten significant MTAs representing five QTLs, four of which were common with that of the entire population. Two most significant QTLs linked with markers TR26.205 and CaM1068.195 were present on LG3 and LG5. Gene ontology search identified 29 candidate genes in the region of significant MTAs on LG3. The present study will be helpful in concentrating on LG3 and LG5 for identification of closely linked markers for protein content in chickpea and for their use in molecular breeding programme for nutritional quality improvement.

  16. Endothelin A receptor/β-arrestin signaling to the Wnt pathway renders ovarian cancer cells resistant to chemotherapy.

    PubMed

    Rosanò, Laura; Cianfrocca, Roberta; Tocci, Piera; Spinella, Francesca; Di Castro, Valeriana; Caprara, Valentina; Semprucci, Elisa; Ferrandina, Gabriella; Natali, Pier Giorgio; Bagnato, Anna

    2014-12-15

    The high mortality of epithelial ovarian cancer (EOC) is mainly caused by resistance to the available therapies. In EOC, the endothelin-1 (ET-1, EDN1)-endothelin A receptor (ETAR, EDNRA) signaling axis regulates the epithelial-mesenchymal transition (EMT) and a chemoresistant phenotype. However, there is a paucity of knowledge about how ET-1 mediates drug resistance. Here, we define a novel bypass mechanism through which ETAR/β-arrestin-1 (β-arr1, ARRB1) links Wnt signaling to acquire chemoresistant and EMT phenotype. We found that ETAR/β-arr1 activity promoted nuclear complex with β-catenin and p300, resulting in histone acetylation, chromatin reorganization, and enhanced transcription of genes, such as ET-1, enhancing the network that sustains chemoresistance. Silencing of β-arr1 or pharmacologic treatment with the dual ETAR/ETBR antagonist macitentan prevented core complex formation and restored drug sensitivity, impairing the signaling pathways involved in cell survival, EMT, and invasion. In vivo macitentan treatment reduced tumor growth, vascularization, intravasation, and metastatic progression. The combination of macitentan and cisplatinum resulted in the potentiation of the cytotoxic effect, indicating that macitentan can enhance sensitivity to chemotherapy. Investigations in clinical specimens of chemoresistant EOC tissues confirmed increased recruitment of β-arr1 and β-catenin to ET-1 gene promoter. In these tissues, high expression of ETAR significantly associated with poor clinical outcome and chemoresistance. Collectively, our findings reveal the existence of a novel mechanism by which ETAR/β-arr1 signaling is integrated with the Wnt/β-catenin pathway to sustain chemoresistance in EOC, and they offer a solid rationale for clinical evaluation of macitentan in combination with chemotherapy to overcome chemoresistance in this setting.

  17. Troglitazone stimulates {beta}-arrestin-dependent cardiomyocyte contractility via the angiotensin II type 1{sub A} receptor

    SciTech Connect

    Tilley, Douglas G.; Nguyen, Anny D.; Rockman, Howard A.

    2010-06-11

    Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists are commonly used to treat cardiovascular diseases, and are reported to have several effects on cardiovascular function that may be due to PPAR{gamma}-independent signaling events. Select angiotensin receptor blockers (ARBs) interact with and modulate PPAR{gamma} activity, thus we hypothesized that a PPAR{gamma} agonist may exert physiologic effects via the angiotensin II type 1{sub A} receptor (AT1{sub A}R). In AT1{sub A}R-overexpressing HEK 293 cells, both angiotensin II (Ang II) and the PPAR{gamma} agonist troglitazone (Trog) enhanced AT1{sub A}R internalization and recruitment of endogenous {beta}-arrestin1/2 ({beta}arr1/2) to the AT1{sub A}R. A fluorescence assay to measure diacylglycerol (DAG) accumulation showed that although Ang II induced AT1{sub A}R-G{sub q} protein-mediated DAG accumulation, Trog had no impact on DAG generation. Trog-mediated recruitment of {beta}arr1/2 was selective to AT1{sub A}R as the response was prevented by an ARB- and Trog-mediated {beta}arr1/2 recruitment to {beta}1-adrenergic receptor ({beta}1AR) was not observed. In isolated mouse cardiomyocytes, Trog increased both % and rate of cell shortening to a similar extent as Ang II, effects which were blocked with an ARB. Additionally, these effects were found to be {beta}arr2-dependent, as cardiomyocytes isolated from {beta}arr2-KO mice showed blunted contractile responses to Trog. These findings show for the first time that the PPAR{gamma} agonist Trog acts at the AT1{sub A}R to simultaneously block G{sub q} protein activation and induce the recruitment of {beta}arr1/2, which leads to an increase in cardiomyocyte contractility.

  18. Injection Drug User Quality of Life Scale (IDUQOL): Findings from a content validation study

    PubMed Central

    Hubley, Anita M; Palepu, Anita

    2007-01-01

    Background Quality of life studies among injection drug users have primarily focused on health-related measures. The chaotic life-style of many injection drug users (IDUs), however, extends far beyond their health, and impacts upon social relationships, employment opportunities, housing, and day to day survival. Most current quality of life instruments do not capture the realities of people living with addictions. The Injection Drug Users' Quality of Life Scale (IDUQOL) was developed to reflect the life areas of relevance to IDUs. The present study examined the content validity of the IDUQOL using judgmental methods based on subject matter experts' (SMEs) ratings of various elements of this measure (e.g., appropriateness of life areas or items, names and descriptions of life areas, instructions for administration and scoring). Methods Six SMEs were provided with a copy of the IDUQOL and its administration and scoring manual and a detailed content validation questionnaire. Two commonly used judgmental measures of inter-rater agreement, the Content Validity Index (CVI) and the Average Deviation Mean Index (ADM), were used to evaluate SMEs' agreement on ratings of IDUQOL elements. Results A total of 75 elements of the IDUQOL were examined. The CVI results showed that all elements were endorsed by the required number of SMEs or more. The ADM results showed that acceptable agreement (i.e., practical significance) was obtained for all elements but statistically significant agreement was missed for nine elements. For these elements, SMEs' feedback was examined for ways to improve the elements. Open-ended feedback also provided suggestions for other revisions to the IDUQOL. Conclusion The results of the study provided strong evidence in support of the content validity of the IDUQOL and direction for the revision of some IDUQOL elements. PMID:17663783

  19. Alterations of Myelin Content in Parkinson’s Disease: A Cross-Sectional Neuroimaging Study

    PubMed Central

    Sojkova, Jitka; Hurley, Samuel; Kecskemeti, Steven; Okonkwo, Ozioma; Bendlin, Barbara B.; Theisen, Frances; Johnson, Sterling C.; Alexander, Andrew L.; Gallagher, Catherine L.

    2016-01-01

    Alterations to myelin may be a core pathological feature of neurodegenerative diseases. Although white matter microstructural differences have been described in Parkinson's disease (PD), it is unknown whether such differences include alterations of the brain’s myelin content. Thus, the objective of the current study is to measure and compare brain myelin content between PD patients and age-matched controls. In this cross-sectional study, 63 participants from the Longitudinal MRI in Parkinson's Disease study underwent brain MRI, Unified Parkinson's Disease Rating Scale (UPDRS) scoring, and cognitive asessments. Subjects were imaged with the mcDEPSOT (multi-component driven equilibrium single pulse observation of T1 and T2), a multicomponent relaxometry technique that quantifies longitudinal and transverse relaxation rates (R1 and R2, respectively) and the myelin water fraction (VFM), a surrogate for myelin content. A voxel-wise approach was used to compare R1, R2, and VFM measures between PD and control groups, and to evaluate relationships with age as well as disease duration, UPDRS scores, and daily levodopa equivalent dose. PD subjects had higher VFM than controls in frontal and temporal white matter and bilateral thalamus. Greater age was strongly associated with lower VFM in both groups, while an age-by-group interaction suggested a slower rate of VFM decline in the left putamen with aging in PD. Within the PD group, measures of disease severity, including UPDRS, daily levodopa equivalent dose, and disease duration, were observed to be related with myelin content in diffuse brain regions. The age-by-group interaction suggests that either PD or dopaminergic therapies allay observed age-related myelin changes. The relationships between VFM and disease severity measures suggests that VFM may provide a surrogate marker for microstructural changes related to Parkinson’s disease. PMID:27706215

  20. Qualitative study of ethanol content in tequilas by Raman spectroscopy and principal component analysis

    NASA Astrophysics Data System (ADS)

    Frausto-Reyes, C.; Medina-Gutiérrez, C.; Sato-Berrú, R.; Sahagún, L. R.

    2005-09-01

    Using Raman spectroscopy, with an excitation radiation source of 514.5 nm, and principal component analysis (PCA) was elaborated a method to study qualitatively the ethanol content in tequila samples. This method is based in the OH region profile (water) of the Raman spectra. Also, this method, using the fluorescence background of the Raman spectra, can be used to distinguish silver tequila from aged tequilas. The first three PCs of the Raman spectra, that provide the 99% of the total variance of the data set, were used for the samples classification. The PCA1 and PCA2 are related with the water (or ethanol) content of the sample, whereas the PCA3 is related with the fluorescence background of the Raman spectra.

  1. In vitro study of antioxidant activity and phenolic content of Chrysanthemum balsamita varieties.

    PubMed

    Benedec, Daniela; Filip, Lorena; Vlase, Laurian; Bele, Constantin; Sevastre, Bogdan; Raita, Oana; Olah, Neli-Kinga; Hanganu, Daniela

    2016-07-01

    The purpose of our study was to identify the phenolic substances of two varieties of Chrysanthemum balsamita (balsamita and tanacetoides) and to measure the overall antioxidant activity. The phenolic compounds were determined by HPLC. The evaluation of the polyphenolic content was performed by colorimetric analysis. The antioxidant activity was measured by three in vitro assay models: the DPPH, the silver nanoparticles antioxidant capacity (SNPAC) and EPR radical detection. Using HPLC-MS analysis, phenolic acids, flavonoids and flavonoid aglycone were detected. The highest antioxidant activity was showed by Chrysanthemum balsamita var. balsamita, while the lowest for the Chrysanthemum balsamita var. tanacetoides extract, in accord with the polyphenolic content. The results show that Chrysanthemum balsamita var. balsamita might be a source of antioxidant flavonoids, especially rutin and isoquercitrin. PMID:27592486

  2. Qualitative study of ethanol content in tequilas by Raman spectroscopy and principal component analysis.

    PubMed

    Frausto-Reyes, C; Medina-Gutiérrez, C; Sato-Berrú, R; Sahagún, L R

    2005-09-01

    Using Raman spectroscopy, with an excitation radiation source of 514.5 nm, and principal component analysis (PCA) was elaborated a method to study qualitatively the ethanol content in tequila samples. This method is based in the OH region profile (water) of the Raman spectra. Also, this method, using the fluorescence background of the Raman spectra, can be used to distinguish silver tequila from aged tequilas. The first three PCs of the Raman spectra, that provide the 99% of the total variance of the data set, were used for the samples classification. The PCA1 and PCA2 are related with the water (or ethanol) content of the sample, whereas the PCA3 is related with the fluorescence background of the Raman spectra.

  3. In vitro study of antioxidant activity and phenolic content of Chrysanthemum balsamita varieties.

    PubMed

    Benedec, Daniela; Filip, Lorena; Vlase, Laurian; Bele, Constantin; Sevastre, Bogdan; Raita, Oana; Olah, Neli-Kinga; Hanganu, Daniela

    2016-07-01

    The purpose of our study was to identify the phenolic substances of two varieties of Chrysanthemum balsamita (balsamita and tanacetoides) and to measure the overall antioxidant activity. The phenolic compounds were determined by HPLC. The evaluation of the polyphenolic content was performed by colorimetric analysis. The antioxidant activity was measured by three in vitro assay models: the DPPH, the silver nanoparticles antioxidant capacity (SNPAC) and EPR radical detection. Using HPLC-MS analysis, phenolic acids, flavonoids and flavonoid aglycone were detected. The highest antioxidant activity was showed by Chrysanthemum balsamita var. balsamita, while the lowest for the Chrysanthemum balsamita var. tanacetoides extract, in accord with the polyphenolic content. The results show that Chrysanthemum balsamita var. balsamita might be a source of antioxidant flavonoids, especially rutin and isoquercitrin.

  4. KIF3A binds to β-arrestin for suppressing Wnt/β-catenin signalling independently of primary cilia in lung cancer.

    PubMed

    Kim, Minsuh; Suh, Young-Ah; Oh, Ju-Hee; Lee, Bo Ra; Kim, Joon; Jang, Se Jin

    2016-01-01

    Aberrant Wnt/β-catenin signalling is implicated in the progression of several human cancers, including non-small cell lung cancer (NSCLC). However, mutations in Wnt/β-catenin pathway components are uncommon in NSCLC, and their epigenetic control remains unclear. Here, we show that KIF3A, a member of the kinesin-2 family, plays a role in suppressing Wnt/β-catenin signalling in NSCLC cells. KIF3A knockdown increases both β-catenin levels and transcriptional activity with concomitant promotion of malignant potential, such as increased proliferation and migration and upregulation of stemness markers. Because KIF3A binds β-arrestin, KIF3A depletion allows β-arrestin to form a complex with DVL2 and axin, stabilizing β-catenin. Although primary cilia, whose biogenesis requires KIF3A, are thought to restrain the Wnt response, pharmacological inhibition of ciliogenesis failed to increase β-catenin activity in NSCLC cells. A correlation between KIF3A loss and a poorer NSCLC prognosis as well as β-catenin and cyclin D1 upregulation further suggests that KIF3A suppresses Wnt/β-catenin signalling and tumourigenesis in NSCLC. PMID:27596264

  5. 2-Deoxyglucose Impairs Saccharomyces cerevisiae Growth by Stimulating Snf1-Regulated and α-Arrestin-Mediated Trafficking of Hexose Transporters 1 and 3

    PubMed Central

    O'Donnell, Allyson F.; McCartney, Rhonda R.; Chandrashekarappa, Dakshayini G.; Zhang, Bob B.; Thorner, Jeremy

    2014-01-01

    The glucose analog 2-deoxyglucose (2DG) inhibits the growth of Saccharomyces cerevisiae and human tumor cells, but its modes of action have not been fully elucidated. Yeast cells lacking Snf1 (AMP-activated protein kinase) are hypersensitive to 2DG. Overexpression of either of two low-affinity, high-capacity glucose transporters, Hxt1 and Hxt3, suppresses the 2DG hypersensitivity of snf1Δ cells. The addition of 2DG or the loss of Snf1 reduces HXT1 and HXT3 expression levels and stimulates transporter endocytosis and degradation in the vacuole. 2DG-stimulated trafficking of Hxt1 and Hxt3 requires Rod1/Art4 and Rog3/Art7, two members of the α-arrestin trafficking adaptor family. Mutations in ROD1 and ROG3 that block binding to the ubiquitin ligase Rsp5 eliminate Rod1- and Rog3-mediated trafficking of Hxt1 and Hxt3. Genetic analysis suggests that Snf1 negatively regulates both Rod1 and Rog3, but via different mechanisms. Snf1 activated by 2DG phosphorylates Rod1 but fails to phosphorylate other known targets, such as the transcriptional repressor Mig1. We propose a novel mechanism for 2DG-induced toxicity whereby 2DG stimulates the modification of α-arrestins, which promote glucose transporter internalization and degradation, causing glucose starvation even when cells are in a glucose-rich environment. PMID:25547292

  6. KIF3A binds to β-arrestin for suppressing Wnt/β-catenin signalling independently of primary cilia in lung cancer

    NASA Astrophysics Data System (ADS)

    Kim, Minsuh; Suh, Young-Ah; Oh, Ju-Hee; Lee, Bo Ra; Kim, Joon; Jang, Se Jin

    2016-09-01

    Aberrant Wnt/β-catenin signalling is implicated in the progression of several human cancers, including non-small cell lung cancer (NSCLC). However, mutations in Wnt/β-catenin pathway components are uncommon in NSCLC, and their epigenetic control remains unclear. Here, we show that KIF3A, a member of the kinesin-2 family, plays a role in suppressing Wnt/β-catenin signalling in NSCLC cells. KIF3A knockdown increases both β-catenin levels and transcriptional activity with concomitant promotion of malignant potential, such as increased proliferation and migration and upregulation of stemness markers. Because KIF3A binds β-arrestin, KIF3A depletion allows β-arrestin to form a complex with DVL2 and axin, stabilizing β-catenin. Although primary cilia, whose biogenesis requires KIF3A, are thought to restrain the Wnt response, pharmacological inhibition of ciliogenesis failed to increase β-catenin activity in NSCLC cells. A correlation between KIF3A loss and a poorer NSCLC prognosis as well as β-catenin and cyclin D1 upregulation further suggests that KIF3A suppresses Wnt/β-catenin signalling and tumourigenesis in NSCLC.

  7. The chemokine CXC4 and CC2 receptors form homo- and heterooligomers that can engage their signaling G-protein effectors and βarrestin.

    PubMed

    Armando, Sylvain; Quoyer, Julie; Lukashova, Viktorya; Maiga, Arhamatoulaye; Percherancier, Yann; Heveker, Nikolaus; Pin, Jean-Philippe; Prézeau, Laurent; Bouvier, Michel

    2014-10-01

    G-protein-coupled receptors have been shown to assemble at least as dimers early in the biosynthetic path, but some evidence suggests that they can also form larger oligomeric complexes. Using the human chemokine receptors CXCR4 and CCR2 as models, we directly probed the existence of higher order homo- and heterooligomers in human embryonic kidney cells. Combining bimolecular fluorescence and luminescence complementation (BiFC, BiLC) with bioluminescence resonance energy transfer (BRET) assays, we show that CXCR4 and CCR2 can assemble as homo- and heterooligomers, forming at least tetramers. Selective activation of CCR2 with the human monocyte chemotactic protein 1 (MCP-1) resulted in trans-conformational rearrangement of the CXCR4 dimer with an EC50 of 19.9 nM, compatible with a CCR2 action. Moreover, MCP-1 promoted the engagement of Gαi1, Gα13, Gαz, and βarrestin2 to the heterooligomer, resulting in calcium signaling that was synergistically potentiated on coactivation of CCR2 and CXCR4, demonstrating that complexes larger than dimers reach the cell surface as functional units. A mutation of CXCR4 (N119K), which prevents Gi activation, also affects the CCR2-promoted engagement of Gαi1 and βarrestin2 by the heterooligomer, supporting the occurrence of transprotomer regulation. Together, the results demonstrate that homo- and heteromultimeric CXCR4 and CCR2 can form functional signaling complexes that have unique properties.

  8. 2-Deoxyglucose impairs Saccharomyces cerevisiae growth by stimulating Snf1-regulated and α-arrestin-mediated trafficking of hexose transporters 1 and 3.

    PubMed

    O'Donnell, Allyson F; McCartney, Rhonda R; Chandrashekarappa, Dakshayini G; Zhang, Bob B; Thorner, Jeremy; Schmidt, Martin C

    2015-03-01

    The glucose analog 2-deoxyglucose (2DG) inhibits the growth of Saccharomyces cerevisiae and human tumor cells, but its modes of action have not been fully elucidated. Yeast cells lacking Snf1 (AMP-activated protein kinase) are hypersensitive to 2DG. Overexpression of either of two low-affinity, high-capacity glucose transporters, Hxt1 and Hxt3, suppresses the 2DG hypersensitivity of snf1Δ cells. The addition of 2DG or the loss of Snf1 reduces HXT1 and HXT3 expression levels and stimulates transporter endocytosis and degradation in the vacuole. 2DG-stimulated trafficking of Hxt1 and Hxt3 requires Rod1/Art4 and Rog3/Art7, two members of the α-arrestin trafficking adaptor family. Mutations in ROD1 and ROG3 that block binding to the ubiquitin ligase Rsp5 eliminate Rod1- and Rog3-mediated trafficking of Hxt1 and Hxt3. Genetic analysis suggests that Snf1 negatively regulates both Rod1 and Rog3, but via different mechanisms. Snf1 activated by 2DG phosphorylates Rod1 but fails to phosphorylate other known targets, such as the transcriptional repressor Mig1. We propose a novel mechanism for 2DG-induced toxicity whereby 2DG stimulates the modification of α-arrestins, which promote glucose transporter internalization and degradation, causing glucose starvation even when cells are in a glucose-rich environment.

  9. KIF3A binds to β-arrestin for suppressing Wnt/β-catenin signalling independently of primary cilia in lung cancer

    PubMed Central

    Kim, Minsuh; Suh, Young-Ah; Oh, Ju-Hee; Lee, Bo Ra; Kim, Joon; Jang, Se Jin

    2016-01-01

    Aberrant Wnt/β-catenin signalling is implicated in the progression of several human cancers, including non-small cell lung cancer (NSCLC). However, mutations in Wnt/β-catenin pathway components are uncommon in NSCLC, and their epigenetic control remains unclear. Here, we show that KIF3A, a member of the kinesin-2 family, plays a role in suppressing Wnt/β-catenin signalling in NSCLC cells. KIF3A knockdown increases both β-catenin levels and transcriptional activity with concomitant promotion of malignant potential, such as increased proliferation and migration and upregulation of stemness markers. Because KIF3A binds β-arrestin, KIF3A depletion allows β-arrestin to form a complex with DVL2 and axin, stabilizing β-catenin. Although primary cilia, whose biogenesis requires KIF3A, are thought to restrain the Wnt response, pharmacological inhibition of ciliogenesis failed to increase β-catenin activity in NSCLC cells. A correlation between KIF3A loss and a poorer NSCLC prognosis as well as β-catenin and cyclin D1 upregulation further suggests that KIF3A suppresses Wnt/β-catenin signalling and tumourigenesis in NSCLC. PMID:27596264

  10. Low-field nuclear magnetic resonance for the in vivo study of water content in trees

    SciTech Connect

    Yoder, Jacob; Malone, Michael W.; Espy, Michelle A.; Sevanto, Sanna

    2014-09-15

    Nuclear magnetic resonance (NMR) and magnetic resonance imaging have long been used to study water content in plants. Approaches have been primarily based on systems using large magnetic fields (∼1 T) to obtain NMR signals with good signal-to-noise. This is because the NMR signal scales approximately with the magnetic field strength squared. However, there are also limits to this approach in terms of realistic physiological configuration or those imposed by the size and cost of the magnet. Here we have taken a different approach – keeping the magnetic field low to produce a very light and inexpensive system, suitable for bulk water measurements on trees less than 5 cm in diameter, which could easily be duplicated to measure on many trees or from multiple parts of the same tree. Using this system we have shown sensitivity to water content in trees and their cuttings and observed a diurnal signal variation in tree water content in a greenhouse. We also demonstrate that, with calibration and modeling of the thermal polarization, the system is reliable under significant temperature variation.

  11. Low-field nuclear magnetic resonance for the in vivo study of water content in trees.

    PubMed

    Yoder, Jacob; Malone, Michael W; Espy, Michelle A; Sevanto, Sanna

    2014-09-01

    Nuclear magnetic resonance (NMR) and magnetic resonance imaging have long been used to study water content in plants. Approaches have been primarily based on systems using large magnetic fields (~1 T) to obtain NMR signals with good signal-to-noise. This is because the NMR signal scales approximately with the magnetic field strength squared. However, there are also limits to this approach in terms of realistic physiological configuration or those imposed by the size and cost of the magnet. Here we have taken a different approach--keeping the magnetic field low to produce a very light and inexpensive system, suitable for bulk water measurements on trees less than 5 cm in diameter, which could easily be duplicated to measure on many trees or from multiple parts of the same tree. Using this system we have shown sensitivity to water content in trees and their cuttings and observed a diurnal signal variation in tree water content in a greenhouse. We also demonstrate that, with calibration and modeling of the thermal polarization, the system is reliable under significant temperature variation.

  12. Tolerance of the carotid-sheath contents to brachytherapy: an experimental study

    SciTech Connect

    Werber, J.L.; Sood, B.; Alfieri, A.; McCormick, S.A.; Vikram, B. )

    1991-06-01

    Tumor invasion of the carotid artery is a potential indication for brachytherapy, which delivers a high dose of irradiation to residual tumor while limiting the dose to adjacent healthy tissues. The tolerance of carotid-sheath contents to varying doses of brachytherapy, however, has not been clearly established. In order to evaluate brachytherapy effects on carotid-sheath contents, after-loading catheters were implanted bilaterally in 3 groups of 6 rabbits each (18 rabbits). Iridium 192 brachytherapy doses of either 5000 cGy (rad), 9000 cGy, or 13,000 cGy were delivered unilaterally, with the contralateral neck serving as a nonirradiated control in each animal. There were no carotid ruptures and wound healing was normal. Two animals from each group were killed at 6, 20, and 48 weeks. Even at the highest dose (13,000 cGy), nerve conduction studies performed on the vagus nerve prior to sacrifice revealed no increased latency, histologic changes were minimal, and carotid arteries were patent. These observations suggest that the carotid-sheath contents in healthy rabbits could tolerate high doses (up to 13,000 cGy) of low-dose-rate interstitial brachytherapy without complications.

  13. Textbooks Content Analysis of Social Studies and Natural Sciences of Secondary School Based on Emotional Intelligence Components

    ERIC Educational Resources Information Center

    Babaei, Bahare; Abdi, Ali

    2014-01-01

    The aim of this study is to analyze the content of social studies and natural sciences textbooks of the secondary school on the basis of the emotional intelligence components. In order to determine and inspect the emotional intelligence components all of the textbooks content (including texts, exercises, and illustrations) was examined based on…

  14. A case study of alternatively trained science teachers: Attainment of pedagogical content knowledge

    NASA Astrophysics Data System (ADS)

    Duncan, Benjamin R.

    Elements essential to effective teaching are closely aligned with the domains of a teacher's pedagogical content knowledge (PCK) (Park & Oliver, 2008). Often, alternatively trained teachers enter the teaching profession lacking exposure to pedagogical events that allow these educators opportunities to reflect on their practice and construction of their PCK (Friedrichsen et al., 2007); yet little is known about the knowledge of experienced alternatively trained educators and the complexities associated with their PCK development. The purpose of this study was to describe the nature and sources of alternatively trained secondary school science teachers' PCK after gaining classroom experience. The Park and Oliver (2008) hexagon PCK model was used as the theoretical framework. A case study of two experienced secondary science teachers at a school in the southeastern region of the United States was conducted. Data were collected from multiple sources, such as interviews, classroom observations, participant field journals, lesson plans, classroom assignments, classroom assessments, and researcher's field notes. Data analysis was conducted using the constant comparative method, qualitative deductive analysis, and a content representation. The results showed that experienced alternatively trained science teachers' PCK development was heavily influenced by each teacher's orientation to science teaching. Alternatively trained science teachers compensated for their lack of pedagogical training by relying heavily upon their content knowledge, their knowledge of students, and past experiences. Even after gaining years of experience in a school setting, alternatively trained teachers still lacked familiarity with traditional educational terminology and practices, rather relying upon instructional approaches and techniques independently acquired while each teacher was in "survival" mode. This study provides several implications for teacher preparation, research, and policy.

  15. DNA content in reactive hyperplasia, precancerosis, and carcinomas of the oral cavity. A cytophotometric study.

    PubMed

    Doseva, D; Christov, K; Kristeva, K

    1984-01-01

    Cytophotometry has been used to study DNA content in oral epithelial cells of Feulgen-stained specimens from a total of 43 patients: 3 with erythema exudativum multiforme (EEM), 5 with pemphigus, 3 with stomatitis aphtosa, 5 with lichen ruber planus, 8 with leukoplakia, and 19 with carcinomas. In contrast to reactive hyperplasia (EEM, pemphigus, stomatitis aphthosa) leukoplakia has histograms closest to those of carcinoma, with a high percentage of cells in the polyploid regions. This emphasizes the significance of cytophotometry for diagnosis of preneoplastic and neoplastic lesions of the oral cavity.

  16. A study of the trace 39Ar content in argon from deep underground sources

    NASA Astrophysics Data System (ADS)

    Xu, J.; Calaprice, F.; Galbiati, C.; Goretti, A.; Guray, G.; Hohman, T.; Holtz, D.; Ianni, An.; Laubenstein, M.; Loer, B.; Love, C.; Martoff, C. J.; Montanari, D.; Mukhopadhyay, S.; Nelson, A.; Rountree, S. D.; Vogelaar, R. B.; Wright, A.

    2015-06-01

    The discovery of argon from deep underground sources with significantly less 39Ar than atmospheric argon was an important step in the development of direct dark matter detection experiments using argon as the active target. We report on the design and operation of a low-background single-phase liquid argon detector that was built to study the 39Ar content of this underground argon. Underground argon from the Kinder Morgan CO2 plant in Cortez, Colorado was determined to have less than 0.65% of the 39Ar activity in atmospheric argon, or 6.6 mBq/kg specific 39Ar activity.

  17. Incorporating formative assessment and science content into elementary science methods---A case study

    NASA Astrophysics Data System (ADS)

    Brower, Derek John

    Just as elementary students enter the science classroom with prior knowledge and experiences, so do preservice elementary teachers who enter the science methods classroom. Elementary science methods instructors recognize the challenges associated with preparing teachers for the science classroom. Two of these challenges include overcoming limited science content understanding and a low science teaching efficacy. Based upon research in science misconceptions, conceptual change theory, formative assessment, and science teaching efficacy, this design experiment explored the use of formative assessment in an authentic learning environment to address some of these challenges. As a case study, the goal was to identify two specific topics in science which the preservice teachers did not understand and to model consistent use of formative assessment to guide instruction in those science topics for six weeks. The research questions for this study sought to explore the design of the class while also exploring students' understanding of the science content and their understanding of formative assessment. One specific question was whether the formative data could differentiate between deeply held student misconceptions in science and incomplete science understanding. In addition, data was collected to measure changes in science teaching efficacy as well as preservice teachers' desire to use formative assessment in their own future classrooms. Based upon student interviews and a final content quiz, the participants in this study did show improved science content understanding in the areas of plant food/energy and plate tectonics. The course design implemented a variety of formative assessment tools including formative assessment probes, student science notebooks, student concept maps, a non-graded quiz, and more. The STEBI-B survey identified improved science teaching efficacy among the participants. Student final essays indicated improved understanding of formative assessment

  18. [Study on estimation of deserts soil total phosphorus content from thermal-infrared emissivity].

    PubMed

    Hou, Yan-jun; Tiyip, Tashpolat; Zhang, Fei; Sawut, Mamat; Nurmemet, Ilyas

    2015-02-01

    Soil phosphorus provides nutrient elements for plants, is one of important parameters for evaluating soil quality. The traditional method for soil total phosphorus content (STPC) measurement is not effective and time-consuming. However, remote sensing (RS) enables us to determine STPC in a fast and efficient way. Studies on the estimation of STPC in near-infrared spectroscopy have been developed by scholars, but model accuracy is still poor due to the low absorption coefficient and unclear absorption peak of soil phosphorus in near-infrared. In order to solve the deficiency which thermal-infrared emissivity estimate desert soil total phosphorus content, and could improve precision of estimation deserts soil total phosphorus. In this paper, characteristics of soil thermal-infrared emissivity are analyzed on the basis of laboratory processing and spectral measurement of deserts soil samples from the eastern Junggar Basin. Furthermore, thermal-infrared emissivity based RS models for STPC estimation are established and accuracy assessed. Results show that: when STPC is higher than 0.200 g x kg(-1), the thermal-infrared emissivity increases with the increase of STPC on the wavelength between 8.00 microm and 13 microm, and the emissivity is more sensitive to STPC on the wavelength between 9.00 and 9.6 microm; the estimate mode based on multiple stepwise regression was could not to estimate deserts soil total phosphorus content from thermal-infrared emissivity because the estimation effects of them were poor. The estimation accuracy of model based on partial least squares regression is higher than the model based on multiple stepwise regression. However, the accuracy of second-order differential estimation model based on partial least square regression is higher than based on multiple stepwise regression; The first differential of continuous remove estimation model based on partial least squares regression is the best model with R2 of correction and verification are up to

  19. The Use of Lesson Study Combined with Content Representation in the Planning of Physics Lessons During Field Practice to Develop Pedagogical Content Knowledge

    NASA Astrophysics Data System (ADS)

    Juhler, Martin Vogt

    2016-08-01

    Recent research, both internationally and in Norway, has clearly expressed concerns about missing connections between subject-matter knowledge, pedagogical competence and real-life practice in schools. This study addresses this problem within the domain of field practice in teacher education, studying pre-service teachers' planning of a Physics lesson. Two means of intervention were introduced. The first was lesson study, which is a method for planning, carrying out and reflecting on a research lesson in detail with a learner and content-centered focus. This was used in combination with a second means, content representations, which is a systematic tool that connects overall teaching aims with pedagogical prompts. Changes in teaching were assessed through the construct of pedagogical content knowledge (PCK). A deductive coding analysis was carried out for this purpose. Transcripts of pre-service teachers' planning of a Physics lesson were coded into four main PCK categories, which were thereafter divided into 16 PCK sub-categories. The results showed that the intervention affected the pre-service teachers' potential to start developing PCK. First, they focused much more on categories concerning the learners. Second, they focused far more uniformly in all of the four main categories comprising PCK. Consequently, these differences could affect their potential to start developing PCK.

  20. A G Protein-biased Designer G Protein-coupled Receptor Useful for Studying the Physiological Relevance of Gq/11-dependent Signaling Pathways.

    PubMed

    Hu, Jianxin; Stern, Matthew; Gimenez, Luis E; Wanka, Lizzy; Zhu, Lu; Rossi, Mario; Meister, Jaroslawna; Inoue, Asuka; Beck-Sickinger, Annette G; Gurevich, Vsevolod V; Wess, Jürgen

    2016-04-01

    Designerreceptorsexclusivelyactivated by adesignerdrug (DREADDs) are clozapine-N-oxide-sensitive designer G protein-coupled receptors (GPCRs) that have emerged as powerful novel chemogenetic tools to study the physiological relevance of GPCR signaling pathways in specific cell types or tissues. Like endogenous GPCRs, clozapine-N-oxide-activated DREADDs do not only activate heterotrimeric G proteins but can also trigger β-arrestin-dependent (G protein-independent) signaling. To dissect the relative physiological relevance of G protein-mediatedversusβ-arrestin-mediated signaling in different cell types or physiological processes, the availability of G protein- and β-arrestin-biased DREADDs would be highly desirable. In this study, we report the development of a mutationally modified version of a non-biased DREADD derived from the M3muscarinic receptor that can activate Gq/11with high efficacy but lacks the ability to interact with β-arrestins. We also demonstrate that this novel DREADD is activein vivoand that cell type-selective expression of this new designer receptor can provide novel insights into the physiological roles of G protein (Gq/11)-dependentversusβ-arrestin-dependent signaling in hepatocytes. Thus, this novel Gq/11-biased DREADD represents a powerful new tool to study the physiological relevance of Gq/11-dependent signaling in distinct tissues and cell types, in the absence of β-arrestin-mediated cellular effects. Such studies should guide the development of novel classes of functionally biased ligands that show high efficacy in various pathophysiological conditions but display a reduced incidence of side effects.

  1. [Study on Contents and Budgets of Cu, Zn and Cd in an Arable Soil Using AAS].

    PubMed

    Zhao, Ying; Jiang, Chun-ming; Ma, Qiang; Zhou, Hua; Xu, Yong-gang; Yu, Wan-tai

    2015-12-01

    Based on a long-term experiment in Shenyang Experimental Station, the effect of manure application on the contents and budgets of Cu, Zn and Cd in the arable soil was studied. The experiment included four treatments: no mature addition (CK), mature addition 10 t · ha⁻¹ year⁻¹(M1), 25 t · ha⁻¹ year⁻¹ (M2), and 50 t · ha⁻¹ year⁻¹(M3). The result showed that Cu, Zn and Cd in soil were accumulated with manure application and prolongation of experiment, and the accumulative magnitude increased with increasing of manure application. The average annual growth rates of the heavy metals in the four treatments (CK, M1, M2, M3) were 2.83%, 6.56%, 7.54%, 8.96%; 0.03%, 3.44%, 4.53%, 6.64% and 1.51%, 8.01%, 10. 27%, 16. 08% for Cu, Zn and Cd, respectively. After six years of the experiment, the content of Cd in the M3 treatment was quite close to the threshold of Chinese Soil Quality Standard Grade III (1 mg · kg⁻¹, GB15618-1995). After 12 years of the experiment, the contents of Cu in the mature-amended treatments fell in the Chinese Soil Quality Standard Grade III, which should be paid more attention. Although the heavy metals in soil were gradually accumulated, the Cu, Zn and Cd levels in crop grain were still below the National Food Contamination Standards (GB2762-2005; GB13106-91; GB15199-94), indicating the contents of heavy metals in crop produced from contaminated soil might not exceed the corresponding standards. The contents of Cu, Zn and Cd in the straw were much greater than those in the grain. The removal of heavy metal by crop was in the order of M3 > M2 > M1 > CK. The average amounts of Cu, Zn and Cd annually removed from the soil in the four treatments (CK, M1, M2 and M3) were 35.68, 47.80, 63.65, 69.64; 249.14, 375.22, 375.16, 444.44, and 0.83, 1.39, 1.64, 1.66 g · ha⁻¹, respectively. The contents of heavy metals in organic manure varied in different years: the contents of Cu and Zn increased year by year, while Cd presented a

  2. Integrating Perioperative Content in Nursing Curricula: A Case Study Approach.

    PubMed

    Byrne, Michelle; Root, Susan; Culbertson, Laurie

    2016-06-01

    Perioperative nursing care requires unique specialty knowledge, skills, and abilities. National initiatives in nursing education and health care support integrating perioperative nursing content into curricular offerings in nursing schools and health care institutions. We provide an overview of the initiatives affecting nursing education, followed by a case study example and a guide to assist educators with incorporating perioperative case studies into their education plans. These resources may enhance the integration of perioperative nursing concepts in undergraduate curricula, internships, and continuing education offerings. The purpose of this article is to provide resources for nurse educators to systematically create case studies and to encourage increased exposure to perioperative concepts and competencies in a myriad of educational environments. PMID:27234794

  3. Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content

    PubMed Central

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit; Saari, Heikki; Ibañez, Elisa Lazaro; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2015-01-01

    Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level. PMID:26649679

  4. Sexual function and depressive symptoms in young women with elevated macroprolactin content: a pilot study.

    PubMed

    Krysiak, Robert; Drosdzol-Cop, Agnieszka; Skrzypulec-Plinta, Violetta; Okopien, Bogusław

    2016-07-01

    Elevated prolactin levels seem to be associated with impaired sexuality. The clinical significance of macroprolactinemia, associated with the predominance of high molecular mass circulating forms of prolactin, is still poorly understood. This study was aimed at investigating sexual function in young women with macroprolactinemia. The study enrolled 14 young women with macroprolactinemia, 14 with increased monomeric prolactin levels, as well as 14 age- and weight-matched healthy women. All patients completed a questionnaire evaluating female sexual function (Female Sexual Function Index-FSFI), as well as a questionnaire assessing the presence and severity of depressive symptoms (Beck Depression Inventory Second Edition-BDI-II). Apart from total prolactin levels and macroprolactin content, circulating levels of thyrotropin, total testosterone, and 17-β estradiol were also measured. Patients with elevated monomeric prolactin levels had a lower total FSFI score, as well as lower scores for all domains: sexual desire, sexual arousal, lubrication, orgasm, sexual satisfaction, and dyspareunia. These scores correlated with total and monomeric prolactin levels. In turn, women with macroprolactinemia were characterized by a lower score for sexual desire, and only this score correlated with total prolactin levels and macroprolactin content. The total score in the BDI-II questionnaire was higher in patients with hyper- and macroprolactinemia than in the control subjects. Contrary to multidimensional impairment of sexual function in women with elevated monomeric prolactin, macroprolactinemia only seems to disturb sexual desire. PMID:26902871

  5. Some recent studies on groundwater radon content as an earthquake precursor

    SciTech Connect

    Teng, T.

    1980-06-10

    Some recent studies of groundwater radon content in relation to seismic activities are reviewed. Results from China and Japan are presented, including laboratory experients and the development of continuous groundwater radon monitoring systems. In addition, groundwater radon monitoring studies conducted at the University of Southern California (USC) since 1974 are presented in some detail. The USC project includes a groundwater radon monitoring network of 14 sampling sites at cold springs, hot springs, deep irrigation wells, and artesian wells that are distributed along a 'locked' stretch of the San Andreas fault from Gorman to San Bernardino in California. Radon contents in weekly samples from these sites are analyzed to the precision of a few percent. No moderate-to-large earthquake has yet occurred in the vicinity, and the results show no clear association of radon anomalies with small (Mapprox.3) earthquakes to provide better test conditions. In contrast to earlier reports from Russia showing a long-term buildup of radon emission before earthquake occurrence, several recent reports from China and Japan show that groundwater radon anomalies can be short in duration and occur only within a few days before the main shocks. Observation of such effects are made possible with the use of continuous monitoring systems. In general, available data suggest that groundwater radon monitoring could sometimes yield precursor information. However, criteria have yet to be established by which sampling sites and sampling frequencies are selected.

  6. Regional mapping of forest canopy water content and biomass using AIRSAR images over BOREAS study area

    NASA Technical Reports Server (NTRS)

    Saatchi, Sasan; Rignot, Eric; Vanzyl, Jakob

    1995-01-01

    In recent years, monitoring vegetation biomass over various climate zones has become the primary focus of several studies interested in assessing the role of the ecosystem responses to climate change and human activities. Airborne and spaceborne Synthetic Aperture Radar (SAR) systems provide a useful tool to directly estimate biomass due to its sensitivity to structural and moisture characteristics of vegetation canopies. Even though the sensitivity of SAR data to total aboveground biomass has been successfully demonstrated in many controlled experiments over boreal forests and forest plantations, so far, no biomass estimation algorithm has been developed. This is mainly due to the fact that the SAR data, even at lowest frequency (P-band) saturates at biomass levels of about 200 tons/ha, and the structure and moisture information in the SAR signal forces the estimation algorithm to be forest type dependent. In this paper, we discuss the development of a hybrid forest biomass algorithm which uses a SAR derived land cover map in conjunction with a forest backscatter model and an inversion algorithm to estimate forest canopy water content. It is shown that unlike the direct biomass estimation from SAR data, the estimation of water content does not depend on the seasonal and/or environmental conditions. The total aboveground biomass can then be derived from canopy water content for each type of forest by incorporating other ecological information. Preliminary results from this technique over several boreal forest stands indicate that (1) the forest biomass can be estimated with reasonable accuracy, and (2) the saturation level of the SAR signal can be enhanced by separating the crown and trunk biomass in the inversion algorithm. We have used the JPL AIRSAR data over BOREAS southern study area to test the algorithm and to generate regional scale water content and biomass maps. The results are compared with ground data and the sources of errors are discussed. Several SAR

  7. Studies on segmented polyetherurethane for biomedical application: effects of composition and hard-segment content on biocompatibility.

    PubMed

    Chen, J H; Wei, J; Chang, C Y; Laiw, R F; Lee, Y D

    1998-09-15

    Segmented polyetherurethane (SPEU) materials based on polytetramethylene oxide (PTMO, Mw 1000 and 2000) with various hard-segment contents were synthesized and their biocompatibilities studied via different tests. The static contact angle data reveal that the higher hard-segment-content SPEU material possesses a lower contact angle, implying that the surface of the higher hard-segment-content SPEU is more hydrophilic than its low hard-segment-content SPEU counterpart. The catalyst- and additive-free PTMO-based SPEU materials in this study possess neither a hemolytic nor a cytotoxic response that could be considered non toxic for biomedical applications. By using L-929 cell lines, a cell-seeding test indicated that the higher hard-segment-content SPEU material possesses quicker cell attachment and proliferation behaviors. In vitro platelet adhesion tests indicated that the lower hard-segment-content SPEU possesses less platelet adhesion than the high hard-segment-content SPEU material. Both ex vivo canine artery-artery (A-A) and arterio-venous (A-V) shunting tests revealed that the extent of platelet adhesion reaction is less for lower hard-segment content SPEU. In addition, the blood compatibility of SPEU material synthesized from PTMO 1000 excels over PTMO 2000 SPEU material by near the same levels as the hard-segment-content SPEU. PMID:9697037

  8. Thiol and metal contents in periphyton exposed to elevated copper and zinc concentrations: a field and microcosm study.

    PubMed

    Le Faucheur, Séverine; Behra, Renata; Sigg, Laura

    2005-10-15

    Phytochelatins are metal-binding polypeptides produced by algae under metal exposure. The aim of this study was to investigate the effects of metal concentration variations in natural systems on periphyton at the biochemical level by analyzing its intracellular thiol content, in particular phytochelatins. To that purpose, two field campaigns were conducted in a stream subject to an increase of dissolved metal concentrations (particularly Cu and Zn) during rain events, which results in an increase of their accumulation in periphyton. At background metal concentrations, several thiols were detectable in periphyton, namely, glutathione (GSH), gamma-glutamylcysteine (gammaGluCys), phytochelatins (PC2), and some unidentified thiols, U1 and U2. Glutathione and gammaGluCys contents were found to vary independently of the rain, as well as U1 and U2, whereas the phytochelatin content increased during the rain events. To investigate whether Cu or Zn may be responsible for this increase, microcosm experiments were carried out with natural water enriched with Cu, Zn, and Cd separately, and Cu and Zn in combination. In this study, GSH, PC2, and U1 were also detected, but not gammaGluCys. An increase in accumulated Cu content did not induce any changes in thiol content, whereas an increase of the Zn content induced a decrease in GSH content and an increase in phytochelatin content. Zinc rather than Cu may thus induce a phytochelatin content increase in periphyton in the field studies. Addition of Cu and Zn in combination also induced an increase in phytochelatin content. Cadmium was found to be the most effective inducer, with the production of larger phytochelatins (PC3-4). This study is the first one to report changes in thiol content in periphyton in response to an increase of the metal concentration in natural freshwaters. PMID:16295881

  9. Comparison of Problem-Based Learning Studies in Science Education in Turkey with the World: Content Analysis of Research Papers

    ERIC Educational Resources Information Center

    Tosun, Cemal; Yasar, M. Diyaddin

    2013-01-01

    Content analysis of the studies of both Turkish and foreign authors on PBL in science education in national and international journals was made considering such variables as the research subject, method, sample, diversity of data collection tools and the data analysis methods. To this end, content analysis of 104 papers - 42 by Turkish authors and…

  10. Outcome Reporting Bias in Government-Sponsored Policy Evaluations: A Qualitative Content Analysis of 13 Studies

    PubMed Central

    2016-01-01

    The reporting of evaluation outcomes can be a point of contention between evaluators and policy-makers when a given reform fails to fulfil its promises. Whereas evaluators are required to report outcomes in full, policy-makers have a vested interest in framing these outcomes in a positive light–especially when they previously expressed a commitment to the reform. The current evidence base is limited to a survey of policy evaluators, a study on reporting bias in education research and several studies investigating the influence of industry sponsorship on the reporting of clinical trials. The objective of this study was twofold. Firstly, it aimed to assess the risk of outcome reporting bias (ORB or ‘spin’) in pilot evaluation reports, using seven indicators developed by clinicians. Secondly, it sought to examine how the government’s commitment to a given reform may affect the level of ORB found in the corresponding evaluation report. To answer these questions, 13 evaluation reports were content-analysed, all of which found a non-significant effect of the intervention on its stated primary outcome. These reports were systematically selected from a dataset of 233 pilot and experimental evaluations spanning three policy areas and 13 years of government-commissioned research in the UK. The results show that the risk of ORB is real. Indeed, all studies reviewed here resorted to at least one of the presentational strategies associated with a risk of spin. This study also found a small, negative association between the seniority of the reform’s champion and the risk of ORB in the evaluation of that reform. The publication of protocols and the use of reporting guidelines are recommended. PMID:27690131

  11. An Analysis of Social Studies Teachers' Perception Levels Regarding Web Pedagogical Content Knowledge

    ERIC Educational Resources Information Center

    Yesiltas, Erkan

    2016-01-01

    Web pedagogical content knowledge generally takes pedagogical knowledge, content knowledge, and Web knowledge as basis. It is a structure emerging through the interaction of these three components. Content knowledge refers to knowledge of subjects to be taught. Pedagogical knowledge involves knowledge of process, implementation, learning methods,…

  12. [Study on the relationship between spectral properties of oilseed rape leaves and their chlorophyll content].

    PubMed

    Fang, Hui; Song, Hai-Yan; Cao, Fang; He, Yong; Qiu, Zheng-Jun

    2007-09-01

    Chlorophyll is the important factors of the crop in its growth stage, and it is the favorable indicator of nutrition stress and photosynthesis. Site-specific crop nutrition diagnosis is the basics of the scientific fertilizer management, and it is essential for the practice of precision agriculture. Spectral properties of the oilseed rape leaves in different nitrogenous fertilizer levels were measured using visible-near infrared reflectance spectroscopy (Vis-NIRS) with natural illumination in the present study. According to the unique spectral properties of the crop, multiple stepwise regression technique was used to find the relationships between chlorophyll content and red edge, green edge. The result shows that the model with two independent variables (red edge, green edge) was better than with the one independeot variable (red edge). The correlation coefficient was 0.863, 0.848, and SEC was 5.273, 5.459, respectively. It can be seen the combination of the red edge and green edge can accurately predict chlorophyll content.

  13. Studies of levels of biogenic amines in meat samples in relation to the content of additives.

    PubMed

    Jastrzębska, Aneta; Kowalska, Sylwia; Szłyk, Edward

    2016-01-01

    The impact of meat additives on the concentration of biogenic amines and the quality of meat was studied. Fresh white and red meat samples were fortified with the following food additives: citric and lactic acids, disodium diphosphate, sodium nitrite, sodium metabisulphite, potassium sorbate, sodium chloride, ascorbic acid, α-tocopherol, propyl 3,4,5-trihydroxybenzoate (propyl gallate) and butylated hydroxyanisole. The content of spermine, spermidine, putrescine, cadaverine, histamine, tyramine, tryptamine and 2-phenylethylamine was determined by capillary isotachophoretic methods in meat samples (fresh and fortified) during four days of storage at 4°C. The results were applied to estimate the impact of the tested additives on the formation of biogenic amines in white and red meat. For all tested meats, sodium nitrite, sodium chloride and disodium diphosphate showed the best inhibition. However, cadaverine and putrescine were characterised by the biggest changes in concentration during the storage time of all the additives. Based on the presented data for the content of biogenic amines in meat samples analysed as a function of storage time and additives, we suggest that cadaverine and putrescine have a significant impact on meat quality. PMID:26515667

  14. Associative Transcriptomics Study Dissects the Genetic Architecture of Seed Glucosinolate Content in Brassica napus

    PubMed Central

    Lu, Guangyuan; Harper, Andrea L.; Trick, Martin; Morgan, Colin; Fraser, Fiona; O'Neill, Carmel; Bancroft, Ian

    2014-01-01

    Breeding new varieties with low seed glucosinolate (GS) concentrations has long been a prime target in Brassica napus. In this study, a novel association mapping methodology termed ‘associative transcriptomics’ (AT) was applied to a panel of 101 B. napus lines to define genetic regions and also candidate genes controlling total seed GS contents. Over 100,000 informative single-nucleotide polymorphisms (SNPs) and gene expression markers (GEMs) were developed for AT analysis, which led to the identification of 10 SNP and 7 GEM association peaks. Within these peaks, 26 genes were inferred to be involved in GS biosynthesis. A weighted gene co-expression network analysis provided additional 40 candidate genes. The transcript abundance in leaves of two candidate genes, BnaA.GTR2a located on chromosome A2 and BnaC.HAG3b on C9, was correlated with seed GS content, explaining 18.8 and 16.8% of phenotypic variation, respectively. Resequencing of genomic regions revealed six new SNPs in BnaA.GTR2a and four insertions or deletions in BnaC.HAG3b. These deletion polymorphisms were then successfully converted into polymerase chain reaction–based diagnostic markers that can, due to high linkage disequilibrium observed in these regions of the genome, be used for marker-assisted breeding for low seed GS lines. PMID:25030463

  15. Studying calcium triggered vesicle fusion in a single vesicle-vesicle content/lipid mixing system

    PubMed Central

    Kyoung, Minjoung; Zhang, Yunxiang; Diao, Jiajie; Chu, Steven; Brunger, Axel T.

    2013-01-01

    This Protocol describes a single vesicle-vesicle microscopy system to study Ca2+-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and SNAP-25, and are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at zero Ca2+-concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca2+-injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-hundred millisecond timescale. Other factors, such as complexin, can be easily added. Our system is unique by monitoring both content and lipid mixing, and by starting from a metastable state of interacting vesicle pairs prior to Ca2+-injection. PMID:23222454

  16. Studies of levels of biogenic amines in meat samples in relation to the content of additives.

    PubMed

    Jastrzębska, Aneta; Kowalska, Sylwia; Szłyk, Edward

    2016-01-01

    The impact of meat additives on the concentration of biogenic amines and the quality of meat was studied. Fresh white and red meat samples were fortified with the following food additives: citric and lactic acids, disodium diphosphate, sodium nitrite, sodium metabisulphite, potassium sorbate, sodium chloride, ascorbic acid, α-tocopherol, propyl 3,4,5-trihydroxybenzoate (propyl gallate) and butylated hydroxyanisole. The content of spermine, spermidine, putrescine, cadaverine, histamine, tyramine, tryptamine and 2-phenylethylamine was determined by capillary isotachophoretic methods in meat samples (fresh and fortified) during four days of storage at 4°C. The results were applied to estimate the impact of the tested additives on the formation of biogenic amines in white and red meat. For all tested meats, sodium nitrite, sodium chloride and disodium diphosphate showed the best inhibition. However, cadaverine and putrescine were characterised by the biggest changes in concentration during the storage time of all the additives. Based on the presented data for the content of biogenic amines in meat samples analysed as a function of storage time and additives, we suggest that cadaverine and putrescine have a significant impact on meat quality.

  17. Associative transcriptomics study dissects the genetic architecture of seed glucosinolate content in Brassica napus.

    PubMed

    Lu, Guangyuan; Harper, Andrea L; Trick, Martin; Morgan, Colin; Fraser, Fiona; O'Neill, Carmel; Bancroft, Ian

    2014-12-01

    Breeding new varieties with low seed glucosinolate (GS) concentrations has long been a prime target in Brassica napus. In this study, a novel association mapping methodology termed 'associative transcriptomics' (AT) was applied to a panel of 101 B. napus lines to define genetic regions and also candidate genes controlling total seed GS contents. Over 100,000 informative single-nucleotide polymorphisms (SNPs) and gene expression markers (GEMs) were developed for AT analysis, which led to the identification of 10 SNP and 7 GEM association peaks. Within these peaks, 26 genes were inferred to be involved in GS biosynthesis. A weighted gene co-expression network analysis provided additional 40 candidate genes. The transcript abundance in leaves of two candidate genes, BnaA.GTR2a located on chromosome A2 and BnaC.HAG3b on C9, was correlated with seed GS content, explaining 18.8 and 16.8% of phenotypic variation, respectively. Resequencing of genomic regions revealed six new SNPs in BnaA.GTR2a and four insertions or deletions in BnaC.HAG3b. These deletion polymorphisms were then successfully converted into polymerase chain reaction-based diagnostic markers that can, due to high linkage disequilibrium observed in these regions of the genome, be used for marker-assisted breeding for low seed GS lines.

  18. Study of arsenic content in mine groundwater commonly used for human consumption in Utah.

    PubMed

    Pawlak, Z; Rauckyte, T; Zak, S; Praveen, P

    2008-02-01

    Of the various sources of arsenic released in to the environment, the presence of arsenic in water probably poses the greatest threat to human health. Arsenic is released in to the environment through water by dissolution of minerals and ores. Natural release is slow, but in some areas the concentration of arsenic in groundwater (commonly referred to as Acid Mine Drainage (or AMD)) is accelerated by mining activity. In fact the presence of arsenic may last a long time even after the mining activity has ceased. Hence it is imperative to study the quality of water (especially for those areas in the vicinity of mines) used for different purposes to identify an appropriate remediation technique for effective pollution control. In this paper, contents of arsenic and other metals in the water were quantified from three different sources: (1) groundwater from the mining tunnel (Judge tunnel), (2) drinking water, and (3) water used in the hydrant-flushed distribution system (Park City) in Utah (USA). The results showed the content of arsenic from the mining tunnel, after chlorination, and in tap water were below 10 microgl(-1). However, significant amounts of arsenic, lead, zinc, iron, manganese and antimony have been found in water samples taken from the distribution systems. In the consideration of the further use of mine groundwater for drinking purposes and the distribution system, Park City should regularly be maintained by a flushing program in the distribution system.

  19. Flavin adenine dinucleotide content of quinone reductase 2: analysis and optimization for structure-function studies.

    PubMed

    Leung, Kevin Ka Ki; Litchfield, David W; Shilton, Brian H

    2012-01-01

    Quinone reductase 2 (NQO2) is a broadly expressed enzyme implicated in responses to a number of compounds, including protein kinase inhibitors, resveratrol, and antimalarial drugs. NQO2 includes a flavin adenine dinucleotide (FAD) cofactor, but X-ray crystallographic analysis of human NQO2 expressed in Escherichia coli showed that electron density for the isoalloxazine ring of FAD was weak and there was no electron density for the adenine mononucleotide moiety. Reversed-phase high-performance liquid chromatography (HPLC) of the NQO2 preparation indicated that FAD was not present and only 38% of the protomers contained flavin mononucleotide (FMN), explaining the weak electron density for FAD in the crystallographic analysis. A method for purifying NQO2 and reconstituting with FAD such that the final content approaches 100% occupancy with FAD is presented here. The enzyme prepared in this manner has a high specific activity, and there is strong electron density for the FAD cofactor in the crystal structure. Analysis of NQO2 crystal structures present in the Protein Data Bank indicates that many may have sub-stoichiometric cofactor content and/or contain FMN rather than FAD. This method of purification and reconstitution will help to optimize structural and functional studies of NQO2 and possibly other flavoproteins.

  20. Biased Gs versus Gq proteins and β-arrestin signaling in the NK1 receptor determined by interactions in the water hydrogen bond network.

    PubMed

    Valentin-Hansen, Louise; Frimurer, Thomas M; Mokrosinski, Jacek; Holliday, Nicholas D; Schwartz, Thue W

    2015-10-01

    X-ray structures, molecular dynamics simulations, and mutational analysis have previously indicated that an extended water hydrogen bond network between trans-membranes I-III, VI, and VII constitutes an allosteric interface essential for stabilizing different active and inactive helical constellations during the seven-trans-membrane receptor activation. The neurokinin-1 receptor signals efficiently through Gq, Gs, and β-arrestin when stimulated by substance P, but it lacks any sign of constitutive activity. In the water hydrogen bond network the neurokinin-1 has a unique Glu residue instead of the highly conserved AspII:10 (2.50). Here, we find that this GluII:10 occupies the space of a putative allosteric modulating Na(+) ion and makes direct inter-helical interactions in particular with SerIII:15 (3.39) and AsnVII:16 (7.49) of the NPXXY motif. Mutational changes in the interface between GluII:10 and AsnVII:16 created receptors that selectively signaled through the following: 1) Gq only; 2) β-arrestin only; and 3) Gq and β-arrestin but not through Gs. Interestingly, increased constitutive Gs but not Gq signaling was observed by Ala substitution of four out of the six core polar residues of the network, in particular SerIII:15. Three residues were essential for all three signaling pathways, i.e. the water-gating micro-switch residues TrpVI:13 (6.48) of the CWXP motif and TyrVII:20 (7.53) of the NPXXY motif plus the totally conserved AsnI:18 (1.50) stabilizing the kink in trans-membrane VII. It is concluded that the interface between position II:10 (2.50), III:15 (3.39), and VII:16 (7.49) in the center of the water hydrogen bond network constitutes a focal point for fine-tuning seven trans-membrane receptor conformations activating different signal transduction pathways.

  1. Inhibitory signaling by CB1 receptors in smooth muscle mediated by GRK5/β-arrestin activation of ERK1/2 and Src kinase.

    PubMed

    Mahavadi, Sunila; Sriwai, Wimolpak; Huang, Jiean; Grider, John R; Murthy, Karnam S

    2014-03-01

    We examined whether CB1 receptors in smooth muscle conform to the signaling pattern observed with other Gi-coupled receptors that stimulate contraction via two Gβγ-dependent pathways (PLC-β3 and phosphatidylinositol 3-kinase/integrin-linked kinase). Here we show that the anticipated Gβγ-dependent signaling was abrogated. Except for inhibition of adenylyl cyclase via Gαi, signaling resulted from Gβγ-independent phosphorylation of CB1 receptors by GRK5, recruitment of β-arrestin1/2, and activation of ERK1/2 and Src kinase. Neither uncoupling of CB1 receptors from Gi by pertussis toxin (PTx) or Gi minigene nor expression of a Gβγ-scavenging peptide had any effect on ERK1/2 activity. The latter was abolished in muscle cells expressing β-arrestin1/2 siRNA. CB1 receptor internalization and both ERK1/2 and Src kinase activities were abolished in cells expressing kinase-deficient GRK5(K215R). Activation of ERK1/2 and Src kinase endowed CB1 receptors with the ability to inhibit concurrent contractile activity. We identified a consensus sequence (102KSPSKLSP109) for phosphorylation of RGS4 by ERK1/2 and showed that expression of a RGS4 mutant lacking Ser103/Ser108 blocked the ability of anandamide to inhibit acetylcholine-mediated phosphoinositide hydrolysis or enhance Gαq:RGS4 association and inactivation of Gαq. Activation of Src kinase by anandamide enhanced both myosin phosphatase RhoA-interacting protein (M-RIP):RhoA and M-RIP:MYPT1 association and inhibited Rho kinase activity, leading to increase of myosin light chain (MLC) phosphatase activity and inhibition of sustained muscle contraction. Thus, unlike other Gi-coupled receptors in smooth muscle, CB1 receptors did not engage Gβγ but signaled via GRK5/β-arrestin activation of ERK1/2 and Src kinase: ERK1/2 accelerated inactivation of Gαq by RGS4, and Src kinase enhanced MLC phosphatase activity, leading to inhibition of ACh-stimulated contraction.

  2. How have researchers studied multiracial populations: A content and methodological review of 20 years of research

    PubMed Central

    Charmaraman, Linda; Woo, Meghan; Quach, Ashley; Erkut, Sumru

    2014-01-01

    The U. S. Census shows that the racial-ethnic make-up of over 9 million people (2.9% of the total population) who self-identified as multiracial is extremely diverse. Each multiracial subgroup has unique social and political histories that may lead to distinct societal perceptions, economic situations, and health outcomes. Despite the increasing academic and media interest in multiracial individuals, there are methodological and definitional challenges in studying the population resulting in conflicting representations in the literature. This content and methods review of articles on multiracial populations provides a comprehensive understanding of which multiracial populations have been included in research and how they have been studied both to recognize the emerging research and to identify gaps for guiding future research on this complex but increasingly visible population. We examine 125 U.S.-based peer-reviewed journal articles published over the past 20 years (1990–2009) containing 133 separate studies focused on multiracial individuals from primarily the fields of psychology, sociology, social work, education, and public health. Findings include (a) descriptive data regarding the sampling strategies, methodologies, and demographic characteristics of studies, including which multiracial subgroups are most studied, gender, age range, region of country, socioeconomic status; (b) major thematic trends in research topics concerning multiracial populations; (c) implications and recommendations for future studies. PMID:25045946

  3. How have researchers studied multiracial populations? A content and methodological review of 20 years of research.

    PubMed

    Charmaraman, Linda; Woo, Meghan; Quach, Ashley; Erkut, Sumru

    2014-07-01

    The U.S. Census shows that the racial-ethnic makeup of over 9 million people (2.9% of the total population) who self-identified as multiracial is extremely diverse. Each multiracial subgroup has unique social and political histories that may lead to distinct societal perceptions, economic situations, and health outcomes. Despite the increasing academic and media interest in multiracial individuals, there are methodological and definitional challenges in studying the population, resulting in conflicting representations in the literature. This content and methods review of articles on multiracial populations provides a comprehensive understanding of which multiracial populations have been included in research and how they have been studied, both to recognize emerging research and to identify gaps for guiding future research on this complex but increasingly visible population. We examine 125 U.S.-based peer-reviewed journal articles published over the past 20 years (1990 to 2009) containing 133 separate studies focused on multiracial individuals, primarily from the fields of psychology, sociology, social work, education, and public health. Findings include (a) descriptive data regarding the sampling strategies, methodologies, and demographic characteristics of studies, including which multiracial subgroups are most studied, gender, age range, region of country, and socioeconomic status; (b) major thematic trends in research topics concerning multiracial populations; and (c) implications and recommendations for future studies.

  4. Assessment study of insight ARTHRO VR (®) arthroscopy virtual training simulator: face, content, and construct validities.

    PubMed

    Bayona, Sofía; Fernández-Arroyo, José Manuel; Martín, Isaac; Bayona, Pilar

    2008-09-01

    The aims of this study were to test the face, content, and construct validities of a virtual-reality haptic arthroscopy simulator and to validate four assessment hypothesis. The participants in our study were 94 arthroscopists attending an international conference on arthroscopy. The interviewed surgeons had been performing arthroscopies for a mean of 8.71 years (σ = 6.94 years). We explained the operation, functionality, instructions for use, and the exercises provided by the simulator. They performed a trial exercise and then an exercise in which performance was recorded. After having using it, the arthroscopists answered a questionnaire. The simulator was classified as one of the best training methods (over phantoms), and obtained a mark of 7.10 out of 10 as an evaluation tool. The simulator was considered more useful for inexperienced surgeons than for surgeons with experience (mean difference 1.88 out of 10, P value < 0.001). The participants valued the simulator at 8.24 as a tool for learning skills, its fidelity at 7.41, the quality of the platform at 7.54, and the content of the exercises at 7.09. It obtained a global score of 7.82. Of the subjects, 30.8% said they would practise with the simulator more than 6 h per week. Of the surgeons, 89.4% affirmed that they would recommend the simulator to their colleagues. The data gathered support the first three hypotheses, as well as face and content validities. Results show statistically significant differences between experts and novices, thus supporting the construct validity, but studies with a larger sample must be carried out to verify this. We propose concrete solutions and an equation to calculate economy of movement. Analogously, we analyze competence measurements and propose an equation to provide a single measurement that contains them all and that, according to the surgeons' criteria, is as reliable as the judgment of experts observing the performance of an apprentice.

  5. Assessment study of insight ARTHRO VR (®) arthroscopy virtual training simulator: face, content, and construct validities.

    PubMed

    Bayona, Sofía; Fernández-Arroyo, José Manuel; Martín, Isaac; Bayona, Pilar

    2008-09-01

    The aims of this study were to test the face, content, and construct validities of a virtual-reality haptic arthroscopy simulator and to validate four assessment hypothesis. The participants in our study were 94 arthroscopists attending an international conference on arthroscopy. The interviewed surgeons had been performing arthroscopies for a mean of 8.71 years (σ = 6.94 years). We explained the operation, functionality, instructions for use, and the exercises provided by the simulator. They performed a trial exercise and then an exercise in which performance was recorded. After having using it, the arthroscopists answered a questionnaire. The simulator was classified as one of the best training methods (over phantoms), and obtained a mark of 7.10 out of 10 as an evaluation tool. The simulator was considered more useful for inexperienced surgeons than for surgeons with experience (mean difference 1.88 out of 10, P value < 0.001). The participants valued the simulator at 8.24 as a tool for learning skills, its fidelity at 7.41, the quality of the platform at 7.54, and the content of the exercises at 7.09. It obtained a global score of 7.82. Of the subjects, 30.8% said they would practise with the simulator more than 6 h per week. Of the surgeons, 89.4% affirmed that they would recommend the simulator to their colleagues. The data gathered support the first three hypotheses, as well as face and content validities. Results show statistically significant differences between experts and novices, thus supporting the construct validity, but studies with a larger sample must be carried out to verify this. We propose concrete solutions and an equation to calculate economy of movement. Analogously, we analyze competence measurements and propose an equation to provide a single measurement that contains them all and that, according to the surgeons' criteria, is as reliable as the judgment of experts observing the performance of an apprentice. PMID:27628252

  6. Pedagogical content knowledge and the gas laws: A multiple case study

    NASA Astrophysics Data System (ADS)

    Sande, Mary Elizabeth

    Pedagogical content knowledge (PCK) has been described as an assemblage of the most powerful analogies, demonstrations, examples and illustrations that make content knowledge understandable to students, together with an understanding of the preconceptions and alternate conceptions that students bring with them to the classroom (Shulman, 1986). In speaking of representations, Johnstone (1991) and Gabel (1993, 1998) suggest that there are three categories of representations in chemistry: the macroscopic, particulate, and symbolic. For the present study, a fourth category has been added, the graphic representation. In addition, Bell, Veal & Tippins (1998) proposed a hierarchy of PCK, a structure wherein the broadest concept (science PCK) is specified by discipline PCK (chemistry PCK) and finally by topic PCK (Gas Law PCK). The present study will investigate the apex of this hierarchy, the intersection of PCK and the specific topic of the Gas Laws. The Gas Law PCK Model was created to illustrate the intersection of subject matter knowledge for teaching and topic-specific PCK. Four chemistry teachers, each holding a degree in chemistry, who had taught high school chemistry for at least three years, and who had taught the Gas Laws during each of the last three years, were given an assessment of their subject matter knowledge for teaching regarding the Gas Laws. Two interviews were conducted to address Gas Law PCK, focusing on representations and student preconceptions and alternate conceptions. Findings of this multiple case study indicate that the participants' subject matter knowledge for teaching, ability to move among representations, i.e. representational competence, and understanding of student alternate conceptions regarding the Gas Laws and how to address those conceptions were limited. Possible influential factors of curricula and lesson planning were also explored. Recommendations for emphasis on specific subject matter knowledge for teaching representations

  7. Iranian nurses’ experience of essential technical competences in disaster response: A qualitative content analysis study

    PubMed Central

    Aliakbari, Fatemeh; Bahrami, Masoud; Aein, Fereshteh; Khankeh, Hamidreza

    2014-01-01

    Background: Today disasters are a part of many people's lives. Iran has a long history of disaster events and nurses are one of the most significant groups within the Iranian disaster relief operations, providing immediate and long-term care for those affected by the disaster. However, the technical competence of Iranian nurses and their training for this work has received little attention. This article presents the results of a study that aims to explore this context. Materials and Methods: A qualitative study was conducted using in-depth interviews to collect data from 30 nurses, who were deliberately selected from the health centers affiliated to the Isfahan University of Medical Sciences. Themes were identified using the conventional qualitative content analysis. The trustworthiness of the study was supported by considering the auditability, neutrality, consistency, and transferability. The study lasted from 2011 to 2012. Results: Data analysis undertaken for the qualitative study resulted in the identification of five main themes, which included: (1) Management competences, (2) ethical and legal competences, (3) team working, and (4) personal abilities and the specific technical competences presented in this report. Conclusions: This report presents an overview of the nursing technical capabilities required for Iranian nurses during disaster relief. It is argued that additional competencies are required for nurses who care in high-risk situations, including disasters. Nurses need to prepare themselves more effectively to be responsible and effective in nursing care. PMID:25558255

  8. Study on fast measurement of sugar content of yogurt using Vis/NIR spectroscopy techniques

    NASA Astrophysics Data System (ADS)

    He, Yong; Feng, Shuijuan; Wu, Di; Li, Xiaoli

    2006-09-01

    In order to measuring the sugar content of yogurt rapidly, a fast measurement of sugar content of yogurt using Vis/NIR-spectroscopy techniques was established. 25 samples selected separately from five different brands of yogurt were measured by Vis/NIR-spectroscopy. The sugar content of yogurt on positions scanned by spectrum were measured by a sugar content meter. The mathematical model between sugar content and Vis/NIR spectral measurements was established and developed based on partial least squares (PLS). The correlation coefficient of sugar content based on PLS model is more than 0.894, and standard error of calibration (SEC) is 0.356, standard error of prediction (SEP) is 0.389. Through predicting the sugar content quantitatively of 35 samples of yogurt from 5 different brands, the correlation coefficient between predictive value and measured value of those samples is more than 0.934. The results show the good to excellent prediction performance. The Vis/NIR spectroscopy technique had significantly greater accuracy for determining the sugar content. It was concluded that the Vis/NIRS measurement technique seems reliable to assess the fast measurement of sugar content of yogurt, and a new method for the measurement of sugar content of yogurt was established.

  9. Studies of Genetic Variation of Essential Oil and Alkaloid Content in Boldo (Peumus boldus).

    PubMed

    Vogel, H; Razmilic, I; Muñoz, M; Doll, U; Martin, J S

    1999-02-01

    Boldo is a tree or shrub with medicinal properties native to Chile. The leaves contain alkaloids and essential oils. Variation of total alkaloid concentration, of the alkaloid boldine, and essential oil components were studied in different populations from northern, central, and southern parts of its geographic range and in their progenies (half-sib families). Total alkaloid concentration showed genetic variation between progenies of the central population but not between populations. Boldine content found in concentrations of 0.007 to 0.009% did not differ significantly between populations. Principal components of the essential oil were determined genetically, with highest values for ascaridole in the population of the north and for P-cymene in the south. Between half-sib families genetic variation was found in the central and northern populations for these components. The high heritability coefficients found indicate considerable potential for successful selection of individuals for these characters. PMID:17260243

  10. Collagen content as a risk factor in breast cancer? A pilot clinical study

    NASA Astrophysics Data System (ADS)

    Pifferi, Antonio; Quarto, Giovanna; Abbate, Francesca; Balestreri, Nicola; Menna, Simona; Cassano, Enrico; Cubeddu, Rinaldo; Taroni, Paola

    2015-07-01

    A retrospective pilot clinical study on time domain multi-wavelength (635 to 1060 nm) optical mammography was exploited to assess collagen as a breast-cancer risk factor on a total of 109 subjects (53 healthy and 56 with malignant lesions). An increased cancer occurrence is observed on the 15% subset of patients with higher age-matched collagen content. Further, a similar clustering based on the percentage breast density leads to a different set of patients, possibly indicating collagen as a new independent breast cancer risk factor. If confirmed statistically and on larger numbers, these results could have huge impact on personalized diagnostics, health care systems, as well as on basic research.

  11. Study of slag content and properties after plasma melting of incineration ash

    NASA Astrophysics Data System (ADS)

    Park, Hyun-Seo

    2011-06-01

    The paper presents the investigation of plasma melting of the mixed bottom and fly incineration ash at various mixing ratios of the components. Chemical compound of the bottom and fly ash as well as the slag after its melting was analyzed by different methods, and the content of toxic components in them was determined. It is demonstrated that the direct disposal of the fly and bottom incineration ash may cause dioxin and heavy metal contamination of the environment. The influence of melted ash basicity on the resulting slag compound was studied. The mass balance of the melting process was defined. The tests were performed to determine the heavy-metals leaching from the ash and slag. It is also shown that the slag after plasma melting is dioxin-free and environmentally friendly.

  12. Determination of nitrogen content in milk by the Kjeldahl method using copper sulfate: interlaboratory study.

    PubMed

    Grappin, R; Horwitz, W

    1988-01-01

    Copper sulfate was substituted for mercury as the catalyst in the International Dairy Federation (IDF) Standard 20A:1986 method for the determination of nitrogen content in milk. The substitution was supported by results obtained in an interlaboratory study by 24 laboratories in 12 countries. Each laboratory analyzed 12 test samples of milk as blind duplicates in a double split level design with high, medium, and low nitrogen concentrations. The method protocol requires the concurrent analyses of an ammonium salt solution and a tryptophan solution as internal quality control standards with a minimum nitrogen recovery between 99 and 100% for the former and at least 98% for the latter. The repeatability and reproducibility relative standard deviations are 0.5 and 1%, respectively, for the range 0.35-0.70 g N/100 g. The performance of the laboratories that did not meet the required quality control specifications was clearly poorer than that of those that did meet the specifications.

  13. Content management systems and E-commerce: a comparative case study

    NASA Astrophysics Data System (ADS)

    Al Rasheed, Amal A.; El-Masri, Samir D.

    2011-12-01

    The need for CMS's to create and edit e-commerce websites has increased with the growing importance of e-commerce. In this paper, the various features essential for e-commerce CMS's are explored. The aim of the paper was to find the best CMS solution for e-commerce which includes the best of both CMS and store management. Accordingly, we conducted a study on three popular open source CMS's for e-commerce: VirtueMart from Joomla!, Ubercart from Drupal, and Magento. We took into account features like hosting and installation, performance, support/community, content management, add on modules and functional features. We concluded with improvements that could be made in order to alleviate problems.

  14. Determination of nitrogen content in milk by the Kjeldahl method using copper sulfate: interlaboratory study

    SciTech Connect

    Grappin, R.; Horwitz, W.

    1988-01-01

    Copper sulfate was substituted for mercury as the catalysts in the International Dairy Federation (IDF) Standard 20A:1986 method for the determination of nitrogen content in milk. The substitution was supported by results obtained in an interlaboratory study by 24 laboratories in 12 countries. Each laboratory analyzed 12 test samples of milk as blind duplicates in a double split level design with high, medium, and low nitrogen concentrations. The method protocol requires the concurrent analyses of an ammonium salt solution and a tryptophan solution as internal quality control standards with a minimum nitrogen recovery between 99 and 100% for the former and at least 98% for the latter. The repeatability and reproducibility relative standard deviations are 0.5 and 1%, respectively, for the range 0.35-0.70 N/100 g. The performance of the laboratories that did not meet the required quality control specifications was clearly poorer than that of those that did meet the specifications.

  15. Pressure ulcer prevention algorithm content validation: a mixed-methods, quantitative study.

    PubMed

    van Rijswijk, Lia; Beitz, Janice M

    2015-04-01

    Translating pressure ulcer prevention (PUP) evidence-based recommendations into practice remains challenging for a variety of reasons, including the perceived quality, validity, and usability of the research or the guideline itself. Following the development and face validation testing of an evidence-based PUP algorithm, additional stakeholder input and testing were needed. Using convenience sampling methods, wound care experts attending a national wound care conference and a regional wound ostomy continence nursing (WOCN) conference and/or graduates of a WOCN program were invited to participate in an Internal Review Board-approved, mixed-methods quantitative survey with qualitative components to examine algorithm content validity. After participants provided written informed consent, demographic variables were collected and participants were asked to comment on and rate the relevance and appropriateness of each of the 26 algorithm decision points/steps using standard content validation study procedures. All responses were anonymous. Descriptive summary statistics, mean relevance/appropriateness scores, and the content validity index (CVI) were calculated. Qualitative comments were transcribed and thematically analyzed. Of the 553 wound care experts invited, 79 (average age 52.9 years, SD 10.1; range 23-73) consented to participate and completed the study (a response rate of 14%). Most (67, 85%) were female, registered (49, 62%) or advanced practice (12, 15%) nurses, and had > 10 years of health care experience (88, 92%). Other health disciplines included medical doctors, physical therapists, nurse practitioners, and certified nurse specialists. Almost all had received formal wound care education (75, 95%). On a Likert-type scale of 1 (not relevant/appropriate) to 4 (very relevant and appropriate), the average score for the entire algorithm/all decision points (N = 1,912) was 3.72 with an overall CVI of 0.94 (out of 1). The only decision point/step recommendation

  16. Pressure ulcer prevention algorithm content validation: a mixed-methods, quantitative study.

    PubMed

    van Rijswijk, Lia; Beitz, Janice M

    2015-04-01

    Translating pressure ulcer prevention (PUP) evidence-based recommendations into practice remains challenging for a variety of reasons, including the perceived quality, validity, and usability of the research or the guideline itself. Following the development and face validation testing of an evidence-based PUP algorithm, additional stakeholder input and testing were needed. Using convenience sampling methods, wound care experts attending a national wound care conference and a regional wound ostomy continence nursing (WOCN) conference and/or graduates of a WOCN program were invited to participate in an Internal Review Board-approved, mixed-methods quantitative survey with qualitative components to examine algorithm content validity. After participants provided written informed consent, demographic variables were collected and participants were asked to comment on and rate the relevance and appropriateness of each of the 26 algorithm decision points/steps using standard content validation study procedures. All responses were anonymous. Descriptive summary statistics, mean relevance/appropriateness scores, and the content validity index (CVI) were calculated. Qualitative comments were transcribed and thematically analyzed. Of the 553 wound care experts invited, 79 (average age 52.9 years, SD 10.1; range 23-73) consented to participate and completed the study (a response rate of 14%). Most (67, 85%) were female, registered (49, 62%) or advanced practice (12, 15%) nurses, and had > 10 years of health care experience (88, 92%). Other health disciplines included medical doctors, physical therapists, nurse practitioners, and certified nurse specialists. Almost all had received formal wound care education (75, 95%). On a Likert-type scale of 1 (not relevant/appropriate) to 4 (very relevant and appropriate), the average score for the entire algorithm/all decision points (N = 1,912) was 3.72 with an overall CVI of 0.94 (out of 1). The only decision point/step recommendation

  17. Content and Contexts: Neglected Elements in Studies of Student Teaching as an Occasion for Learning to Teach.

    ERIC Educational Resources Information Center

    Zeichner, Kenneth M.

    1986-01-01

    This article argues that studies of student teaching as an occasion for learning to teach have mistakenly ignored the role of program content and contexts in the socialization of prospective teachers. (Author/MT)

  18. Socialization Content in Schools and Education for Sustainable Development--II. A Study of Students' Apprehension of Teachers' Companion Meanings in ESD

    ERIC Educational Resources Information Center

    Sund, Per; Wickman, Per-Olof

    2011-01-01

    Subject content is always studied within an educational context. This context is constituted by the socialization content, which can be regarded as an educational content beyond the subject content. This is the third article of three studies (this article; Sund and Wickman 2011; Sund 2008) that together form a triangulation of possible…

  19. Instructional Developer as Content Specialist: Three Case Studies Utilizing the Instructional Development-Operations Research Model.

    ERIC Educational Resources Information Center

    Faust, Stephen M.

    1980-01-01

    Presents a 3-phase model (content research, specification, delivery) for instructional development-operations research and describes its application in developing courses in zoology, geology, and paleontology. (MER)

  20. Opsin1-2, G(q)α and arrestin levels at Limulus rhabdoms are controlled by diurnal light and a circadian clock.

    PubMed

    Battelle, Barbara-Anne; Kempler, Karen E; Parker, Alexander K; Gaddie, Cristina D

    2013-05-15

    Dark and light adaptation in photoreceptors involve multiple processes including those that change protein concentrations at photosensitive membranes. Light- and dark-adaptive changes in protein levels at rhabdoms have been described in detail in white-eyed Drosophila maintained under artificial light. Here we tested whether protein levels at rhabdoms change significantly in the highly pigmented lateral eyes of wild-caught Limulus polyphemus maintained in natural diurnal illumination and whether these changes are under circadian control. We found that rhabdomeral levels of opsins (Ops1-2), the G protein activated by rhodopsin (G(q)α) and arrestin change significantly from day to night and that nighttime levels of each protein at rhabdoms are significantly influenced by signals from the animal's central circadian clock. Clock input at night increases Ops1-2 and G(q)α and decreases arrestin levels at rhabdoms. Clock input is also required for a rapid decrease in rhabdomeral Ops1-2 beginning at sunrise. We found further that dark adaptation during the day and the night are not equivalent. During daytime dark adaptation, when clock input is silent, the increase of Ops1-2 at rhabdoms is small and G(q)α levels do not increase. However, increases in Ops1-2 and G(q)α at rhabdoms are enhanced during daytime dark adaptation by treatments that elevate cAMP in photoreceptors, suggesting that the clock influences dark-adaptive increases in Ops1-2 and G(q)α at Limulus rhabdoms by activating cAMP-dependent processes. The circadian regulation of Ops1-2 and G(q)α levels at rhabdoms probably has a dual role: to increase retinal sensitivity at night and to protect photoreceptors from light damage during the day. PMID:23393287

  1. Specific α-arrestins negatively regulate Saccharomyces cerevisiae pheromone response by down-modulating the G-protein-coupled receptor Ste2.

    PubMed

    Alvaro, Christopher G; O'Donnell, Allyson F; Prosser, Derek C; Augustine, Andrew A; Goldman, Aaron; Brodsky, Jeffrey L; Cyert, Martha S; Wendland, Beverly; Thorner, Jeremy

    2014-07-01

    G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate responses to extracellular stimuli by mediating ligand-dependent activation of cognate heterotrimeric G proteins. In yeast, occupancy of GPCR Ste2 by peptide pheromone α-factor initiates signaling by releasing a stimulatory Gβγ complex (Ste4-Ste18) from its inhibitory Gα subunit (Gpa1). Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest, including upregulation of the expression of an α-factor-degrading protease (Bar1), a regulator of G-protein signaling protein (Sst2) that stimulates Gpa1-GTP hydrolysis, and Gpa1 itself. Ste2 is also downregulated by endocytosis, both constitutive and ligand induced. Ste2 internalization requires its phosphorylation and subsequent ubiquitinylation by membrane-localized protein kinases (Yck1 and Yck2) and a ubiquitin ligase (Rsp5). Here, we demonstrate that three different members of the α-arrestin family (Ldb19/Art1, Rod1/Art4, and Rog3/Art7) contribute to Ste2 desensitization and internalization, and they do so by discrete mechanisms. We provide genetic and biochemical evidence that Ldb19 and Rod1 recruit Rsp5 to Ste2 via PPXY motifs in their C-terminal regions; in contrast, the arrestin fold domain at the N terminus of Rog3 is sufficient to promote adaptation. Finally, we show that Rod1 function requires calcineurin-dependent dephosphorylation.

  2. Loss of retinoschisin (RS1) cell surface protein in maturing mouse rod photoreceptors elevates the luminance threshold for light-driven translocation of transducin but not arrestin.

    PubMed

    Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bush, Ronald A; Sieving, Paul A

    2012-09-19

    Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1-KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1-KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1-KO mice as a marker for functional abnormalities in maturing rod photoreceptors. We found a progressive reduction in luminance threshold for transducin translocation in wild-type (WT) retinas between postnatal days P18 and P60. At P21, the threshold in Rs1-KO retinas was 10-fold higher than WT, but it decreased to <2.5-fold higher by P60. Light-activated arrestin translocation and re-translocation of transducin in the dark were not affected. Rs1-KO rod outer segment (ROS) length was significantly shorter than WT at P21 but was comparable with WT at P60. These findings suggested a delay in the structural and functional maturation of Rs1-KO ROS. Consistent with this, transcription factors CRX and NRL, which are fundamental to maturation of rod protein expression, were reduced in ROS of Rs1-KO mice at P21 but not at P60. Expression of transducin was 15-30% lower in P21 Rs1-KO ROS and transducin GTPase hydrolysis was nearly twofold faster, reflecting a 1.7- to 2.5-fold increase in RGS9 (regulator of G-protein signaling) level. Transduction protein expression and activity levels were similar to WT at P60. Transducin translocation threshold elevation indicates photoreceptor functional abnormalities in young Rs1-KO mice. Rapid reduction in threshold coupled with age-related changes in transduction protein levels and transcription factor expression are consistent with delayed maturation of Rs1-KO photoreceptors.

  3. Specific α-Arrestins Negatively Regulate Saccharomyces cerevisiae Pheromone Response by Down-Modulating the G-Protein-Coupled Receptor Ste2

    PubMed Central

    Alvaro, Christopher G.; O'Donnell, Allyson F.; Prosser, Derek C.; Augustine, Andrew A.; Goldman, Aaron; Brodsky, Jeffrey L.; Cyert, Martha S.; Wendland, Beverly

    2014-01-01

    G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate responses to extracellular stimuli by mediating ligand-dependent activation of cognate heterotrimeric G proteins. In yeast, occupancy of GPCR Ste2 by peptide pheromone α-factor initiates signaling by releasing a stimulatory Gβγ complex (Ste4-Ste18) from its inhibitory Gα subunit (Gpa1). Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest, including upregulation of the expression of an α-factor-degrading protease (Bar1), a regulator of G-protein signaling protein (Sst2) that stimulates Gpa1-GTP hydrolysis, and Gpa1 itself. Ste2 is also downregulated by endocytosis, both constitutive and ligand induced. Ste2 internalization requires its phosphorylation and subsequent ubiquitinylation by membrane-localized protein kinases (Yck1 and Yck2) and a ubiquitin ligase (Rsp5). Here, we demonstrate that three different members of the α-arrestin family (Ldb19/Art1, Rod1/Art4, and Rog3/Art7) contribute to Ste2 desensitization and internalization, and they do so by discrete mechanisms. We provide genetic and biochemical evidence that Ldb19 and Rod1 recruit Rsp5 to Ste2 via PPXY motifs in their C-terminal regions; in contrast, the arrestin fold domain at the N terminus of Rog3 is sufficient to promote adaptation. Finally, we show that Rod1 function requires calcineurin-dependent dephosphorylation. PMID:24820415

  4. Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of β-Arrestins.

    PubMed

    Smith, Thomas H; Coronel, Luisa J; Li, Julia G; Dores, Michael R; Nieman, Marvin T; Trejo, JoAnn

    2016-08-26

    Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of β-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of β-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation. PMID:27402844

  5. Study of total electron content variations over equatorial and low latitude ionosphere during extreme solar minimum

    NASA Astrophysics Data System (ADS)

    Sharma, Kavita; Dabas, R. S.; Ravindran, Sudha

    2012-10-01

    In recent years with the advancement in satellite based navigational applications, study of Total Electron Content (TEC) has gained significant importance. It is well known that due to dynamical behaviour of equatorial and low latitude ionosphere, the levels of ionization is relatively high herein. The sustained decrease in solar extreme ultraviolet radiations during the current minimum is greater than any in recent history. This gives us the opportunity to study the observations of global positioning system total electron content (GPS-TEC) dual frequency signals from the GPS satellites continuously recorded at Trivandrum (an equatorial station) and Delhi (a low latitude station) during the extremely low solar activity period from January 2007 to June 2009. This study illustrates the diurnal, seasonal and annual variations of TEC during the extended solar minimum period. This study also investigates the behaviour of daytime ionosphere around spring and autumn equinoxes at low solar activity period. The results clearly reveal the presence of equinoctial asymmetry which is more pronounced at equatorial station Trivandrum. The diurnal variation of TEC shows a short-lived day minimum which occurs between 0500 to 0600 LT at both the stations. Delhi TEC values show its steep increase and reach at its peak value between 1200 and 1400 LT, while at the equator the peak is broad and occurs around 1600 LT. Further, the daily maximum TEC ranges from about 5 to 40 TEC units at Trivandrum and about 10 to 40 TEC units at Delhi, which correspond to range delay variations of about 1 to 8 m at the GPS L1 frequency of 1.575 GHz. The Maximum values of TEC were observed during spring equinox rather than autumn equinox, showing presence of semi annual variation at both the locations. The minimum values of TEC were observed during the summer solstice at Trivandrum indicating the presence of winter anomaly at equatorial region while Delhi TEC values were minimum during winter solstice

  6. BRET biosensors to study GPCR biology, pharmacology, and signal transduction.

    PubMed

    Salahpour, Ali; Espinoza, Stefano; Masri, Bernard; Lam, Vincent; Barak, Larry S; Gainetdinov, Raul R

    2012-01-01

    Bioluminescence resonance energy transfer (BRET)-based biosensors have been extensively used over the last decade to study protein-protein interactions and intracellular signal transduction in living cells. In this review, we discuss the various BRET biosensors that have been developed to investigate biology, pharmacology, and signaling of G protein-coupled receptors (GPCRs). GPCRs form two distinct types of multiprotein signal transduction complexes based upon their inclusion of G proteins or β-arrestins that can be differentially affected by drugs that exhibit functional selectivity toward G protein or β-arrestin signaling. BRET has been especially adept at illuminating the dynamics of protein-protein interactions between receptors, G proteins, β-arrestins, and their many binding partners in living cells; as well as measuring the formation and accumulation of second messengers following receptor activation. Specifically, we discuss in detail the application of BRET to study dopamine and trace amine receptors signaling, presenting examples of an exchange protein activated by cAMP biosensor to measure cAMP, β-arrestin biosensors to determine β-arrestin recruitment to the receptor, and dopamine D2 receptor and trace amine-associated receptor 1 biosensors to investigate heterodimerization between them. As the biochemical spectrum of BRET biosensors expands, the number of signaling pathways that can be measured will concomitantly increase. This will be particularly useful for the evaluation of functional selectivity in which the real-time BRET capability to measure distinct signaling modalities will dramatically shorten the time to characterize new generation of biased drugs. These emerging approaches will further expand the growing application of BRET in the screening for novel pharmacologically active compounds.

  7. An Experimental Study on the Iso-Content-Based Angle Similarity Measure.

    ERIC Educational Resources Information Center

    Zhang, Jin; Rasmussen, Edie M.

    2002-01-01

    Retrieval performance of the iso-content-based angle similarity measure within the angle, distance, conjunction, disjunction, and ellipse retrieval models is compared with retrieval performance of the distance similarity measure and the angle similarity measure. Results show the iso-content-based angle similarity measure achieves satisfactory…

  8. Pedagogical Content Knowledge and the Gas Laws: A Multiple Case Study

    ERIC Educational Resources Information Center

    Sande, Mary Elizabeth

    2010-01-01

    Pedagogical content knowledge (PCK) has been described as an assemblage of the most powerful analogies, demonstrations, examples and illustrations that make content knowledge understandable to students, together with an understanding of the preconceptions and alternate conceptions that students bring with them to the classroom (Shulman, 1986). In…

  9. A Study to Determine Through Content Analysis Selected Criteria for Open-End Examinations.

    ERIC Educational Resources Information Center

    McNally, Elaine F.

    Content analysis was used to determine the evaluation criteria of high school and college teachers and college seniors in grading essay tests. Content analysis is defined as a way of asking a fixed set of questions unfalteringly of all of a predetermined body of writings, in such a way as to produce quantitative results. Four reponses to a…

  10. A Learning Content Authoring Approach Based on Semantic Technologies and Social Networking: An Empirical Study

    ERIC Educational Resources Information Center

    Nesic, Sasa; Gasevic, Dragan; Jazayeri, Mehdi; Landoni, Monica

    2011-01-01

    Semantic web technologies have been applied to many aspects of learning content authoring including semantic annotation, semantic search, dynamic assembly, and personalization of learning content. At the same time, social networking services have started to play an important role in the authoring process by supporting authors' collaborative…

  11. Technical Communications in OSS Content Management Systems: An Academic Institutional Case Study

    ERIC Educational Resources Information Center

    Cripps, Michael J.

    2011-01-01

    Single sourcing through a content management system (CMS) is altering technical communication practices in many organizations, including institutions of higher education. Open source software (OSS) solutions are currently among the most popular content management platforms adopted by colleges and universities in the United States and abroad. The…

  12. Investigating Correspondence between Language Proficiency Standards and Academic Content Standards: A Generalizability Theory Study

    ERIC Educational Resources Information Center

    Lin, Chih-Kai; Zhang, Jinming

    2014-01-01

    Research on the relationship between English language proficiency standards and academic content standards serves to provide information about the extent to which English language learners (ELLs) are expected to encounter academic language use that facilitates their content learning, such as in mathematics and science. Standards-to-standards…

  13. [Study on hair Hg and Pb content distribution of traffic polices, Guilin].

    PubMed

    Qian, Jian-Ping; Zhang, Li; Li, Cheng-Chao; Huang, Dong

    2013-06-01

    95 hair samples from traffic polices and 110 hair samples from ordinary people were collected from 6 areas of Guilin, China, and Hg, Pb contents in hairs were determined. The result shows that the heavier the traffic was, the higher hair Hg, Pb contents of traffic polices are. Hair Hg, Pb contents of traffic polices also increase with their working time. Average hair Hg, Pb contents of outdoor polices are higher than those of indoor polices. The average hair Hg content (1.340 microg x g(-1)) of traffic polices is 1.74 times as high as the Chinese average value (0.77 microg x g(-1)), while the average hair Pb content (2.877 microg x g(-1)) is below the Chinese average value (6.60 microg x g(-1)). The use of unleaded petrol reduced the air Pb pollution, but Hg pollution still exists. The average hair Hg content (1.504 microg x g(-1)) in male traffic polices is higher than that (1.176 microg x g(-1)) of female traffic polices,while the average hair Pb content (2.852 microg x g(-1) in male traffic polices lower than that (2. 902 microg x g(-1)) of female traffic polices.

  14. Fathers’ Representation in Observational Studies on Parenting and Childhood Obesity: A Systematic Review and Content Analysis

    PubMed Central

    Gicevic, Selma; Aftosmes-Tobio, Alyssa; Ganter, Claudia; Simon, Christine L.; Newlan, Sami; Manganello, Jennifer A.

    2016-01-01

    Background. The involvement of fathers in caregiving has increased substantially over the past 30 years. Yet in child and adolescent psychopathology, few studies include fathers as research participants and few present results for fathers separate from those for mothers. We test for the first time whether a similar pattern exists in research on parenting and childhood obesity. Objectives. To conduct a systematic review and quantitative content analysis of observational studies on parenting and childhood obesity to (1) document the inclusion of fathers, relative to mothers, as research participants and (2) examine characteristics of studies that did and did not include fathers. This study presents new data on the number and gender of parent research participants. Search methods. We searched title, abstract, and Medical Subject Headings term fields in 5 research databases (PubMed, EMBASE, Academic Search Premier, PsycINFO, and CINAHL) using terms combining parents or parenting (e.g., mother, father, caregiver, parenting style, food parenting) and obesity (e.g., obesity, body weight, overweight) or obesity-related lifestyle behaviors (e.g., diet, snacking, physical activity, outdoor play, exercise, media use). Selection criteria. We identified and screened studies as per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) published between January 2009 and December 2015, examining links between parenting and childhood obesity, including parents or caregivers as research participants, and written in English. We excluded interventions, nonhuman studies, dissertations, conference abstracts, and studies on youths with specific medical conditions. Of 5557 unique studies, 667 studies were eligible. Data collection and analysis. For each of the 667 studies, 4 coders were trained to code characteristics of the study (e.g., publication year, geographic region, journal, study focus) and parent research participants (e.g., parent gender, demographic

  15. Nighttime increases in ionosphere electron content (a statistical and experimental study)

    NASA Astrophysics Data System (ADS)

    Gordienko, G. I.; Zachateiskiy, D. E.; Kaliev, M. Z.; Mukasheva, S. N.; Vodyannikov, V. V.

    2001-04-01

    Variations of nighttime ionosphere total electron content (TEC) have been studied using the Faraday rotation angle data obtained at middle latitudes (Almaty, Kazakhstan) from the geostationary satellite ETS-II during the years 1985-1986. Critical frequency values of the ionosphere F2-layer shown in the vertical sounding ionograms measured at Alma-Ata station (43.25/°N, 76.92/°E) have been used throughout the study. Morphological and statistical analyses are used to investigate the anomalous nighttime increases (ANI) in TEC that occurred during the years. The ANI events are looked at in terms of their amplitude and probability of occurrence, and their duration and peak time. Special features in the events are found to interpret the ANI as a travelling ionosphere disturbance. By examining the hourly, nightly values foF2 and TEC over the two years it has been suggested that the ANI events exist on an ionosphere background which contains regular quasi-periodical oscillations in the period range of 4-30 days.

  16. Comparative studies on the immunoregulatory effects of three polysaccharides using high content imaging system.

    PubMed

    Lv, Xiaocheng; Chen, Dandan; Yang, Liecheng; Zhu, Ning; Li, Jingling; Zhao, Jian; Hu, Zhibi; Wang, Fu-Jun; Zhang, Leshuai W

    2016-05-01

    In this study, polysaccharides were isolated from Astragalus membranaceus, Ganoderma lucidum and Radix ophiopogonis and named APSII, GLPII and OGPII for comparison of their immunoactivities. MTT assay indicated that these polysaccharides increased the metabolic activity of Raw264.7 macrophages and induced cell differentiation to dendritic like cells. High content screening and mathematical modeling were used to quantify the cell irregularity, a hallmark of cell differentiation by polysaccharides. The results showed that GLPII increased cell irregularity, but APSII and OGPII had slightly less effects. Imaging analysis also revealed that polysaccharides inhibited cell proliferation while inducing the cell differentiation. In addition, APSII and GLPII but not OGPII induced NO production and enhanced cell phagocytic ability. Interestingly, inducible nitric oxide synthase inhibitor blocked polysaccharide-enhanced phagocytosis, indicating NO production is crucial for macrophages to acquire phagocytic ability, which was further confirmed by correlation studies. APSII and GLPII significantly promoted the maturation of macrophages by the increase in the expression of MHCII, CD40, CD80 and CD86, while OGPII had less effects. In summary, we have suggested a practical and economical method to quantify macrophage differentiation (irregularity) induced by polysaccharides for quality assurance and have found the role of NO production on macrophage phagocytic ability. PMID:26783639

  17. Measurement of moisture content in photovoltaic panel encapsulants using spectroscopic optical coherence tomography: a feasibility study

    NASA Astrophysics Data System (ADS)

    Rashtchi, Shabnam; Ruiz, Pablo D.; Wildman, Ricky; Ashcroft, Ian

    2012-10-01

    EVA, a copolymer of ethylene and vinyl acetate, is a common encapsulant material used in silicon-based PV modules. It contributes to the structural integrity of the modules, provides electrical insulation and also acts as an environmental barrier. However, water can diffuse through EVA into the modules, leading to swelling and chemical degradation, which can impact interfacial bonds, leading to delamination and allowing more ingress to occur that can eventually end up in accelerated corrosion and device failure. Fourier Transform infrared spectroscopy (FTIR) and gravimetric techniques have been used to quantify water concentration and the diffusion coefficient in free standing EVA films. However, these techniques cannot be applied to measure water content in PV modules deployed in the field, as the encapsulant is usually between a glass front sheet and a back sheet made of glass or multilayered films. In this paper we study the feasibility of combining FTIR and spectroscopic optical coherence tomography (SOCT) to measure water concentration of the EVA layer inside the modules. SOCT provides depth resolved spectral information and thus has the potential of measuring water absorption at different layers in the PV module. These depth-resolved measurements are necessary to inform predictive models developed to study the structural integrity, stability and durability of PV modules. The fundamental principle of the technique is explained, the optimum spectral ranges are identified and the feasibility of a SOCT system is discussed based on light source and detector characteristics. Other strategies are also considered.

  18. Study of optical properties and proteoglycan content of tendons by polarization sensitive optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Yang, Ying; Rupani, Asha; Bagnaninchi, Pierre; Wimpenny, Ian; Weightman, Alan

    2012-08-01

    The highly orientated collagen fibers in tendons play a critical role for transferring tensile stress, and they demonstrate birefringent optical properties. However, the influence that proteoglycans (PGs) have on the optical properties of tendons is yet to be fully elucidated. PGs are the essential components of the tendon extracellular matrix; the changes in their quantities and compositions have been associated with tendinopathies. In this study, polarization sensitive optical coherence tomography (PS-OCT) has been used to reveal the relationship between PG content/location and birefringence properties of tendons. Fresh chicken tendons were imaged at regular intervals by PS-OCT and polarization light microscopy during the extraction of PGs, using guanidine hydrochloride (GuHCl). Complementary time-lapsed images taken from the two modalities mutually demonstrated that the extraction of PGs disturbed the local organization of collagen bundles. This corresponded with a decrease in birefringence and associated banding pattern observed by PS-OCT. Furthermore, this study revealed there was a higher concentration of PGs in the outer sheath region than in the fascicles, and therefore the change in birefringence was reduced when extraction was performed on unsheathed tendons. The results provide new insights of tendon structure and the role of PGs on the structural stability of tendons, which also demonstrates the great potential for using PS-OCT as a diagnostic tool to examine tendon pathology.

  19. Study of optical properties and proteoglycan content of tendons by polarization sensitive optical coherence tomography.

    PubMed

    Yang, Ying; Rupani, Asha; Bagnaninchi, Pierre; Wimpenny, Ian; Weightman, Alan

    2012-08-01

    The highly orientated collagen fibers in tendons play a critical role for transferring tensile stress, and they demonstrate birefringent optical properties. However, the influence that proteoglycans (PGs) have on the optical properties of tendons is yet to be fully elucidated. PGs are the essential components of the tendon extracellular matrix; the changes in their quantities and compositions have been associated with tendinopathies. In this study, polarization sensitive optical coherence tomography (PS-OCT) has been used to reveal the relationship between PG content/location and birefringence properties of tendons. Fresh chicken tendons were imaged at regular intervals by PS-OCT and polarization light microscopy during the extraction of PGs, using guanidine hydrochloride (GuHCl). Complementary time-lapsed images taken from the two modalities mutually demonstrated that the extraction of PGs disturbed the local organization of collagen bundles. This corresponded with a decrease in birefringence and associated banding pattern observed by PS-OCT. Furthermore, this study revealed there was a higher concentration of PGs in the outer sheath region than in the fascicles, and therefore the change in birefringence was reduced when extraction was performed on unsheathed tendons. The results provide new insights of tendon structure and the role of PGs on the structural stability of tendons, which also demonstrates the great potential for using PS-OCT as a diagnostic tool to examine tendon pathology.

  20. A Content Analysis of Internet Sessions Presented at the National Council for Social Studies Annual Meeting, 1995-2002

    ERIC Educational Resources Information Center

    VanFossen, Phillip J.; Shiveley, James M.

    2003-01-01

    The Internet has been touted as a useful resource for social studies teachers. This study used content analysis methods to examine trends in sessions focused on the use of the Internet presented at the National Council for the Social Studies (NCSS) annual meeting (1995-2002). Session abstracts were analyzed for: type of Internet sessions…

  1. Study of enzyme activities and protein content of beluga (Huso huso) semen before and after cryopreservation.

    PubMed

    Aramli, M S

    2015-02-01

    Knowledge gained regarding the biochemical processes that occur during sperm collection, processing and freezing-thawing might improve current sperm cryopreservation techniques. In our present study, we determined the effect of cryopreservation on the total protein concentration (TP) and the activities of certain enzymes in semen samples from the beluga (Huso huso). The TP content of the seminal plasma of fresh semen was 0.47 ± 0.026 g/l, and the TP after cryopreservation was 1.86 ± 0.6 g/l. The activities of acid phosphatase (0.82 ± 0.042 U/l), lactate dehydrogenase (234.4 ± 19.4 U/l), arylsulfatase (143.1 ± 32.5 U/l) and β-N-acetylglucosaminidase (58.39 ± 4.14 U/l) in the seminal plasma of fresh semen were significantly lower than those in the supernatant of frozen-thawed semen samples (7.43 ± 0.64, 3224.6 ± 167.2, 422.6 ± 21.3 and 90.2 ± 5.37 U/l respectively). These parameters may be useful as biomarkers for estimating damage to the cell membrane of spermatozoa caused by freezing-thawing.

  2. Impact of the volume of aneurysmal contents on intraaneurysmal pressure after endovascular grafting (experimental studies).

    PubMed

    Volodos', S N; Sayers, R D; Bell, P R F; Gostelow, J P

    2005-01-01

    With the purpose of clarifying the nature and outlining certain constituents of such an undesirable condition designated as "endotension", that emerges after transluminal grafting of the aneurysmally changed aorta, a basic theoretical model was worked out for explanation of "endotension". Also, there was designed and constructed in vitro an original experimental model using which the authors carried out a study into the relationship between the pressure in the aneurysm after its complete exclusion from the blood flow by an endovascular graft and the volume of the aneurysmal contents. Some factors described in the literature as influencing the pressure level in the aneurysm were at the given stage purposefully excluded. The volume of the "aneurysm" reproduced in our model was equal to 675 ml; each of five vascular grafts implanted into the aneurysm measured 110 mm in length. The pulsating liquid flow was reproduced in the system using a serial appliance for extracorporeal circulation. The liquid was aspirated from the aneurysm by means of a syringe with concurrent pressure guidance in the sac. To start pressure lowering, it was necessary to evacuate 0.4-1.6 ml of the liquid. The real clinical situations were considered from the standpoint of our results.

  3. [Study on the Identification of Geographical Indication Wuchang Rice Based on the Content of Inorganic Elements].

    PubMed

    Li, Yong-le; Zheng, Yan-jie; Tang, Lu; Su, Zhi-yi; Xiong, Cen

    2016-03-01

    Wuchang rice is a geographical indication product in China. Due to its high quality and low production, the phenome- non of fake is more and more serious. An effective identification method of Wuchang rice is urgent needed, for the maintenance of its brand image and interest of consumers. Base on the content of inorganic elements which are analyzed by ICP-AES and ICP-MS in rice, the identification model of Wuchang rice is studied combining with principal component analysis (PCA), Fisher discrimination and artificial neural network (ANN) in this paper. The effect on the identification of samples is poor through PCA, while the samples from Wuchang area and other areas can be identified accurately through Fisher discrimination and ANN. The average accurate identification ratio of training and verification set through Fisher discrimination is 93.5%, while the average accurate identification ratio through ANN is 96.4%. The ability to identify of ANN is better than Fisher discrimination. Wuchang rice can be identified accurately through the result of this research which provides a technology for the protection of geographical indications of this product. PMID:27400533

  4. [Study of contents of nitrates, fluorides, aluminium in dialysis water in Tunisia].

    PubMed

    Jennen, Faycal; Gorbel, Hayette; Hmida, Jalel; Cherni, Noureddine; Abderrazek, Hedhili

    2005-12-01

    Nitrates, fluorides and aluminum have often been incriminated in cases of poisoning sometimes life threatening such as methaemoglobinaemia, dental and skeletal fluorosis, and myoclonal encephalopathy in patients undergoing haemodialysis. The three elements mentioned have something in common: their water origin (drinking water and dialysis water). To study the situation in Tunisia in this respect we propose to determinate the contents of nitrates, fluorides, and aluminum in drinking water and dialysis water supplied by the water board (SONEDE). 540 samples of drinking water and dialysis water were taken from 95 centers of haemodialysis scattered all over the country, between January 2000 and December 2001. These samples are divided in three parts (drinking water, soften water, and dialysis water ). Results show levels of fluorides < 1.5 ppm in most areas with the exception of zones rich in phosphate where the mean level is 1.61 +/- 1.22. the levels of nitrates are in most regions < 50mg/l and those of aluminum < 100microg/l. The levels of fluorides, nitrates and aluminum in dialysis water were within acceptable national and international limits excepting a few centers where the levels of the these contaminants are high, exposing patients on haemodialysis to acute and chronic intoxication. This contamination is caused in majority of cases by a dysfunction of water dialysis treatment chain, therefore caution is necessary and strict measures of control should be introduced.

  5. Non-invasive quantification of small bowel water content by MRI: a validation study

    NASA Astrophysics Data System (ADS)

    Hoad, C. L.; Marciani, L.; Foley, S.; Totman, J. J.; Wright, J.; Bush, D.; Cox, E. F.; Campbell, E.; Spiller, R. C.; Gowland, P. A.

    2007-12-01

    Substantial water fluxes across the small intestine occur during digestion of food, but so far measuring these has required invasive intubation techniques. This paper describes a non-invasive magnetic resonance imaging (MRI) technique for measuring small bowel water content which has been validated using naso-duodenal infusion. Eighteen healthy volunteers were intubated, with the tube position being verified by MRI. After a baseline MRI scan, each volunteer had eight 40 ml boluses of a non-absorbable mannitol and saline solution infused into their proximal small bowel with an MRI scan being acquired after each bolus. The MRI sequence used was an adapted magnetic resonance cholangiopancreatography sequence. The image data were thresholded to allow for intra- and inter-subject signal variations. The MRI measured volumes were then compared to the known infused volumes. This MRI technique gave excellent images of the small bowel, which closely resemble those obtained using conventional radiology with barium contrast. The mean difference between the measured MRI volumes and infused volumes was 2% with a standard deviation of 10%. The maximum 95% limits of agreement between observers were -15% to +17% while measurements by the same operator on separate occasions differed by only 4%. This new technique can now be applied to study alterations in small bowel fluid absorption and secretion due to gastrointestinal disease or drug intervention.

  6. Study of fluoride content in groundwater of Nawa Tehsil in Nagaur, Rajasthan.

    PubMed

    Gautam, Radha; Bhardwaj, Nagendra; Saini, Yashoda

    2011-01-01

    There is a severe fluoride problem in Nawa tehsil of Nagaur district. Villagers are suffering from dental fluorosis and skeletal fluorosis. So an extensive geochemical study of 27 villages of eastern, south-eastern and southern zone of Nawa tehsil was done. Total 46 ground water samples were collected and analyzed for various physicochemical parameters as well as fluoride content. The ground water samples collected in clean polyethylene plastic containers were analyzed for pH, electrical conductivity, total dissolved salts, calcium, magnesium, total hardness, chloride and alkalinity. The fluoride concentration in the three different zones ranged from 0.64 to 14.62 mg l(-1) where 13.04% samples were found within permissible limit while 86.96% had fluoride beyond permissible limit (> 1.5 mg l(-1)). It was found that among the three different zones south-eastern zone was under serious fluoride contamination where fluoride concentration ranged between 1.10 to 14.62 mg l(-1). In the eastern zone fluoride concentration was recorded from 1.52 to 5.13 mg l(-1) whereas in the southern zone it was found between 0.64 to 3.63 mg l(-1).

  7. Iranian nurses’ perception of spirituality and spiritual care: a qualitative content analysis study

    PubMed Central

    Mahmoodishan, Gholamreza; Alhani, Fatemeh; Ahmadi, Fazlollah; Kazemnejad, Anoshirvan

    2010-01-01

    The purpose of the present study was to explore nurses’ perception about spirituality and spiritual care. A qualitative content analysis approach was conducted on 20 registered nurses interviewed using unstructured strategy in 2009. Three themes emerged from the data analysis: 1) “meaning and purpose of work and life” including ‘spiritualistic view to profession’, ‘commitment and professional responsibility’, and ‘positive attitude’; 2) “religious attitude” including ‘God approval’, ‘spiritual reward’, ‘taking advice’, ‘inner belief in the Supreme Being’, ‘faith-based interactions and altruism’; 3) “transcendence-seeking” including ‘need for respect’ and ‘personal–professional transcendence’. Therefore, the spirituality produces maintenance, harmony and balance in nurses in relation to God. Spiritual care focuses on respecting patients, friendly and sympathetic interactions, sharing in rituals and strengthening patients and nurses’ inner energy. This type of spirituality gives a positive perspective to life and profession, peaceful interactions, a harmonious state of mind, and acts as a motivator among nurses to promote nursing care and spirituality. PMID:23908741

  8. Study of the Total Electron Content in Mars ionosphere from MARSIS data set

    NASA Astrophysics Data System (ADS)

    Bergeot, Nicolas; Witasse, Olivier; Kofman, Wlodek; Grima, Cyril; Mouginot, Jeremie; Peter, Kerstin; Pätzold, Martin; Dehant, Véronique

    2016-04-01

    Centimeter level accuracy on the signal delay will be required on X-band radio link for future Mars landers such as InSIGHT, aiming at better determining the interior structure of Mars. One of the main error sources in the estimated signal delay is directly linked to the Total Electron Content (TEC) values at Earth and Mars ionosphere level. While the Earth ionosphere is now well modeled and monitored at regional and global scales, this is not the case concerning the Mars' upper atmosphere. The present paper aims at establishing the basis to model the climatological behavior of the TEC on a global scale in the Mars' ionosphere. For that we analyzed ˜8.5 years of data (mid-2005 to 2014) of the vertical Total Electron Content (vTEC) expressed in TEC units (1 TECu = 1016e-.m-2) from the Mars Advanced Radar for Subsurface and Ionospheric Sounding (MARSIS) radar. Our study takes advantage of the double data set of EUV solar index and Mars vTEC data to develop an empirical Model of Mars Ionosphere (MoMo). The finality of this model is to predict the vTEC at a given latitude, solar zenith angle and season taking only F10.7P solar index as input. To minimize the differences during the least-square adjustment between the modeled and observed vTEC, we considered (1) a 4th-order polynomial function to describe the vTEC diurnal behavior (2) a discretization with respect to Mars seasons (depending on Ls) and (3) two latitudinal sectors (North and South hemispheres). The mean of the differences between the model and the observations is 0.00±0.07 TECu with an error of the model around 0.1 TECu depending on the Solar Zenith Angle (SZA), season and hemisphere of interest (e.g. rms 0.12 TECu for SZA equal to 50°±5° in the Northern hemisphere during the spring season). Additionally, comparison with 250 Mars Express radio occultation data shows differences with MoMo predictions of 0.02±0.06 TECu for solar zenith angles below 50 degrees. Using the model we (1) highlighted

  9. Effect of intracellular P content on phosphate removal in Scenedesmus sp. Experimental study and kinetic expression.

    PubMed

    Ruiz-Martínez, A; Serralta, J; Romero, I; Seco, A; Ferrer, J

    2015-01-01

    The present work determines the effect of phosphorus content on phosphate uptake rate in a mixed culture of Chlorophyceae in which the genus Scenedesmus dominates. Phosphate uptake rate was determined in eighteen laboratory batch experiments, with samples taken from a progressively more P-starved culture in which a minimum P content of 0.11% (w/w) was achieved. The results obtained showed that the higher the internal biomass P content, the lower the phosphate removal rate. The highest specific phosphate removal rate was 6.5mgPO4-PgTSS(-1)h(-1). Microalgae with a P content around 1% (w/w) attained 10% of this highest removal rate, whereas those with a P content of 0.6% (w/w) presented 50% of the maximum removal rate. Different kinetic expressions were used to reproduce the experimental data. Best simulation results for the phosphate uptake process were obtained combining Steele equation and Hill function to represent the effect of light and intracellular phosphorus content, respectively.

  10. An XAS experimental approach to study low Pt content electrocatalysts operating in PEM fuel cells.

    PubMed

    Principi, Emiliano; Witkowska, Agnieszka; Dsoke, Sonia; Marassi, Roberto; Di Cicco, Andrea

    2009-11-21

    We present an X-ray absorption spectroscopy (XAS) study of a low Pt content catalyst layer (Pt loading 0.1 mg cm(-2)) operating at the cathode of a proton exchange membrane fuel cell (PEMFC). This catalyst is based on the use of a mesoporous inorganic matrix as a support for the catalyst Pt nanoparticles. Due to the high Pt dilution, in situ measurements of its structural properties by XAS are challenging and suitable experimental strategies must be devised for this purpose. In particular, we show that accurate XAS in situ fluorescence measurements can be obtained using an optimized fuel cell, suitable protocols for alignment of a focused X-ray beam and an appropriate filter for the background signal of the other atomic species contained in the electrodes. Details, advantages and limitations of the XAS technique for in situ measurements are discussed. Analysis of the near-edge XAS and EXAFS (extended X-ray absorption fine structure) data, corroborated by a HRTEM (high-resolution transmission electron microscopy) study, shows that the Pt particles have a local structure compatible with that of bulk Pt (fcc) and coordination numbers match those expected for particles with typical sizes in the 1.5-2.0 nm range. Substantial changes in the oxidation state and in local atomic arrangement of the Pt particles are found for different applied potentials. The catalyst support, containing W atoms, exhibits a partial reduction upon PEMFC activation, thus mimicking the catalyst behavior. This indicates a possible role of the mesoporous matrix in favouring the oxygen reduction reaction (ORR) and stimulates further research on active catalyst supports.

  11. Developing Pre-Service Teachers' Technological Pedagogical Content Knowledge for Teaching Mathematics with the Geometer's Sketchpad through Lesson Study

    ERIC Educational Resources Information Center

    Meng, Chew Cheng; Sam, Lim Chap

    2013-01-01

    The purpose of this study was to develop pre-service secondary teachers' technological pedagogical content knowledge (TPACK) for teaching mathematics with The Geometer's Sketchpad (GSP) through Lesson Study (LS). Specifically, a single-group pretest-posttest design was employed to examine whether there was a significant difference in the…

  12. Singapore Pre-Service Secondary Mathematics Teachers' Content Knowledge: Findings from an International Comparative Study

    ERIC Educational Resources Information Center

    Toh, Tin Lam; Kaur, Berinderjeet; Koay, Phong Lee

    2013-01-01

    In this article, we explore the mathematical content knowledge of one entire cohort of pre-service teachers (N = 107) through analysing their performance in a Secondary Mathematics Audit that was developed for the International Comparative Studies in Mathematics Teacher Training that was initiated by the University of Plymouth. We study how their…

  13. Meeting the Dual Goals of Content Knowledge and English Language Learning: A Study of the CCUEI Curriculum Materials

    ERIC Educational Resources Information Center

    Huang, Xiaodan; Trube, Barbara; Yi, Chunlan

    2011-01-01

    This article reports a study on the China-Canada-United States English Immersion (CCUEI) Moral Education and Social Studies (MESS) curriculum materials for elementary classes (Grades 3-6) with the aim of learning how the curriculum addressed the dual goals of MESS content and English language learning. An analysis comparing the CCUEI third grade…

  14. Content Analysis of 1998-2012 Empirical Studies in Science Reading Using a Self-Regulated Learning Lens

    ERIC Educational Resources Information Center

    Hsu, Ying-Shao; Yen, Miao-Hsuan; Chang, Wen-Hua; Wang, Chia-Yu; Chen, Sufen

    2016-01-01

    There is an increasing interest in conducting reading-related studies in science education using a self-regulated learning (SRL) lens. This exploration involved a content analysis of 34 articles (38 studies in total) in highly regarded journals from 1998 to 2012 using an SRL interpretative framework to reveal critical features and relationships in…

  15. Integrating Science and Technology: Using Technological Pedagogical Content Knowledge as a Framework to Study the Practices of Science Teachers

    NASA Astrophysics Data System (ADS)

    Pringle, Rose M.; Dawson, Kara; Ritzhaupt, Albert D.

    2015-10-01

    In this study, we examined how teachers involved in a yearlong technology integration initiative planned to enact technological, pedagogical, and content practices in science lessons. These science teachers, engaged in an initiative to integrate educational technology in inquiry-based science lessons, provided a total of 525 lesson plans for this study. While our findings indicated an increase in technology-related practices, including the use of sophisticated hardware, very little improvements occurred with fostering inquiry-based science and effective science-specific pedagogy. In addition, our conceptual framework, technological pedagogical content knowledge, as a lens to examine teachers' intentions as documented in their lesson plans, provided an additional platform from which to investigate technology integration practices within the ambit of reform science teaching practices. This study, therefore, contributes knowledge about the structure and agenda of professional development initiatives that involve educational technology and integration into content knowledge disciplines such as science.

  16. A Study in Blue: The Baryon Content of Isolated Low-mass Galaxies

    NASA Astrophysics Data System (ADS)

    Bradford, Jeremy D.; Geha, Marla C.; Blanton, Michael R.

    2015-08-01

    We study the baryon content of low-mass galaxies selected from the Sloan Digital Sky Survey (SDSS DR8), focusing on galaxies in isolated environments where the complicating physics of galaxy–galaxy interactions are minimized. We measure neutral hydrogen (HI) gas masses and line widths for 148 isolated galaxies with stellar mass between 107 and {10}9.5{M}ȯ . We compare isolated low-mass galaxies to more massive galaxies and galaxies in denser environments by remeasuring HI emission lines from the Arecibo Legacy Fast ALFA survey 40% data release. All isolated low-mass galaxies either have large atomic gas fractions or large atomic gas fractions cannot be ruled out via their upper limits. We measure a median atomic gas fraction of {f}{gas}=0.81+/- 0.13 for our isolated low-mass sample with no systems below 0.30. At all stellar masses, the correlations between galaxy radius, baryonic mass, and velocity width are not significantly affected by environment. Finally, we estimate a median baryon to total dynamical mass fraction of {f}{baryon,{disk}}=0.15+/- 0.17. We also estimate two different median baryon to halo mass fractions using the results of semi-analytic models ({f}{baryon,{halo}}=0.04+/- 0.06) and abundance matching ({f}{baryon,{halo}}=0.04+/- 0.02). Baryon fractions estimated directly using HI observations appear independent of environment and maximum circular velocity, while baryon fractions estimated using abundance matching show a significant depletion of baryons at low maximum circular velocities.

  17. The study of optical properties and proteoglycan content of tendons by PS-OCT

    NASA Astrophysics Data System (ADS)

    Yang, Ying; Rupani, Asha; Weightman, Alan; Wimpenny, Ian; Bagnaninchi, Pierre; Ahearne, Mark

    2011-03-01

    Tendons are load-bearing collagenous tissues consisting mainly of type I collagen and various proteoglycans (PGs) including decorin and versican. It is widely accepted that highly orientated collagen fibers in tendons a play critical role for transferring tensile stress and demonstrate birefringent optical properties. However, the influence that proteoglycans have on the optical properties of tendons is yet to be fully elucidated. Tendinopathy (defined as a syndrome of tendon pain, tenderness and swelling that affects the normal function of the tissue) is a common disease associated with sporting injuries or degeneration. PG's are the essential components of the tendon extracellular matrix; changes in their quantities and compositions have been associated with tendinopathy. In this study, polarization sensitive optical coherence tomography (PS-OCT) has been used to reveal the relationship between proteoglycan content/location and birefringent properties of tendons. Tendons dissected from freshly slaughtered chickens were imaged at regular intervals by PS-OCT and polarizing light microscope during the extraction of PGs or glycosaminoglycans using established protocols (guanidine hydrochloride (GuHCl) or proteinase K solution). The macroscopic and microscopic time lapsed images are complimentary; mutually demonstrating that there was a higher concentration of PG's in the outer sheath region than in the fascicles; and the integrity of the sheath affected extraction process and the OCT birefringence bands. Extraction of PGs using GuHCl disturbed the organization of local collagen bundles, which corresponded to a reduction in the frequency of birefringence bands and the band width by PS-OCT. The feature of OCT penetration depth helped us to define the heterogeneous distribution of PG's in tendon, which was complimented by polarizing light microscopy. The results provide new insight of tendon structure and also demonstrate a great potential for using PS-OCT as a

  18. [Comparative study on hyperspectral inversion accuracy of soil salt content and electrical conductivity].

    PubMed

    Peng, Jie; Wang, Jia-Qiang; Xiang, Hong-Ying; Teng, Hong-Fen; Liu, Wei-Yang; Chi, Chun-Ming; Niu, Jian-Long; Guo, Yan; Shi, Zhou

    2014-02-01

    The objective of the present article is to ascertain the mechanism of hyperspectral remote sensing monitoring for soil salinization, which is of great importance for improving the accuracy of hyperspectral remote sensing monitoring. Paddy soils in Wensu, Hetian and Baicheng counties of the southern Xinjiang were selected. Hyperspectral data of soils were obtained. Soil salt content (S(t)) an electrical conductivity of 1:5 soil-to-water extracts (EC(1:5)) were determined. Relationships between S(t) and EC(1:5) were studied. Correlations between hyperspectral indices and S(t), and EC(1:5) were analyzed. The inversion accuracy of S(t) using hyperspectral technique was compared with that of EC(1:5). Results showed that: significant (p<0.01) relationships were found between S(t) and EC(1:5) for soils in Wensu and Hetian counties, and correlation coefficients were 0.86 and 0.45, respectively; there was no significant relationship between S(t) and EC(1:5) for soils in Baicheng county. Therefore, the correlations between S(t) and EC(1:5) varied with studied sites. S(t) and EC(1:5) were significantly related with spectral reflectance, first derivative reflectance and continuum-removed reflectance, respectively; but correlation coefficients between S(t) and spectral indices were higher than those between EC(1:5) and spectral indices, which was obvious in some sensitive bands for soil salinization such as 660, 35, 1229, 1414, 1721, 1738, 1772, 2309 nm, and so on. Prediction equations of St and EC(1:5) were established using multivariate linear regression, principal component regression and partial least-squares regression methods, respectively. Coefficients of determination, determination coefficients of prediction, and relative analytical errors of these equations were analyzed. Coefficients of determination and relative analytical errors of equations between S(t) and spectral indices were higher than those of equations between EC(1:5) and spectral indices. Therefore, the

  19. A comparative study of body potassium content in males and females at Kalpakkam (India).

    PubMed

    Brindha, J Thulasi; Rajaram, S; Kannan, V

    2007-01-01

    A group of 300 subjects of both sexes were measured for total body potassium content using a shadow shield whole-body counting system at Kalpakkam (India). The mean body potassium content for males was 1.7 +/- 0.5 g kg(-1) body weight and 1.5 +/- 0.5 g kg(-1) body weight for females. The body potassium content varies inversely with age as well as with the body weight for both the sexes. However, the total body potassium varies directly with body-build index for both males and females. The mean annual dose due to (40)K for males and females was found to be 149 +/- 44 and 132 +/- 41 uGy, respectively.

  20. Exploring elementary school teachers' perception of their role in teaching content literacy in the elementary science and social studies classrooms: A mixed methods study

    NASA Astrophysics Data System (ADS)

    Jones-Moore, Lisa Michelle

    2011-12-01

    This mixed-methods study explored third, fourth, and fifth grade teachers' perceptions of their role in teaching content literacy in the elementary science and social studies classroom. The rationale for this study was the growing number of studies questioning the reliance on the inoculation theory for content area literacy comprehension. The study was a mixed methods study so as to provide insight into the participants' thought processes in decision making and instructional planning. Data sources included timed instructional observations, tiered checklist to identify strategy instruction, and prompted critical reflections. The three-tiered observation instrument categorized strategies used by teachers in tiers according to the focus of the strategy. Tier I strategies were those identified as strategies good readers use, typically taught with narrative text. The inoculation theory posits these skills transfer to reading informational and expository text. Tier II strategies were those identified as strategies appropriate for informational or expository text. Use of these strategies acknowledged that narrative and informational/expository text require different strategies, but does not differentiate between expository text drawn from particular content area. Tier III strategies were those identified as strategies particularly suited to informational or expository text drawn from specific content areas. These strategies embody cognitive processes used to comprehend text drawn from specific content areas. The findings showed the participating teachers used a preferential Tier of strategy instruction. Some participants felt that reading comprehension was more important than content. They viewed reading as a subject instead of an integral part of science and social studies instruction.

  1. An ergonomic study on the navigation structure and information units of websites with multimedia content. A case study of the Xbox 360 promotional website.

    PubMed

    Ariel, Eduardo; de Moraes, Anamaria

    2012-01-01

    This paper presents an ergonomic study on the navigation structures and information units of entertainment sites with multimedia content. This research is a case study on the XBOX 360 promotional website. It analyzes the presentation of the content on a grid that simulates the spatial displacement of the screen's elements and evaluates the interaction that the page allows for, from the users' point of view.

  2. Using Reading Strategies To Reduce the Failure Rate in the Content Area. Subject: Social Studies. Grade Level: 6-7-8.

    ERIC Educational Resources Information Center

    Dobbs, Olivett

    Content area reading instruction includes two elements: the information presented in subject matter text, and the plan that teachers use to help students understand the content. According to research and interviews with social studies teachers, there is a high failure rate in the social studies content area because children have problems…

  3. Studying Challenges in Integrating Technology in Secondary Mathematics with Technological Pedagogical and Content Knowledge (TPACK)

    ERIC Educational Resources Information Center

    Stoilescu, Dorian

    2014-01-01

    This paper describes challenges encountered by two secondary mathematics teachers when they try to integrate ICT devices in their classes. These findings are based on using the Technological Pedagogical and Content Knowledge (TPACK) context, the four dimension framework developed by Niess: 1) overarching conceptions of integrating ICT, 2)…

  4. Podcasting in Engineering Education: A Preliminary Study of Content, Student Attitudes, and Impact

    ERIC Educational Resources Information Center

    Berger, Edward

    2007-01-01

    Edward Berger describes a pilot project implemented in an undergraduate engineering mechanics course, entitled Strength of Materials to investigate whether and how students would perceive a benefit from podcasting as a pedagogical tool. Three types of podcasting content were produced: (a) video problem solutions, (b) roundtable discussions, and…

  5. A Case Study of Alternatively Trained Science Teachers: Attainment of Pedagogical Content Knowledge

    ERIC Educational Resources Information Center

    Duncan, Benjamin R.

    2013-01-01

    Elements essential to effective teaching are closely aligned with the domains of a teacher's pedagogical content knowledge (PCK) (Park & Oliver, 2008). Often, alternatively trained teachers enter the teaching profession lacking exposure to pedagogical events that allow these educators opportunities to reflect on their practice and construction…

  6. Children, Multimedia Content and Technological Artefacts: An Exploratory Study Using Text Analysis Tools

    ERIC Educational Resources Information Center

    Mazzoni, Elvis; Nicolò, Enrico; Sapio, Bartolomeo

    2015-01-01

    Purpose: This paper aims to investigate the user experience of young people with video content accessed through different technological artefacts. Design/methodology/approach: To this purpose, an essay has been assigned to the pupils of two lower secondary school classes (mean age 12 years) to know their diverse types of usage of multimedia…

  7. The dielectric behaviour of snow: A study versus liquid water content

    NASA Technical Reports Server (NTRS)

    Ambach, W.; Denoth, A.

    1980-01-01

    Snow is treated as a heterogeneous dielectric material consisting of ice, air, and water. The greater difference in the high frequency relative permittivity of dry snow and water allows to determine the liquid water content by measurements of the relative permittivity of snow. A plate condenser with a volume of about 1000 cv cm was used to measure the average liquid water content in a snow volume. Calibration was carried out using a freezing calorimeter. In order to measure the liquid water content in thin snow layers, a comb-shaped condenser was developed, which is the two dimensional analogon of the plate condenser. With this moisture meter the liquid water content was measured in layers of a few millimeters in thickness, whereby the effective depth of measurement is given by the penetration depth of electric field lines which is controlled by the spacing of the strip lines. Results of field measurements with both moisture meters, the plate condenser and the comb-shaped condenser, are given.

  8. The Next Generation of Psychological Autopsy Studies: Part I. Interview Content

    ERIC Educational Resources Information Center

    Conner, Kenneth R.; Beautrais, Annette L.; Brent, David A.; Conwell, Yeates; Phillips, Michael R.; Schneider, Barbara

    2011-01-01

    The psychological autopsy (PA) is a systematic method to understand the psychological and contextual circumstances preceding suicide. The method requires interviews with one or more proxy respondents (i.e., informants) of decedents. The methodological challenges that need to be addressed when determining the content of these research interviews…

  9. Design of Online Professional Development in Science Content and Pedagogy: A Pilot Study in Florida

    ERIC Educational Resources Information Center

    Cavanaugh, Cathy; Dawson, Kara

    2010-01-01

    The Exploring Florida Science project goals were: (1) increasing content knowledge of secondary science teachers, specifically in topics that are important to the future of the state, and (2) providing secondary science students with digital media for use in project-based learning. A team of instructional designers, educators, scientists, web…

  10. Content Analysis of Introductory Interior Design College Textbooks: A Study Revisited

    ERIC Educational Resources Information Center

    Temple, Julie A.; Potthoff, Joy K.

    2013-01-01

    Introductory interior design texts adopted by design educators present information relevant to both historical and contemporary issues in interior design. According to one author, they provide a "survey of the field of interior design as it now exists" (Pile, 2007). A comparison of the content of contemporary texts with those of more…

  11. Accuracy of stated energy contents of restaurant foods in a multi-site study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Context National recommendations for prevention and treatment of obesity emphasize reducing energy intake. Foods purchased in restaurants provide approximately 35% of daily energy intake, but the accuracy of information on the energy contents of these foods is unknown. Objective To examine the a...

  12. Pedagogical Content Knowledge Development and Pre-Service Physics Teacher Education: A Case Study

    ERIC Educational Resources Information Center

    Sperandeo-Mineo, R. M.; Fazio, C.; Tarantino, G.

    2006-01-01

    This paper addresses the question of how to develop prospective teachers' pedagogical content knowledge (PCK) in science teacher education. The main focus is on the knowledge transformation process and on the cognitive strategies used to shift prospective teachers' explanations within the domain of modelling thermal physical phenomena. This study…

  13. Nutrient Content of Consumed Elementary School Lunches: A Pilot Study from Sweden

    ERIC Educational Resources Information Center

    Rosander, Ulla; Rumpunen, Kimmo; Lindmark-Mansson, Helena; Gullberg, Bo; Paulsson, Marie; Holm, Ingvar

    2013-01-01

    Purpose/Objectives: Purpose was to investigate the nutrient content of Swedish school meals consumed by students in the context of national recommendations regarding food composition and intake. Methods: Composite samples of lunch meals consumed by six students during a five-day period were collected using the double portion method and analyzed…

  14. Content and Language Integrated Learning through an Online Game in Primary School: A Case Study

    ERIC Educational Resources Information Center

    Dourda, Kyriaki; Bratitsis, Tharrenos; Griva, Eleni; Papadopoulou, Penelope

    2014-01-01

    In this paper an educational design proposal is presented which combines two well established teaching approaches, that of Game-based Learning (GBL) and Content and Language Integrated Learning (CLIL). The context of the proposal was the design of an educational geography computer game, utilizing QR Codes and Google Earth for teaching English…

  15. Pilot study: tendency of increasing iodine content in human milk and cow's milk.

    PubMed

    Bader, N; Möller, U; Leiterer, M; Franke, K; Jahreis, G

    2005-01-01

    The iodine supply in Germany has improved throughout the last decade, albeit with enormous differences between individuals and regions. In the Thuringian city of Jena, analyses of the iodine content of human milk have been undertaken regularly since 1982. Significantly increasing iodine concentrations in human and cow's milk have been found. Therefore, the current situation and the effectiveness of measures to prevent iodine deficiency demands re-evaluation. The iodine content of human milk from 32 lactating mothers was analysed on the 5th day (mean) postpartum and mothers' dietary iodine intake during the last two months of pregnancy was assessed by means of a food frequency questionnaire. To corroborate the assumption that the increasing iodine levels of cow's milk are one of the main reasons for the improved iodine supply, the iodine concentration of 34 cow's milk bulk-samples was also determined. Both human and cow's milk samples were analysed by the ICP-MS method. Twenty women took iodine supplements (mean daily intake = 175 microg). The average daily iodine intake of the 20 supplemented and 12 non-supplemented women was 258 microg and 116 microg, respectively. Daily iodine intake from food and beverages was significantly lower in supplemented women (83 microg/day). The average iodine content of human milk was 169 +/- 88 microg/l with a range of 33 - 348 microg/l. This content is two times higher than levels from 1994 in the same area. There was no difference in the human milk iodine content between mothers taking supplements and those who did not. Cow's milk samples showed a mean iodine concentration of 178 +/- 131 microg/l (range 48 - 661 microg/l).

  16. Loss of Retinoschisin (RS1) Cell Surface Protein in Maturing Mouse Rod Photoreceptors Elevates the Luminance Threshold for Light-Driven Translocation of Transducin But Not Arrestin

    PubMed Central

    Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bush, Ronald A.

    2012-01-01

    Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1–KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1–KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1–KO mice as a marker for functional abnormalities in maturing rod photoreceptors. We found a progressive reduction in luminance threshold for transducin translocation in wild-type (WT) retinas between postnatal days P18 and P60. At P21, the threshold in Rs1–KO retinas was 10-fold higher than WT, but it decreased to <2.5-fold higher by P60. Light-activated arrestin translocation and re-translocation of transducin in the dark were not affected. Rs1–KO rod outer segment (ROS) length was significantly shorter than WT at P21 but was comparable with WT at P60. These findings suggested a delay in the structural and functional maturation of Rs1–KO ROS. Consistent with this, transcription factors CRX and NRL, which are fundamental to maturation of rod protein expression, were reduced in ROS of Rs1–KO mice at P21 but not at P60. Expression of transducin was 15–30% lower in P21 Rs1–KO ROS and transducin GTPase hydrolysis was nearly twofold faster, reflecting a 1.7- to 2.5-fold increase in RGS9 (regulator of G-protein signaling) level. Transduction protein expression and activity levels were similar to WT at P60. Transducin translocation threshold elevation indicates photoreceptor functional abnormalities in young Rs1–KO mice. Rapid reduction in threshold coupled with age-related changes in transduction protein levels and transcription factor expression are consistent with delayed maturation of Rs1–KO photoreceptors. PMID:22993419

  17. A Study of K-12 Teacher Interns' Incorporation of Multicultural Content and Theory into Their Teaching Practices

    ERIC Educational Resources Information Center

    Dzoole, Edith Mechelle

    2012-01-01

    This research study examined 394 K-12 teacher interns' incorporation of multicultural content and theory into their teaching practices during a 16-week internship in schools, mostly located within a 30-mile radius of Mississippi State University. The teacher interns had completed all coursework and practicum hours required by their teacher…

  18. Analysis of High School Newspaper Editorials Before and After "Hazelwood School District v. Kuhlmeier:" A Content Analysis Case Study.

    ERIC Educational Resources Information Center

    Lomicky, Carol S.

    2000-01-01

    In "Hazelwood" the U.S. Supreme Court said public school officials can censor school-sponsored expression for legitimate educational purposes. A content-analysis case study of student-written newspaper editorials found that more than three times as many editorials of criticism were published prior to the Court's decision. Argues that since the…

  19. Individual Differences in Navigation between Sharable Content Objects--An Evaluation Study of a Learning Module Prototype.

    ERIC Educational Resources Information Center

    Gauss, Boris; Urbas, Leon

    2003-01-01

    Reports the design and evaluation of a prototype for learning modules compliant to the SCORM (Sharable Content Object Reference Model) standard for use with hypermedia systems in Web-based instruction. Discusses a study of undergraduates that considered relations between individual differences in learner characteristics, including intrinsic…

  20. The Influence of Content Knowledge on Teaching and Learning in Traditional and Sport Education Contexts: An Exploratory Study

    ERIC Educational Resources Information Center

    Iserbyt, Peter; Ward, Phillip; Martens, Jonas

    2016-01-01

    Background: Our understanding of the role in which content knowledge (CK) can strengthen instructional models and how that knowledge matters for professional development is limited. It is contended that mere use of an instructional model is insufficient to impact psychomotor learning in meaningful ways. Purpose: This study was conducted to…

  1. National Education Longitudinal Study of 1988: Second Follow-up Questionnaire Content Areas and Research Issues. Working Paper Series.

    ERIC Educational Resources Information Center

    Ingels, Steven; Dowd, Katy

    The National Education Longitudinal Study of 1988 (NELS:88) provides a wealth of information about factors that influence academic performance and social development of students. The focus of this content overview is the research issues addressed by the NELS:88 second followup in 1992. In 1988, by surveying nearly 25,000 eighth graders, their…

  2. Effects of decreasing resolution on spectral and spatial information content in an agricultural area. [Pottawatmie study site, Iowa and Nebraska

    NASA Technical Reports Server (NTRS)

    1983-01-01

    The effects of decreasing spatial resolution from 6 1/4 miles square to 50 miles square are described. The effects of increases in cell size is studied on; the mean and variance of spectral data; spatial trends; and vegetative index numbers. Information content changes on cadastral, vegetal, soil, water and physiographic information are summarized.

  3. The Convergence of Content, Pedagogy, and Technology in Online Professional Development for Teachers of German: An Intrinsic Case Study

    ERIC Educational Resources Information Center

    Bustamante, Carolina; Moeller, Aleidine J.

    2013-01-01

    This qualitative case study describes a unique online professional development program utilizing Web 2.0 technologies for teachers of German using the Technological Pedagogical Content Knowledge (TPACK) model as a theoretical framework to promote technology literacy, expand German language proficiency and cultural knowledge, and integrate…

  4. Scientific Evidence as Content Knowledge: A Replication Study with English and Turkish Pre-Service Primary Teachers

    ERIC Educational Resources Information Center

    Roberts, Ros; Sahin-Pekmez, Esin

    2012-01-01

    Pre-service teachers around the world need to develop their content knowledge of scientific evidence to meet the requirements of recent school curriculum developments which prepare pupils to be scientifically literate. This research reports a replication study in Turkey of an intervention originally carried out with pre-service primary teachers in…

  5. Introduction to Computers & Introduction to Word Processing: Integrating Content Area Coursework into College Reading/Study Skills Curricula Using Microcomputers.

    ERIC Educational Resources Information Center

    Balajthy, Ernest; And Others

    A study examined the planning, implementation, and evaluation of a curriculum designed to teach 60 college level developmental reading students to use microcomputers (Apple) as learning tools and to improve their content area reading ability. The textbook from a biology course in which all but three of the subjects were enrolled was the source for…

  6. Content Analysis of Essays from a Cross-National Survey: Implications for Teaching Strategies in Holocaust Studies.

    ERIC Educational Resources Information Center

    McRoy, James J.

    The content of essays written by randomly selected samples of 1500 U.S. and 500 British secondary students on the topic "What have I learned about Adolf Hitler?" were partitioned into theme-related assertions and analyzed. An experimental group of 150 9th- and 11th-grade male students who had studied the Holocaust also contributed papers that were…

  7. Integrating Science and Technology: Using Technological Pedagogical Content Knowledge as a Framework to Study the Practices of Science Teachers

    ERIC Educational Resources Information Center

    Pringle, Rose M.; Dawson, Kara; Ritzhaupt, Albert D.

    2015-01-01

    In this study, we examined how teachers involved in a yearlong technology integration initiative planned to enact technological, pedagogical, and content practices in science lessons. These science teachers, engaged in an initiative to integrate educational technology in inquiry-based science lessons, provided a total of 525 lesson plans for this…

  8. A Content Analytic Study of "People's Daily" in Its Socialization Process: A Case in Cross-Cultural Communication.

    ERIC Educational Resources Information Center

    Marr, Theodore J.

    The author outlines a study comparing the modes of communication in two newspapers: "Jen Min Jih Pao" (People's Daily) and the Des Moines"Register." He applies a content-category system, based on Hayakawa's trichotomy of sentence types, to the reporting of these two newspapers on five major international events: the 1967 Arab-Israeli conflict, the…

  9. The Effects of Topic Familiarity, Author Expertise, and Content Relevance on Norwegian Students' Document Selection: A Mixed Methods Study

    ERIC Educational Resources Information Center

    McCrudden, Matthew T.; Stenseth, Tonje; Bråten, Ivar; Strømsø, Helge I.

    2016-01-01

    This mixed methods study investigated the extent to which author expertise and content relevance were salient to secondary Norwegian students (N = 153) when they selected documents that pertained to more familiar and less familiar topics. Quantitative results indicated that author expertise was more salient for the less familiar topic (nuclear…

  10. Free-Response and Card-Sort Techniques for Assessing Cognitive Content: Two Studies Concerning their Stability, Validity, and Utility.

    ERIC Educational Resources Information Center

    Hirschman, Elizabeth C.; Wallendorf, Melanie R.

    1982-01-01

    Free-response and card-sort techniques are criticized as to their application to investigating cognitive content. Two studies are presented which examine the validity and reliability of these two techniques when they are used concurrently with college students. (Author)

  11. Delivering Educational Multimedia Contents through an Augmented Reality Application: A Case Study on Its Impact on Knowledge Acquisition and Retention

    ERIC Educational Resources Information Center

    Perez-Lopez, David; Contero, Manuel

    2013-01-01

    This paper presents a study to analyze the use of augmented reality (AR) for delivering multimedia content to support the teaching and learning process of the digestive and circulatory systems at the primary school level, and its impact on knowledge retention. Our AR application combines oral explanations and 3D models and animations of anatomical…

  12. [Study of Determination of Oil Mixture Components Content Based on Quasi-Monte Carlo Method].

    PubMed

    Wang, Yu-tian; Xu, Jing; Liu, Xiao-fei; Chen, Meng-han; Wang, Shi-tao

    2015-05-01

    Gasoline, kerosene, diesel is processed by crude oil with different distillation range. The boiling range of gasoline is 35 ~205 °C. The boiling range of kerosene is 140~250 °C. And the boiling range of diesel is 180~370 °C. At the same time, the carbon chain length of differentmineral oil is different. The carbon chain-length of gasoline is within the scope of C7 to C11. The carbon chain length of kerosene is within the scope of C12 to C15. And the carbon chain length of diesel is within the scope of C15 to C18. The recognition and quantitative measurement of three kinds of mineral oil is based on different fluorescence spectrum formed in their different carbon number distribution characteristics. Mineral oil pollution occurs frequently, so monitoring mineral oil content in the ocean is very important. A new method of components content determination of spectra overlapping mineral oil mixture is proposed, with calculation of characteristic peak power integrationof three-dimensional fluorescence spectrum by using Quasi-Monte Carlo Method, combined with optimal algorithm solving optimum number of characteristic peak and range of integral region, solving nonlinear equations by using BFGS(a rank to two update method named after its inventor surname first letter, Boyden, Fletcher, Goldfarb and Shanno) method. Peak power accumulation of determined points in selected area is sensitive to small changes of fluorescence spectral line, so the measurement of small changes of component content is sensitive. At the same time, compared with the single point measurement, measurement sensitivity is improved by the decrease influence of random error due to the selection of points. Three-dimensional fluorescence spectra and fluorescence contour spectra of single mineral oil and the mixture are measured by taking kerosene, diesel and gasoline as research objects, with a single mineral oil regarded whole, not considered each mineral oil components. Six characteristic peaks are

  13. [Study of Determination of Oil Mixture Components Content Based on Quasi-Monte Carlo Method].

    PubMed

    Wang, Yu-tian; Xu, Jing; Liu, Xiao-fei; Chen, Meng-han; Wang, Shi-tao

    2015-05-01

    Gasoline, kerosene, diesel is processed by crude oil with different distillation range. The boiling range of gasoline is 35 ~205 °C. The boiling range of kerosene is 140~250 °C. And the boiling range of diesel is 180~370 °C. At the same time, the carbon chain length of differentmineral oil is different. The carbon chain-length of gasoline is within the scope of C7 to C11. The carbon chain length of kerosene is within the scope of C12 to C15. And the carbon chain length of diesel is within the scope of C15 to C18. The recognition and quantitative measurement of three kinds of mineral oil is based on different fluorescence spectrum formed in their different carbon number distribution characteristics. Mineral oil pollution occurs frequently, so monitoring mineral oil content in the ocean is very important. A new method of components content determination of spectra overlapping mineral oil mixture is proposed, with calculation of characteristic peak power integrationof three-dimensional fluorescence spectrum by using Quasi-Monte Carlo Method, combined with optimal algorithm solving optimum number of characteristic peak and range of integral region, solving nonlinear equations by using BFGS(a rank to two update method named after its inventor surname first letter, Boyden, Fletcher, Goldfarb and Shanno) method. Peak power accumulation of determined points in selected area is sensitive to small changes of fluorescence spectral line, so the measurement of small changes of component content is sensitive. At the same time, compared with the single point measurement, measurement sensitivity is improved by the decrease influence of random error due to the selection of points. Three-dimensional fluorescence spectra and fluorescence contour spectra of single mineral oil and the mixture are measured by taking kerosene, diesel and gasoline as research objects, with a single mineral oil regarded whole, not considered each mineral oil components. Six characteristic peaks are

  14. A synthesis and meta-analysis of reading interventions using social studies content for students with learning disabilities.

    PubMed

    Swanson, Elizabeth; Hairrell, Angela; Kent, Shawn; Ciullo, Stephen; Wanzek, Jeanne A; Vaughn, Sharon

    2014-01-01

    A synthesis and meta-analysis of the extant research on the effects of reading interventions delivered using social studies content for students with learning disabilities in kindergarten through Grade 12 is provided. A total of 27 studies met criteria for the synthesis, with 16 studies providing sufficient data for inclusion in the meta-analysis. Reading interventions implemented within the context of social studies have employed the use of graphic organizers, mnemonics, reading and answering questions, guided notes, and multicomponent comprehension instruction. The overall mean effect size for interventions included in the meta-analysis was 1.02, indicating that reading interventions delivered using social studies content have a substantial positive effect on outcomes among students with learning disabilities.

  15. Free water content and monitoring of healing processes of skin burns studied by microwave dielectric spectroscopy in vivo

    NASA Astrophysics Data System (ADS)

    Hayashi, Yoshihito; Miura, Nobuhiro; Shinyashiki, Naoki; Yagihara, Shin

    2005-02-01

    We have investigated the dielectric properties of human skin in vivo at frequencies up to 10 GHz using a time-domain reflectometry method with open-ended coaxial probes. Since γ-dispersion results from the reorientation of free water molecules, the free water content of skin is quantitatively determined by dielectric measurements. The free water content of finger skin increased by about 10% after soaking in 37 °C water for 30 min, and it systematically decreased again through the drying process, as expected. Thus this analytical method has been applied to the study of skin burns. The free water content of burned human cheek skin due to hydrofluoric acid was significantly lower than that of normal skin, and the burned skin recovered through the healing process. In the case of a human hand skin burn due to heat, although the free water content was almost the same as that of normal skin at the beginning, it decreased during the healing process for the first 10 days, then began to increase. Although the number of test subjects was one for each experiment, it was shown that free water content is a good indicator for evaluating skin health and can be well monitored by dielectric spectroscopy.

  16. [Application study of the thermal infrared emissivity spectra in the estimation of salt content of saline soil].

    PubMed

    Xia, Jun; Tashpolat, Tiyip; Mamat, Sawut; Zhang, Fei; Han, Gui-Hong

    2012-11-01

    Studying of soil salinization is of great significance for agricultural production in arid area oasis, thermal infrared remote sensing technology provides a new technology and method in this field. Authors used Fourier transform infrared spectrometer to measure the oasis saline soil in field, employed iterative spectrally smooth temperature/emissivity separation algorithm (ISSTES) to separate temperature and emissivity, and acquired the thermal infrared emissivity data of the saline soil. Through researching the emissivity spectral feature of saline soil, and concluded that soil emissivity will reduce with the increasing of salt content from 8 to 13 microm, so emissivity spectra is more sensitive to salt factor from 8 to 9.5 microm. Then, analyzed the correlation between original emissivity spectra and its first derivative, second derivative and normalized ratio with salt content, the result showed that they have a negative correlation relationship between soil emissivity and salt content, and the correlation between emissivity first derivative and salt content is highest, reach to 0.724 2, the corresponding bands are from 8.370 745-8.390 880 microm. Finally, established the quadratic function regression model, its determination coefficient is 0.741 4, and root mean square error is 0.235 5, the result explained that the approach of using thermal infrared emissivity to retrieve the salt content of saline soil is feasible.

  17. Dream Recall Frequencies and Dream Content in Wilson's Disease with and without REM Sleep Behaviour Disorder: A Neurooneirologic Study

    PubMed Central

    Trindade, Mateus C.; Schredl, Michael; Pires, Joana; Reinhard, Iris; Bittencourt, Thais; Lorenzi-Filho, Geraldo; Alves, Rosana Cardoso; de Andrade, Daniel Ciampi; Fonoff, Erich T.; Bor-Seng-Shu, Edson; Machado, Alexandre A.; Teixeira, Manoel J.; Barbosa, Egberto R.

    2016-01-01

    Objective. Violent dream content and its acting out during rapid eye movement sleep are considered distinctive for rapid eye movement sleep behaviour disorder (RBD). This study reports first quantitative data on dreaming in a cohort of patients with treated Wilson's disease (WD) and in patients with WD with RBD. Methods. Retrospective questionnaires on different dimensions of dreaming and a prospective two-week home dream diary with self-rating of emotions and blinded, categorical rating of content by an external judge. Results. WD patients showed a significantly lower dream word count and very few other differences in dream characteristics compared to age- and sex-matched healthy controls. Compared to WD patients without RBD, patients with WD and RBD reported significantly higher nightmare frequencies and more dreams with violent or aggressive content retrospectively; their prospectively collected dream reports contained significantly more negative emotions and aggression. Conclusions. The reduction in dream length might reflect specific cognitive deficits in WD. The lack of differences regarding dream content might be explained by the established successful WD treatment. RBD in WD had a strong impact on dreaming. In accordance with the current definition of RBD, violent, aggressive dream content seems to be a characteristic of RBD also in WD. PMID:27051076

  18. Dream Recall Frequencies and Dream Content in Wilson's Disease with and without REM Sleep Behaviour Disorder: A Neurooneirologic Study.

    PubMed

    Tribl, Gotthard G; Trindade, Mateus C; Schredl, Michael; Pires, Joana; Reinhard, Iris; Bittencourt, Thais; Lorenzi-Filho, Geraldo; Alves, Rosana Cardoso; de Andrade, Daniel Ciampi; Fonoff, Erich T; Bor-Seng-Shu, Edson; Machado, Alexandre A; Teixeira, Manoel J; Barbosa, Egberto R

    2016-01-01

    Objective. Violent dream content and its acting out during rapid eye movement sleep are considered distinctive for rapid eye movement sleep behaviour disorder (RBD). This study reports first quantitative data on dreaming in a cohort of patients with treated Wilson's disease (WD) and in patients with WD with RBD. Methods. Retrospective questionnaires on different dimensions of dreaming and a prospective two-week home dream diary with self-rating of emotions and blinded, categorical rating of content by an external judge. Results. WD patients showed a significantly lower dream word count and very few other differences in dream characteristics compared to age- and sex-matched healthy controls. Compared to WD patients without RBD, patients with WD and RBD reported significantly higher nightmare frequencies and more dreams with violent or aggressive content retrospectively; their prospectively collected dream reports contained significantly more negative emotions and aggression. Conclusions. The reduction in dream length might reflect specific cognitive deficits in WD. The lack of differences regarding dream content might be explained by the established successful WD treatment. RBD in WD had a strong impact on dreaming. In accordance with the current definition of RBD, violent, aggressive dream content seems to be a characteristic of RBD also in WD. PMID:27051076

  19. Dream Recall Frequencies and Dream Content in Wilson's Disease with and without REM Sleep Behaviour Disorder: A Neurooneirologic Study.

    PubMed

    Tribl, Gotthard G; Trindade, Mateus C; Schredl, Michael; Pires, Joana; Reinhard, Iris; Bittencourt, Thais; Lorenzi-Filho, Geraldo; Alves, Rosana Cardoso; de Andrade, Daniel Ciampi; Fonoff, Erich T; Bor-Seng-Shu, Edson; Machado, Alexandre A; Teixeira, Manoel J; Barbosa, Egberto R

    2016-01-01

    Objective. Violent dream content and its acting out during rapid eye movement sleep are considered distinctive for rapid eye movement sleep behaviour disorder (RBD). This study reports first quantitative data on dreaming in a cohort of patients with treated Wilson's disease (WD) and in patients with WD with RBD. Methods. Retrospective questionnaires on different dimensions of dreaming and a prospective two-week home dream diary with self-rating of emotions and blinded, categorical rating of content by an external judge. Results. WD patients showed a significantly lower dream word count and very few other differences in dream characteristics compared to age- and sex-matched healthy controls. Compared to WD patients without RBD, patients with WD and RBD reported significantly higher nightmare frequencies and more dreams with violent or aggressive content retrospectively; their prospectively collected dream reports contained significantly more negative emotions and aggression. Conclusions. The reduction in dream length might reflect specific cognitive deficits in WD. The lack of differences regarding dream content might be explained by the established successful WD treatment. RBD in WD had a strong impact on dreaming. In accordance with the current definition of RBD, violent, aggressive dream content seems to be a characteristic of RBD also in WD.

  20. [NEUROCHEMICAL STUDY OF TROPOXIN EFFECTS ON THE CONTENT OF MONOAMINES AND ITS METABOLITES IN WISTAR RAT BRAIN STRUCTURES].

    PubMed

    Naplekova, P L; Shevchenko, S V; Narkevich, V B; Gan'shina, T S; Kostochka, L M; Mirzoyan, R S; Kudrin, V S; Voronina, T A

    2016-01-01

    The influence of perspective anti-migraine drug tropoxin on the content of monoamines and related metabolites in Wistar rat brain structures, including frontal cortex (FC), hypothalamus, nucleus accumbens (NA), striatum, and hippocampus, has been studied using HPLC/ED technique. Tropoxin (10 mg/kg) induced a 30% decrease (p < 0.05) in dopamine (DA) level in FC as well as norepinephrine content in NA, while the concentrations of DA metabolites DOPAC and HVA in the hypothalamus were found to increase. The injection of tropoxin in a dose of 20 mg/kg led to an increase in HVA level in hypothalamus as well as seroto- nin metabolite 5-HIAA content in NA. The obtained data provide evidence that tropoxin predominantly influenced the activity of dopaminergic system while the drug effects on the parameters of serotoninergic link seem to be rather mild.

  1. Design of Online Professional Development in Science Content and Pedagogy: A Pilot Study in Florida

    NASA Astrophysics Data System (ADS)

    Cavanaugh, Cathy; Dawson, Kara

    2010-10-01

    The Exploring Florida Science project goals were: (1) increasing content knowledge of secondary science teachers, specifically in topics that are important to the future of the state, and (2) providing secondary science students with digital media for use in project-based learning. A team of instructional designers, educators, scientists, web designers and teacher educators designed an online professional development environment. Needs analysis included identification of the high incidence topics on the state science achievement test and topics of socio-scientific importance in the state. Development followed tenets of design-based research, and was guided by E-Learning for Educators standards and evaluated for content, pedagogy and usability using rubrics based on established guidelines. This paper details design, development, and evaluation frameworks, and summarizes pilot testing outcomes.

  2. NMR study on mechanisms of ionic polymer-metal composites deformation with water content

    NASA Astrophysics Data System (ADS)

    Zhu, Zicai; Chen, Hualing; Wang, Yongquan; Luo, Bin; Chang, Longfei; Li, Bo; Chen, Luping

    2011-10-01

    Ionic polymer-metal composites (IPMCs) exhibit a large dynamic bending deformation under exterior electric field. The states and proportions of water within the IPMCs have great effect on the IPMCs deformation properties. This letter investigates the influence of the proportion changes of different types of water on the deformation, which may disclose the working mechanisms of the IPMCs. We give a deformation trend of IPMCs with the reduction of water content firstly. Then by the method of nuclear magnetic resonance, various water types (water bonded to sulfonates, loosely bound water and free water) of IPMCs and their proportions are investigated in the drying process which corresponds to their different deformation states. It is obtained that the deformation properties of IPMCs depend strongly on their water content and the excess free water is responsible for the relaxation deformation.

  3. Combustion studies of high moisture content waste in a fluidised bed.

    PubMed

    Suksankraisorn, K; Patumsawad, S; Fungtammasan, B

    2003-01-01

    The combustion of three high moisture content waste materials in a fluidised bed combustor has been investigated and a comparison with co-firing of these materials with coal in the same combustor has been made. Waste materials burnt were olive oil waste, municipal solid waste and potato, which is representative of vegetable waste. Mixtures of up to 20% mass concentration water in the waste were fed to the combustor. Above that value the moisture content was too high to sustain combustion without addition of coal. Measurements of CO, NOx, SO2 temperatures were made and the carbon combustion efficiency evaluated. Co-firing with coal resulted in markedly higher combustion efficiencies with an increase of approximately 10-80% when burning the simulated MSW. However, this was much lower than the value of 93% when coal was burnt on its own. It was also much lower than the value obtained, average 90%, when co-firing potato and olive oil waste with coal and there was little difference in the combustion efficiency between the two types of waste and with increasing moisture content. It was concluded that the high ash content of the simulated MSW 26%, compared with 5% in the other two waste materials resulted in slower burning and consequently the char particles were elutriated from the bed without being fully burnt. In term of gaseous emissions during co-combustion, CO emission is relatively insensitive to change in waste fraction. While emission of SO2 can be reduced as the waste fraction increases as a result of fuel-S dilution. But in terms of percent fuel-S converted, it is actually increased by increasing waste fraction. Emissions of NO and N2O increase slightly with MSW fraction.

  4. Health promotion at local level: a case study of content, organization and development in four Swedish municipalities

    PubMed Central

    2010-01-01

    Background Several health determinants are related to local conditions and prerequisites at community level. For this reason, strengthening community action has been one of five strategies implemented in health promotion since the end of the 1980s. Such action includes setting priorities, making decisions, planning strategies, and implementing them to achieve better health. The aim of this paper is to obtain a deeper understanding of content, organization and processes in the development of local health promotion. Methods A qualitative multiple case study of four Swedish municipalities. The cases were analyzed in accordance with the principles of cross-case study analysis, and a content analysis of documents and interviews was conducted in two steps. First, a manifest content analysis was performed to identify present and former actors and measures. Thereafter, a latent content analysis was performed to investigate structures and processes in local contexts. Results The results of the inductive content analysis showed development of local health promotion in three phases: initiation, action, and achievement. Strengthening factors were local actors, health statistics and events. Hindering factors were lack of resources and vague objectives. External factors, e.g. national policies, were not perceived as prominent influencing factors. Media reports were regarded as having had an influence, but only to some extent. The content of local health promotion has developed from ad-hoc lifestyle and behaviour-related actions into structural, intersectoral actions related to determinants of health. Conclusions The municipalities have organized and developed their health promotion targets, actions and priorities on the basis of local needs and prerequisites. The three phases in the identified health promotion processes were experienced and documented as being subject to greater influence from internal rather than external strengthening and hindering factors in their local

  5. Comparative study on the vitamin C contents of the food legume seeds.

    PubMed

    Moriyama, Michie; Oba, Kazuko

    2008-02-01

    We found that dehydrated legume seeds (6 genera, 19 species and cultivated varieties) contained considerable amounts of vitamin C (VC). The average value of total VC content per 100 g of dry weight in dehydrated seeds varied from 0.24 mg (kidney beans) to 4.14 mg (green peas). Yard beans showed highest values among all legumes examined here in the both dehydrated and rehydrated forms (3.19 and 10.8 mg, respectively). By soaking for 16 h in the dark at 20(o)C, total VC contents of black grams and mung beans increased to 3.1- and 4.5-fold, respectively. However, three varieties of green peas (Hakuryu, Kurumeyutaka, and Nankaimidori) significantly lost their VC during the same soaking treatment. Total VC content of a rehydrated and cooked mung beans was higher than that of a dehydrated form. Appreciable amounts of total VC were detected in the immature seeds of six different genera such as yard beans, kidney beans, broad beans, green peas, soybeans and peanuts. Except for mung beans, 70-100% of VC in dehydrated seeds of adzuki beans, broad beans, green peas, black soybeans, and soybeans was lost by boiling. Total VC and L-ascorbic acid in mung beans, green peas, broad beans, black soybeans, and adzuki beans remained even after boiling, suggesting that it is possible to obtain VC from the cooked forms of these legume seeds.

  6. Ethnobotanical study, antifungal activity, phytochemical screening and total phenolic content of Algerian Aristolochia longa

    PubMed Central

    Benarba, Bachir; Meddah, Boumedienne

    2014-01-01

    Aim: Aristolochia longa (from the family Aristolochiaceae) is widely used in Algerian traditional medicine. Here, we document ethnomedicinal uses by local population of Mascara province (West Algeria) and we evaluate the antifungal activity, the phytochemical composition and total phenolic content of aqueous extract (decoction) of the roots of A. longa from Algeria. Materials and Methods: The ethnobotanical investigation was carried out in Mascara Province (West Algeria). Antifungal activity was assessed against Saccharomyces cerevisiae. Total phenolic content was measured using the Folin–Ciocalteu’s reagent. Results: Our results showed that A. longa is widely used to treat several ailments such as cancer (38%), skin infections (14%), and diabetes (11%). Crushed roots are commonly used (89%) mixed with honey, milk, water or other medicinal plants. A. longa aqueous extract induced growth inhibition of S. cerevisiae cells in a dose - and time - dependent manner. An effective suppression of S. cerevisiae (97.06% inhibition of proliferation) was obtained at the 500 µg/mL after 72 h. Results of the phytochemical screening revealed that A. longa aqueous extract contained various bioactive compounds, including polyphenols and flavonoids. Total phenolic content in A. longa aqueous extract was found to be 6.07 ± 0.12 mg (gallic acid equivalents)/g. Conclusion: A. longa may be considered as a promising source of new drugs for treating cancer and as a good antifungal agent. PMID:26401365

  7. A preliminary study of a Peruvian diet using dietary analysis and hair mineral content as indicators.

    PubMed

    Tueller, Daniel J; Eggett, Dennis L; Parker, Tory L

    2013-11-01

    Observations among former American residents living long-term in Peru suggested that hair health improved while in Peru. To determine if a Peruvian diet correlates with hair composition, dietary intake of nutrients and mineral content of hair were measured among Peruvian and matched US residents. Selected foods from Peru were also analyzed for mineral and antioxidant content and compared with equivalent foods available in the USA. Statistically significant differences between Peruvian and US residents' hair were found for sodium (decreased in Peru, p = 0.007) and vanadium (decreased in Peru, p = 0.03). Differences in hair composition between residencies may be explained by lower dietary sodium and vanadium intake among Peruvian residents or by lower concentrations of these minerals in Peruvian drinking water. Many significant mineral differences were also identified between Peruvian foods and their US equivalents. Although no statistically significant correlations between dietary intake and hair mineral content were found, results indicate that a Peruvian diet contributes differently to hair composition than a US diet. More research is needed to elucidate the link between a Peruvian diet and specific aspects of hair health.

  8. Regulation of cardiac myosin synthesis: Studies of RNA content in cultured heart cells

    SciTech Connect

    McDermott, P.; Whitaker-Dowling, P.; Klein, I. Cornell Univ., New York, NY )

    1987-11-01

    Contraction regulates the myosin content and the rate of myosin synthesis in cultured neonatal rat heart cells. To further explore the mechanism for this regulation the authors examined various parameters of RNA content and RNA synthesis in contracting versus noncontracting myocytes. While contraction stimulated myosin heavy chain (MHC) synthesis by 72% compared to that of KCl-arrested cells, simultaneous analyses of polysome profiles were no different under the two culture conditions. Incorporation of ({sup 3}H) uridine monophosphate into cellular RNA revealed no change in the rate of total RNA or ribosomal subunits synthesis. In vitro translation of cellular RNA yielded similar incorporation of ({sup 35}S) methionine not trichloroacetic acid precipitable protein. Specific transcription of the MHC gene was examined by dot-blot analysis and was unaltered by contraction. Northern blot analysis of the MHC sequences detected by a cDNA probe revealed an mRNA sequence corresponding to a molecular weight of approximately 30 S. These data suggest that RNA synthesis and RNA content are unaltered by contraction in cultured heart cells and therefore the changes in myosin synthesis may be mediated at a post-transcriptional control level.

  9. Comparative study on the vitamin C contents of the food legume seeds.

    PubMed

    Moriyama, Michie; Oba, Kazuko

    2008-02-01

    We found that dehydrated legume seeds (6 genera, 19 species and cultivated varieties) contained considerable amounts of vitamin C (VC). The average value of total VC content per 100 g of dry weight in dehydrated seeds varied from 0.24 mg (kidney beans) to 4.14 mg (green peas). Yard beans showed highest values among all legumes examined here in the both dehydrated and rehydrated forms (3.19 and 10.8 mg, respectively). By soaking for 16 h in the dark at 20(o)C, total VC contents of black grams and mung beans increased to 3.1- and 4.5-fold, respectively. However, three varieties of green peas (Hakuryu, Kurumeyutaka, and Nankaimidori) significantly lost their VC during the same soaking treatment. Total VC content of a rehydrated and cooked mung beans was higher than that of a dehydrated form. Appreciable amounts of total VC were detected in the immature seeds of six different genera such as yard beans, kidney beans, broad beans, green peas, soybeans and peanuts. Except for mung beans, 70-100% of VC in dehydrated seeds of adzuki beans, broad beans, green peas, black soybeans, and soybeans was lost by boiling. Total VC and L-ascorbic acid in mung beans, green peas, broad beans, black soybeans, and adzuki beans remained even after boiling, suggesting that it is possible to obtain VC from the cooked forms of these legume seeds. PMID:18388400

  10. Information content and cross-talk in biological signal transduction: An information theory study

    NASA Astrophysics Data System (ADS)

    Prasad, Ashok; Lyons, Samanthe

    2014-03-01

    Biological cells respond to chemical cues provided by extra-cellular chemical signals, but many of these chemical signals and the pathways they activate interfere and overlap with one another. How well cells can distinguish between interfering extra-cellular signals is thus an important question in cellular signal transduction. Here we use information theory with stochastic simulations of networks to address the question of what happens to total information content when signals interfere. We find that both total information transmitted by the biological pathway, as well as its theoretical capacity to discriminate between overlapping signals, are relatively insensitive to cross-talk between the extracellular signals, until significantly high levels of cross-talk have been reached. This robustness of information content against cross-talk requires that the average amplitude of the signals are large. We predict that smaller systems, as exemplified by simple phosphorylation relays (two-component systems) in bacteria, should be significantly much less robust against cross-talk. Our results suggest that mammalian signal transduction can tolerate a high amount of cross-talk without degrading information content, while smaller bacterial systems cannot.

  11. Sensitivity of global ocean heat content from reanalyses to the atmospheric reanalysis forcing: A comparative study

    NASA Astrophysics Data System (ADS)

    Storto, Andrea; Yang, Chunxue; Masina, Simona

    2016-05-01

    The global ocean heat content evolution is a key component of the Earth's energy budget and can be consistently determined by ocean reanalyses that assimilate hydrographic profiles. This work investigates the impact of the atmospheric reanalysis forcing through a multiforcing ensemble ocean reanalysis, where the ensemble members are forced by five state-of-the-art atmospheric reanalyses during the meteorological satellite era (1979-2013). Data assimilation leads the ensemble to converge toward robust estimates of ocean warming rates and significantly reduces the spread (1.48 ± 0.18 W/m2, per unit area of the World Ocean); hence, the impact of the atmospheric forcing appears only marginal for the global heat content estimates in both upper and deeper oceans. A sensitivity assessment performed through realistic perturbation of the main sources of uncertainty in ocean reanalyses highlights that bias correction and preprocessing of in situ observations represent the most crucial component of the reanalysis, whose perturbation accounts for up to 60% of the ocean heat content anomaly variability in the pre-Argo period. Although these results may depend on the single reanalysis system used, they reveal useful information for the ocean observation community and for the optimal generation of perturbations in ocean ensemble systems.

  12. LCA case study on lawn establishment and maintenance with various peat and compost contents in substrates.

    PubMed

    Silvenius, Frans; Niemeläinen, Oiva; Kurppa, Sirpa

    2016-07-01

    The environmental impacts of the establishment and maintenance of lawn, including the production and use of various substrates, were analyzed by life cycle assessment (LCA). The project focused on comparing substrates with different peat and compost contents using pilot substrates and developed a calculation tool to optimize landscaping from an ecological perspective. The impact categories were climate change, aquatic eutrophication, acidification, and use of primary energy. Life cycle assessment methodology and ISO standards 14040 and 14044 were used. Two thousand tons of substrates per hectare of lawn area were assumed to be needed; this large amount explains the importance of the substrate properties for all of the impact categories. Degradation of peat was the most significant factor of the influence of climate; thus, the most effective means of reducing the impact of landscaping on climate is to replace peat with compost. Nitrous oxide and methane emissions were related to the use of compost, but most of these emissions will occur regardless of how the sludge or biowaste is treated. Ammonia emissions from composting were the most important factor for acidification. The significance of fuel consumption by machinery in lawn establishment and mowing was low. The high contents of N and P in compost-based substrates may lead to high nutrient emissions into water systems, which can have significant local impact. The tool helps optimize substrate contents to minimize the environmental effects. Integr Environ Assess Manag 2016;12:459-464. © 2016 SETAC.

  13. Developing a Conceptual Framework for Evaluation of E-Content of Virtual Courses: E-Learning Center of an Iranian University Case Study

    ERIC Educational Resources Information Center

    Akhavan, Peyman; Arefi, Majid Feyz

    2014-01-01

    The purpose of this study is to obtain suitable quality criteria for evaluation of electronic content for virtual courses. We attempt to find the aspects which are important in developing e-content for virtual courses and to determine the criteria we need to judge for the quality and efficiency of learning objects and e-content. So we can classify…

  14. First Aid and Transportation Course Contents Based on Experience gained in the Iran-Iraq War: a Qualitative Study

    PubMed Central

    Sarhangi, Forogh; Gholami, Hamid Reza; Khaghanizade, Morteza; Najafi Mehri, Soheil

    2015-01-01

    Background: Effective first aid and transportation influences injury-induced mortality. But few qualitative studies have been conducted so far in this area. Objectives: The aim of this study was to identify the content of the first aid and patient transportation course based on experience gained from the Iran-Iraq war. Patients and Methods: This was a conventional qualitative content analysis study; a purposeful sample of 14 first aid and transportation experts who had worked during the Iran-Iraq war was recruited. We collected and analyzed the study data by using the semi-structured interview method and the conventional content analysis approach respectively. Each interview transcript was reviewed several times. Words, sentences, and paragraphs were labeled with codes. Codes were compared with each other and categorized according to their similarities. Similar sub-categories and categories were also grouped together and formed themes. Results: Study participants’ experiences of wartime first aid and transportation (FAT) education fell into two main themes including ‘the congruence of education and educational needs’ and ‘managers’ engagement in FAT education. The four main categories of these two themes were use of appropriate educational facilities, adopting effective teaching strategies, universal FAT education and specialized training skills. Conclusions: The two key requirements of the first aid and transportation courses are practicality and managerial engagement. We developed and provided specific guidance of FAT curriculum by using the study findings. This curriculum is recommended for educating FAT staffs, paramedics, emergency technicians, and military nurses. PMID:25825700

  15. A modelling study of the effects of neutral air winds on electron content at mid-latitudes in winter

    NASA Astrophysics Data System (ADS)

    Sethia, G. C.; Hargreaves, J. K.; Bailey, G. J.; Moffett, R. J.

    1984-05-01

    A modelling study of the effects of neutral air winds on the electron content of the mid-latitude ionosphere protonosphere in winter has been made. The theoretical models are based on solutions of time dependent momentum and continuity equations for oxygen and hydrogen ions. The computations are compared with results from slant path observations of the ATS-6 radio beacon made at Lancaster (U.K.) and Boulder, Colorado (U.S.A.). It is found that the magnitude of the poleward neutral air wind velocity has a strong effect on the general magnitude of the electron content, but that the daily pattern of electron content variation is relatively insensitive to changes in the magnitude and phase of the wind pattern. These results are in contrast with the behavior reported previously (Sethia et al., 1983) for summer conditions. However, the night-time electron content is increased by advancing the phase of the neutral air wind and decreased by retarding it. It appears that day-to-day variations in the electron content pattern in winter cannot be explained as effects of changing neutral air winds, which again contrasts with the findings for summer. As in summer, the wind has a major effect on the filling of the protonosphere, but in opposite sense. It is argued that the effect of the neutral air wind on the ionospheric and the protonospheric electron contents depends on the duration of the poleward wind in relation to daylight and on whether or not the wind reverses direction whilst the ionosphere is sunlit.

  16. The neural time course of art perception: an ERP study on the processing of style versus content in art.

    PubMed

    Augustin, M Dorothee; Defranceschi, Birgit; Fuchs, Helene K; Carbon, Claus-Christian; Hutzler, Florian

    2011-06-01

    A central prerequisite to understand the phenomenon of art in psychological terms is to investigate the nature of the underlying perceptual and cognitive processes. Building on a study by Augustin, Leder, Hutzler, and Carbon (2008) the current ERP study examined the neural time course of two central aspects of representational art, one of which is closely related to object- and scene perception, the other of which is art-specific: content and style. We adapted a paradigm that has repeatedly been employed in psycholinguistics and that allows one to examine the neural time course of two processes in terms of when sufficient information is available to allow successful classification. Twenty-two participants viewed pictures that systematically varied in style and content and conducted a combined go/nogo dual choice task. The dependent variables of interest were the Lateralised Readiness Potential (LRP) and the N200 effect. Analyses of both measures support the notion that in the processing of art style follows content, with style-related information being available at around 224 ms or between 40 and 94 ms later than content-related information. The paradigm used here offers a promising approach to further explore the time course of art perception, thus helping to unravel the perceptual and cognitive processes that underlie the phenomenon of art and the fascination it exerts.

  17. Learning science content through socio-scientific issues-based instruction: a multi-level assessment study

    NASA Astrophysics Data System (ADS)

    Sadler, Troy D.; Romine, William L.; Sami Topçu, Mustafa

    2016-07-01

    Science educators have presented numerous conceptual and theoretical arguments in favor of teaching science through the exploration of socio-scientific issues (SSI). However, the empirical knowledge base regarding the extent to which SSI-based instruction supports student learning of science content is limited both in terms of the number of studies that have been conducted in this area and the quality of research. This research sought to answer two questions: (1) To what extent does SSI-based instruction support student learning of science content? and (2) How do assessments at variable distances from the curriculum reveal patterns of learning associated with SSI-based instruction? Sixty-nine secondary students taught by three teachers participated in the study. Three teachers implemented an SSI intervention focused on the use of biotechnology for identifying and treating sexually transmitted diseases. We found that students demonstrated statistically and practically significant gains in content knowledge as measured by both proximal and distal assessments. These findings support the claim that SSI-based teaching can foster content learning and improved performance on high-stakes tests.

  18. FT-IR study of the effect of zinc exposure on the biochemical contents of the muscle of Labeo rohita

    NASA Astrophysics Data System (ADS)

    Palaniappan, PL. RM.; Renju, V. B.

    2009-01-01

    Heavy metal pollution is a major environmental problem in the modern world due to increasing human activities. Zinc is an essential element involved in a wide variety of cellular processes. However, it becomes toxic when elevated concentrations are introduced into the environment. The goal of the present study is to investigate the effect of zinc exposure on the biochemical contents of the muscle tissues of freshwater species Labeo rohita using Fourier transform infrared (FT-IR) spectroscopy. Since the muscle constitutes the greatest mass of the fish that is consumed, the present study has paid particular attention to muscle component. The result reveals that the zinc exposure causes significant changes in the biochemical contents of the L. rohita muscle tissues. In addition, it causes an alteration in the protein secondary structures by decreasing the α-helix and increasing the β-sheet contents of muscle tissues. Further, it has been observed that the administration of chelating agent D-penicillamine improves the protein and lipid contents in the muscle tissues compared to zinc exposed tissues. This result shows that D-penicillamine is the effective chelator of zinc in reducing the body burden of L. rohita fingerlings.

  19. Chronic loss of noradrenergic tone produces β-arrestin2-mediated cocaine hypersensitivity and alters cellular D2 responses in the nucleus accumbens.

    PubMed

    Gaval-Cruz, Meriem; Goertz, Richard B; Puttick, Daniel J; Bowles, Dawn E; Meyer, Rebecca C; Hall, Randy A; Ko, Daijin; Paladini, Carlos A; Weinshenker, David

    2016-01-01

    Cocaine blocks plasma membrane monoamine transporters and increases extracellular levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT). The addictive properties of cocaine are mediated primarily by DA, while NE and 5-HT play modulatory roles. Chronic inhibition of dopamine β-hydroxylase (DBH), which converts DA to NE, increases the aversive effects of cocaine and reduces cocaine use in humans, and produces behavioral hypersensitivity to cocaine and D2 agonism in rodents, but the underlying mechanism is unknown. We found a decrease in β-arrestin2 (βArr2) in the nucleus accumbens (NAc) following chronic genetic or pharmacological DBH inhibition, and overexpression of βArr2 in the NAc normalized cocaine-induced locomotion in DBH knockout (Dbh -/-) mice. The D2/3 agonist quinpirole decreased excitability in NAc medium spiny neurons (MSNs) from control, but not Dbh -/- animals, where instead there was a trend for an excitatory effect. The Gαi inhibitor NF023 abolished the quinpirole-induced decrease in excitability in control MSNs, but had no effect in Dbh -/- MSNs, whereas the Gαs inhibitor NF449 restored the ability of quinpirole to decrease excitability in Dbh -/- MSNs, but had no effect in control MSNs. These results suggest that chronic loss of noradrenergic tone alters behavioral responses to cocaine via decreases in βArr2 and cellular responses to D2/D3 activation, potentially via changes in D2-like receptor G-protein coupling in NAc MSNs.

  20. Chronic loss of noradrenergic tone produces β-arrestin2-mediated cocaine hypersensitivity and alters cellular D2 responses in the nucleus accumbens.

    PubMed

    Gaval-Cruz, Meriem; Goertz, Richard B; Puttick, Daniel J; Bowles, Dawn E; Meyer, Rebecca C; Hall, Randy A; Ko, Daijin; Paladini, Carlos A; Weinshenker, David

    2016-01-01

    Cocaine blocks plasma membrane monoamine transporters and increases extracellular levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT). The addictive properties of cocaine are mediated primarily by DA, while NE and 5-HT play modulatory roles. Chronic inhibition of dopamine β-hydroxylase (DBH), which converts DA to NE, increases the aversive effects of cocaine and reduces cocaine use in humans, and produces behavioral hypersensitivity to cocaine and D2 agonism in rodents, but the underlying mechanism is unknown. We found a decrease in β-arrestin2 (βArr2) in the nucleus accumbens (NAc) following chronic genetic or pharmacological DBH inhibition, and overexpression of βArr2 in the NAc normalized cocaine-induced locomotion in DBH knockout (Dbh -/-) mice. The D2/3 agonist quinpirole decreased excitability in NAc medium spiny neurons (MSNs) from control, but not Dbh -/- animals, where instead there was a trend for an excitatory effect. The Gαi inhibitor NF023 abolished the quinpirole-induced decrease in excitability in control MSNs, but had no effect in Dbh -/- MSNs, whereas the Gαs inhibitor NF449 restored the ability of quinpirole to decrease excitability in Dbh -/- MSNs, but had no effect in control MSNs. These results suggest that chronic loss of noradrenergic tone alters behavioral responses to cocaine via decreases in βArr2 and cellular responses to D2/D3 activation, potentially via changes in D2-like receptor G-protein coupling in NAc MSNs. PMID:25123018